CLINICAL EVALUATION OF AMNIOTIC MEMBRANE GRAFT FOR

MANAGEMENT OF CORNEAL ULCER IN DOGS

THESIS

Submitted
in the partial fulfillment of the requirements for the Degree of

MASTER OF VETERINARY SCIENCE

IN
VETERINAY SURGERY AND RADIOLOGY
BY

ATTAR TAYYAB ISMAIL

Enrolment No: V/13/066

Krantisinh Nana Patil College of Veterinary Science,

Shirwal, Dist- Satara (M.S.)

MAHARASHTRA ANIMAL AND FISHERY SCIENCES

UNIVERSITY, NAGPUR – 440 001
(INDIA)

2022
 DECLARATION OF THE STUDENT

I hereby declare that the experimental research work and

interpretation of the thesis entitled “CLINICAL EVALUATION OF
AMNIOTIC MEMBRANE GRAFT FOR MANAGEMENT OF
CORNEAL ULCER IN DOGS” or part
thereof has not been submitted for any of the other degree or diploma of
any university, nor the data have been derived from any thesis or
publications of any university or scientific organization. The sources of
material used and all assistance
 DECLARATION OF ADVISORY COMMITTEE

Mr. ATTAR TAYYAB ISMAIL has satisfactorily prosecuted his course

of research for a period of not less than one semester for M. V. Sc. and that the
thesis entitled “CLINICAL EVALUATION OF AMNIOTIC MEMBRANE
GRAFT FOR MANAGEMENT OF CORNEAL ULCER IN DOGS”
submitted
by him is the result of research work is sufficient to warrant its presentation to the
examination in subject of Veterinary Surgery and Radiology for the award of
Master of Veterinary Science degree by the Maharashtra Animal and Fishery
Sciences University, Nagpur.
We also certify that the thesis or part thereof has not been previously
submitted by him for a degree of any other University.

Date: / /2022 (Dr. A. H. Ulemale)

Advisor/Guide
Associate Professor
Dept. of Veterinary Surgery & Radiology
.
Advisory Committee

Name Designation Signature

1. Dr. S. P. Salvekar, Assistant Professor
Dept. of Veterinary
Surgery & Radiology

2. Dr. R. V. Suryawanshi, Hospital Registrar

Dept. of Veterinary
Surgery and Radiology, VCC

3. Dr. V. S. Dhaygude, Assistant Professor & Head

Dept. of Veterinary Pathology

4. Dr. A. B. Mali, Hospital Registrar

Dept. of Animal Reproduction,
Gynaecology and Obstetrics, VCC
 CERTIFICATE

This is to certify that the thesis entitled “CLINICAL EVALUATION OF

AMNIOTIC MEMBRANE GRAFT FOR MANAGEMENT OF CORNEAL
ULCER IN DOGS” submitted by Mr. .ATTAR TAYYAB ISMAIL to the
Maharashtra Animal and Fishery Sciences University, Nagpur, in partial
fulfillment of the requirement for the degree of Master of Veterinary Science
has been approved by the Student’s Advisory Committee after examination in
collaboration

Dr. V. D. Aher Dr. A. H. Ulemale

Name and Signature of Professor and Advisor/Guide
External Examiner Head of Associate Professor

Advisory Committee

Name Designation Signature

1. Dr. S. P. Salvekar Assistant Professor

Dept. of Veterinary
Surgery & Radiology

2. Dr. R. V. Suryawanshi Hospital Registrar

Dept. of Veterinary
Surgery & Radiology, VCC

3. Dr. V. S. Dhaygude Assistant Professor & Head

Dept. of Veterinary Pathology

4. Dr. A. B. Mali Hospital Registrar

Dept. Animal Reproduction,
Gynaecology and Obstetrics, VCC

Associate Dean
å

Dedicated to
My
Family….
Acknowledgement
 ACKNOWLEDGEMENT

At this moment, I wish to express my feelings for all those who were involved in my journey. I
want to thank each and every person who made a difference in my life and want to say that everything was
possible through the invaluable contribution of a number of people. There are not enough to express
my gratitude.
My deepest gratitude goes to my family for their untiring love and support throughout my life. I
fall short of words to acknowledge in so formal manner. I am what I am today because of the sacrifice,
inspiration, silent prayers, support and indefinite love rendered by my beloved family.
I also want to take a moment to thank my guru and inspiration, Dr. Sangeeta
Vengsarkar Shah (small animal cardiologist).
I would like to acknowledge my indebtedness and render my warmest thanks to my major advisor
Dr A. H. Ulemale , Associate Professor, Department of Veterinary Surgery and Radiology
who made this work possible, his friendly guidance ,time to time valuable suggestions and his legacy to
bring out the best in his students both in studies as well in life.
I am deeply indebted to Dr. S.P. Salvekar Assistant Professor, Dept. of
Surgery and Radiology, KNPCVS for her inspiration, guidance, expert advice, timely
supervision and constructive criticism. Without her pat on my back, it would have been impossible to
accomplish this milestone in my life.
I would like to thank Dr. R.V. Suryawanshi, Hospital registrar Dept. of
VSR,VCC,KNPCVS for introducing me to the world of surgery and piquing my
interest in the subject even as an undergraduate student.
I also want to express my gratitude to worthy member of my advisory Committee namely Dr.
V.S. Dhaygude, Assistant Professor, Department of Veterinary Pathology for their
encouraging attitude, meticulously scrutinizing this manuscript and offering valuable Suggestions.
I also want to express my gratitude to another worthy member of my advisory Committee,
Dr.A.B Mali, Hospital registrar department of ARGO,VCC.
I am grateful to Dr. V.D. Aher Associate Dean KNPCVS and also
Head Dept. of Veterinary Surgery and Radiology,for providing me with all the facilities to carry
out my research work.

i
 It is my privilege to express my gratitude and sincere thanks from the bottom of my heart to
Dr Kasturi Bhadsavle for constant Support and enthusiasm which encouraged me to undertake this valuable
research work. I will always remain indebted to her for her untiring efforts in successful completion of this
research work.
. I owe a special word of thanks to Dr Kamalkumar Jiddimani, for his invaluable help
throughout my post-graduation and my research work. I wish to express a special gratitude to
my seniors Dr. Mahesh Dhangar, Dr.Amol Kawhale , Dr. Rahul Gondake, Dr. Uday
Bagal and Dr. Pushpnath Chaugule for believing in me and providing me with all the help.
I sincerely appreciate the ever-willing co-operation of my departmental colleagues Dr Shubham
Pant, Dr Toufik Shaikh because of whose help I was able to complete my degree and made it
an unforgettable journey of my life. I also want to thank Dr Roshan Mehar and my junior
colleagues, Dr. Kiran K.S, Dr Pratik Sudrik, Dr. Devika Deshpande and especially Dr.
Jayant Sukare .I also want to put on record special thanks to chetan Pawar who helped me to
accomplish my research work.
My sense of gratitude to one and all those who have helped me either directly or indirectly in
this venture. It is extremely difficult to thank all of them individually by name. This shortcoming may be
pardoned.
I express my sincere gratitude to my alma mater Krantisinh Nana Patil College
of Veterinary Science, Shirwal for giving me this opportunity to learn and grow. I will always cherish
the time I spent in this institute and made my dream come true.

Place: Shirwal Mr. ATTAR TAYYAB

ISMAIL Date: / / 2022 V/13/0FF

ii
 TABLE OF CONTENTS

SR. NO. TITLE PAGE NO.

I INTRODUCTION 1-4

II REVIEW OF LITERATURE 5-33

III MATERIALS AND METHODS 34-42

IV RESULTS AND DISCUSSION 43-59

V SUMMARY AND CONCLUSIONS 60-63

A BIBLIOGRAPHY i-xv

B VITA xvi

C THESIS ABSTRACT xvii-xviii

D गोषवारा xix-xx
 LIST OF TABLES

SR. BETWEEN
NO. CONTENTS PAGES

44-45
4.1 Signalment and anamnesis of dogs from group-I and group-II
(n=12).
46-47
4.2 Mean (±S.E) physiological parameters recorded in dogs from
group-I and group-II (n=12).

46-47
4.3 Preoperative evaluation of vision test viz: Menace response
(M), Dazzle reflex (D), Pupillary light reflex (PLR), corneal
edema and corneal opacity in dogs from group-I and group-II
(n=12).
48-49
4.4 Classification of Ulcers on the basis of depth in dogs from
group-I and group-II (n=12).

50-51
4.5 Postoperative evaluation of vision tests viz: Menace
Response, Dazzle reflex Pupillary Light reflex on day 4, 8,
and 12 in dogs with corneal ulcers (n=12).
52-53
4.6. Evaluation of Schirmer Tear Test on day 0, 4, 8 and 12 in
dogs from group-I and group-II (n=12).

54-55
4.7 Evaluation of Fluorescein Dye Test on day 0, 4, 8 and 12th
day in dogs from group-I and group-II (n=12).

54-55
4.8 Mean (±S.E) of Hematological parameters on preoperative
day and on 12th post- operative day in dogs from group-I and
group-II (n=12).
56-57
4.9 Postoperative appearance of cornea in dogs treated with dry
& wet human amniotic membrane graft on 4th , 8th and 12th
post-operative day in dogs from group-I and group-II (n=12).
 LIST OF PLATES

Sr. No. CONTENTS Page

3.1 Photograph showing lateral positioning of dog under operating A
microscope.
Photograph showing placing of eye speculum along with stay A
3.2 suture.
3.3 Photograph showing debridement of loose epithelium with the B
help of sterile cotton bud.
4.1 Photograph showing corneal ulcer in Shih Tzu breed of dog. C

4.2 Photograph showing corneal ulcer in Rottweiler breed of dog. C

4.3a Photograph showing centrally located corneal ulcer in Dog no. D

2.
4.3b Photograph showing centrally located corneal ulcer in Dog D
no.8.
4.4 Photograph showing ventro nasally located corneal ulcer in D
Dog no. 5.
4.5 Photograph showing ventro temporally located corneal ulcer in E
Dog no. 12.
4.6 Photograph showing dorso nasally located corneal ulcer in E
Dog no.4.
4.7 Photograph showing dorso temporally located corneal ulcer in E
Dog no.7.
4.8 (A) Photograph showing corneal edema and opacity noticed by F
direct ophthalmoscopy on day 0 prior to membrane grafting.
4.8 (B) Photograph showing corneal edema and opacity noticed by G
direct ophthalmoscopy on day 0 prior to membrane grafting
4.9 Photograph showing site of retrobulbar nerve block. H

4.10 Photograph showing preparation of ulcer bed for grafting. H

4.11 Photograph showing ulcer bed prepared for grafting. H

4.12 Photograph showing grafting of cornea with dry human I

amniotic membrane.
4.13 Photograph showing grafting of cornea with wet human I
amniotic membrane.
4.14 Photograph showing Tarsorrhaphy suture. I

4.15 Photograph showing Schirmer tear test. J

4.16 Photograph showing clear patch at the center in case of J

descemetocele.
4.17 Photograph showing staining of u l c e r patch by u s i n g J
fluorescein dye.
4.18 (a) Photograph showing Neutrophils as predominant cell type K
along with few epithelial cells on day 12, in Dog no. 2
(Giemsa stain 1000x).
4.18 (b) Photograph showing only epithelial cells on day 0, in Dog no. K
2 (Giemsa stain 1000x).
4.19 (a) Photograph showing neutrophils as predominant cell type K
along with few epithelial cells on day 12, in Dog no. 5
(Giemsa stain 1000x).
4.19 (b) Photograph showing only epithelial cells on day 0, in Dog no. K
5 (Giemsa stain 1000x).
4.20 (a) Photograph showing neutrophils as predominant cell type L
along with few epithelial cells on day 12, in Dog no. 7
(Giemsa stain 1000x).
4.20 (b) Showing only epithelial cells on day 0, in Dog no. 7 (Giemsa L
stain 1000x).
4.21 (a) Photograph showing neutrophils as predominant cell type L
along with few epithelial cells on Day 12 i n Dog no. 10
4.21 (b) Photograph showing only epithelial cells on day 0, in Dog no. L
10 (Giemsa stain 1000x).
4.23 Photograph showing post-operative appearance viz: corneal M
(A) edema, corneal opacity and corneal healing in Dog no. 1.
4.23 Photograph showing post-operative appearance viz: corneal N
(B) edema, corneal opacity and corneal healing in dog no. 3.
4.24 Photograph showing post-operative appearance viz: corneal O
(A) edema, corneal opacity and corneal healing in Dog no. 7.
 LIST OF ABBREVIATIONS

No Abbreviation
% Percent
μ Micro
μg Microgram
ºC Degree Celsius
AM Amniotic membrane
BID Bis in die
CBC Complete blood count
dl Decilitre
DLC Differential leukocyte count
EDTA Ethylene diamine tetra acetic acid
FGF Fibroblast growth factor
hAm Human amniotic membrane
Hb Hemoglobin
Hrs Hours
IOP Intraocular pressure
Kg Kilogram
MCV Mean corpuscular volume
MCHC Mean corpuscular hemoglobin
Mg Milligram
Min Minute
ml Milliliter
mm Millimeter
PO Per os
PLR Papillary light reflex
SIS Small Intestine Sub Mucosa
STT Schirmer tear test
QID Quater in die
TID Ter in die
TLC Total leukocyte count
VCC Veterinary Clinical Complex
Introduction
 CHAPTER I

INTRODUCTION

The eye is a gateway to the outside world and is referred to as the only organ
of vision and a biological camera (Dutta, 2006). Good vision serves as a crucial
component for companion animals as an indication of well-being, maintaining their
independence and quality of life (Warren, 2004). The pet owners nowadays are more
concerned about the health of their pet’s eye and show keen interest in treatment of
conditions affecting vision of their pets.

Corneal ulcers,iris prolapse, cataract, dermoid, proptosis, cherry eye,

keratoconjunctivitis sicca, glaucoma, lid lacerations, neoplasms of the globe and
adnexa are the most frequent ocular disorders affecting vision in dogs. Amongst
them, corneal ulcers are the most prevalent (Gilger et al., 2007).

The playful and vibrant nature of young dogs (1-3 years) predisposes them to
the higher incidence of corneal ulcers than any other eye affection (Ramani et al.,
2013). (Moore, 2003) recorded breed wise predilection of corneal ulcers and
attributed it to anatomical differences of craniofacial and eyelid confirmation
especially in brachycephalic breeds like Pug, Shih Tzu, Boxer and Rottweiler.

Cornea acts as a portal for light to enter and generates the image. It is a
structural and physiological border to the outside world that must fulfill unique
requirements such as light refraction, transparency and resistance to external hazards
(Nautscher et al., 2015). Anatomically, canine cornea has five different layers
consisting of the precorneal tear film, anterior epithelium, central stroma, descemet's
membrane and the posterior endothelium. Special features such as lack of blood
vessels, lamellar nature of stromal collagen fibrils, non-keratinized nature of the
anterior epithelium and rich supply of sensory nerves makes the cornea transparent
and clear. Anything that interferes with this normal stromal architecture, disrupts the
physio-anatomical and functional aspects of the cornea at a varying severity viz.
blood vessel migration, increased corneal pigmentation, corneal edema and corneal
scaring

1
that affects the corneal transparency and eventually results in vision loss (Gelatt and
Brooks 2011).

Slatter (2001) described corneal ulcers as a break in continuity of the corneal

epithelium, with or without loss of corneal stroma. (Moore, 2003) classified corneal
ulcers based its depth and severity, viz: superficial, profound descemetocele or
perforated corneal ulcers. The ulcers are also classified on the basis of etiology as,
infectious (bacterial, viral, or mycotic) or noninfectious. Noninfectious causes include
recurrent erosions, trauma induced superficial ulcer, ulcers due to distichiasis,
trichiasis, entropion and prominent facial folds (Gilger et al., 2007).

Corneal ulcers could be best diagnosed clinically with fluorescein dye test
along with ancillary tests like Schirmer tear test, corneal cytology, microbiological
investigations and direct ophthalmoscopy. Fluorescein dye test is one of the most
effective methods for detecting corneal defects, conjunctival epithelial defects and
precorneal tear-film inadequacies. The hydrophilic fluorescein dye does not stain the
intact and healthy corneal epithelium or Descemet's membrane. When the corneal
integrity is compromised, dye enters the water-soluble corneal stroma and stains only
the intracellular compartments (Slatter, 1990).

Schirmer tear test measures the increased or decreased tear production thus,
proving to be useful guide for diagnosis of keratoconjunctivitis sicca (Anoop et al.,
2015).Corneal cytology reveals the causative agents responsible for corneal affections
externally, whereas inside of the cornea can be visualized with direct ophthalmoscopy
while exploring the anterior chamber of the eye. Kern (2004) expressed that for
determining the best course of treatment for different types of corneal ulcers
necessitates the exact diagnosis.

Damaged epithelial wall frequently allows invasion of secondary infection,

followed by progressive stromal ulceration by collagenases and other proteolytic
enzymes risking the perforation. Continuous leakage of aqueous humor occurs
through the perforation causing enlargement of anterior chamber. Ultimately all these
events lead to anterior synechiae, glaucoma, cataracts, endophthalmitis and
vision loss

2
(Startup, 1984). Therefore preservation of eyesight necessitates a timely and
appropriate treatment of corneal ulcers.
The principles of treatment for corneal ulcer includes removal of fundamental
cause, reducing inflammation, controlling infection, improving corneal healing,
minimizing corneal scarring and replacing damaged corneal tissue (Gelatt, 1991).
While treating deep corneal ulcers, it is essential to provide mechanical support to the
damaged cornea in conjunction with effective medical therapy (Boruchoff and Foulks
1990; Dawson and Sanchez 2015). Suturing, tissue adhesives, a soft contact lens, as
well as conjunctival flaps and grafts can be used to accelerate ulcer healing (Severin,
1995).
Whitley (1991) strongly recommended surgical therapy for all the deep
corneal ulcers, recurring superficial corneal ulcers and stromal melting cases.
Keratotomy and epithelial debridement can be used to treat superficial corneal ulcers
or epithelial erosions. The choice of surgical treatment employed depends on the
depth and size of the corneal defect (Wilkie and Whittaker 1997).
Various surgical techniques have been successfully tried for treating corneal
ulcers by using third eyelid flap, corneo-scleral grafting, conjunctival grafts, corneal
transplantation by porcine small intestinal sub mucosa (SIS) graft and autologous
lamellar corneal graft. Reconstructive techniques by using cryopreserved amniotic
membrane grafts of various species viz canine, equine, bovine and human for
accelerated healing of corneal ulcers have also been used successfully.
Considering the reconstructive methods, (Wichayacoop et al., 2005) reported
that human amniotic membrane (HAM) grafting had excellent results over healing
of induced corneal ulcers in dogs. The amniotic membrane functions as a substrate
for epithelial growth and also enhances the axonal regeneration. It also helps in
epithelialization by facilitating the migration of the epithelial cells, strengthening the
adhesion of basal epithelial cells by promoting its differentiation and by preventing
apoptosis (Barros et al., 2005).
Preserved/processed HAM has the ability to inhibit proteinase activity and
infiltration of inflammatory cells. Fresh and cryopreserved HAM possess anti-

3
inflammatory and anti angiogenic activity (Hao et al., 2000 and kim et al., 2000).
Stromal matrix of preserved amniotic membrane suppresses the expression of the
inflammatory cytokines and interleukins Iα (IL-α), IL-β, IL-2, IL-8, Interferon γ,
TNF- α, platelet derived growth factor and β-fibroblast growth factor (Solomon et al.,
2001) resulting in minimal scar formation, neovascularization and fibrosis.
Considering the high prevalence and clinical relevance of corneal ulcers,
more research is needed to develop improved treatment options in small animal
practice. Hence, the present study is designed to assess the efficacy of dry and wet
human amniotic membrane grafts for repair of corneal ulcerations in canine with the
following objectives.

1. Identification of corneal ulcers.

2. To evaluate the efficacy of amniotic membrane graft in the healing of corneal ulcers
in canine.
3.Comparison of dry human amniotic membrane graft with wet human
amniotic membrane graft for management of corneal ulcers.

4
Review of Literature
 CHAPTER II

REVIEW OF LITERATURE

The present study, entitled "Clinical Evaluation of Amniotic Membrane Graft

for Management of Corneal Ulcer in Dogs," was conducted at a veterinary clinical
complex, KNPCVS Shirwal, in clinical cases of dogs that seem to have a history of
corneal ulcer. The available literature on the etiology, diagnosis, and treatment of
corneal ulcers in dogs is reviewed under the topics listed below.

2.1 Anatomy and Physiology of Cornea.

2.2 Incidence of Corneal Ulcers:-
2.2.1 Age wise Distribution
2.2.2 Sex wise Distribution
2.2.3 Breed wise Distribution
2.3 Grading and Classification of Corneal Ulcers
2.3.1 Superficial Ulcers
2.3.2 Deep Stromal Ulcers
2.3.3 Melting Ulcers
2.3.4 Indolent Ulcers
2.3.5 Descemetocele
2.4 Causative Factors and Etiology of Corneal Ulcers
2.5 Clinical Signs Associated With Corneal Ulcers
2.5.1 Corneal Edema
2.5.2 Corneal Vascularization
2.5.3. Corneal Pigmentation
2.6 Diagnostic Techniques for Corneal Ulcers
2.6.1 Ophthalmic Examination
2.6.2 Visual Function Tests
2.6.2.1 Ocular Reflexes

5
 2.6.2.2 Menace Response
2.6.2.3. Dazzle Reflex
2.6.2.4 Pupillary Light Reflex (PLR)
2.6.3 Direct Ophthalmoscopy
2.6.4 Schirmer Tear Test
2.6.5 Fluorescein Dye Test
2.6.6 Corneal Cytology
2.7 Hematology
2.8 Medical Management of Corneal Ulcers
2.9 Surgical Management of Corneal Ulcers
2.10 Human Amniotic Membrane Graft for Healing of Corneal Ulcers
2.11 Complications Associated with Corneal Ulcers

2.1 Anatomy and Physiology of Cornea

Renwick (1996) stated that the cornea is made up of one-fifth of the fibrous
tunic of the eye and that its key features are avascularity, non-myelinated nerves,
non- keratinized epithelium and regular organization of collagen lamellae in corneal
stroma and a relatively dehydrated state.

Marfurt et al., (2001) observed that the anterior stroma of the canine cornea
was densely innervated, but the posterior stroma was free of innervations except for a
few nerve fibres located next to the corneal endothelium.

Robert et al., (2001) found that, the cornea is a highly differentiated tissue
that is rich in extracellular collagen matrix (ECM), which ensures its dual functions
of transparency and protection of inner eye structures. The precise regulation of the
diameter and orientation of collagen fibers and interfibrillar gaps revealed
relationships between glycosaminoglycan and collagen formation, which alter with
age (Maillard reaction) and in numerous pathological circumstances such as corneal
dystrophies and wound healing.

6
 Abrams et al. (2002) studied the epithelial basement membranes and
endothelial (Descemet's) basement membranes of the canine cornea and reported that
the surface of the epithelial and endothelial basement membranes was a complex
meshwork of pores and fibers.
Xie et al. (2002) observed that corneal components were highly organized and
avascular, resulting in minimum light scattering and rendering the cornea clear.
Because of its curvature and physio-anatomical characteristics, the cornea contributed
two-thirds of the overall refractive power of the eye's optical system. Corneal health
was maintained not only by aqueous humour, which supplies it with glucose, amino
acids, and electrolytes, but also by tears, which serves as a vehicle for the provision
of air oxygen.

Andrew (2008) stated that, cornea consists of stratified squamous epithelium,

epithelial basement membrane, corneal stroma, descemet's membrane and
endothelium. The only documented inflammatory cells in the cornea are a few
lymphocytes in the epithelium and a few leukocytes in the stroma. The central cornea,
long assumed to be immune-privileged, was shown to be composed of epithelial cells,
langerhans cells and stromal precursor dendritic cells.

2.2 Incidence of Corneal Ulcers.

2.2.1 Age wise Distribution
Turner and Blogg (1997) reported that the incidence of corneal ulcer was
most predominant in dogs of average 6.5 years of age.
Wilkie and Whittaker (1997) reported that older dogs and particular dog
breeds such as Boxers, Golden Retrievers, Corgis and Miniature Poodles appeared to
be predisposed to corneal ulcers.
Murphy et al. (2001) recorded that the age group between 1-3 years had the
highest incidence of corneal ulcers (50%) followed by 4-7 years (29%) and above 8
years (21 %).

7
 Moore (2003) reported that indolent corneal ulceration occurred more
frequently in middle to older-aged dogs with a mean age of 8.2 years.
Sood and Mohindroo (2005) observed that dogs with a mean age of 5.46±
1.03 yrs. were more prone to corneal affections.
Kim et al. (2009) found that the incidence of ulcerative keratitis was 47 %, 28
%, 14 %, and 9 % in dogs aged 3 years, 3-6 years, 6-9 years, and 9-12 years,
respectively.
Sale et al. (2013) conducted a study on the occurrence of ocular affections in
different age groups of less than one year, one to two years, two to five years, five to
ten years, and more than ten years. They revealed that ocular diseases occurred in
animals of all ages.

2.2.2 Sex wise Distribution

Turner and Blogg (1997) reported that sex wise incidence of corneal ulcer
was highest in males (80%) compared to females (20%).

Moore (2003) observed that chronic corneal epithelial defects have no sex
predilection.
Ramani et al. (2012) showed that male dogs had a greater frequency of
corneal ulcers than female canines. The number of instances recorded in male dogs
(54.67 percent) is slightly higher than the number of cases reported in female dogs
(45.33 percent).
Ramani et al. (2013) investigated the surgical bacteriology and grading of
corneal ulcers in dogs and concluded that corneal ulcers occurred in 67 percent of
male dogs and 33 percent of female dogs.

2.2.3 Breed wise Distribution

Holmberg (1981) studied the incidence of corneal erosions in dogs and

reported highest incidence in Shih Tzu (73%) followed by Boxers (20%). Dogs below

8
6 months of age were more predisposed towards the development of corneal
ulceration. The incidence was found more in males (80%).

Wolfer and Grahn (1994) noted that brachycephalic breeds are predisposed
to corneal trauma due to globe prominence, lagophthalmos and decreased corneal
sensitivity which leads to ulcerative keratitis and melting ulcers.

Moore (2003) noticed that the boxer is the most prevalent breed susceptible to
developing indolent corneal ulcers. Boxers had a 24.56 percent incidence rate, while
the other 45 breeds included mixed breeds, poodles and poodle crosses, Golden
retrievers, Corgis, Labrador retrievers, Springer spaniels, and German shepherd and
Sheppard crosses.

Ancheril (2004) observed that the incidence of corneal ulcer was 51.85
percent in Spitz, 22.22 percent in Non-Descript, 7.41 percent in Lhasa Apso, and 3.7
percent in Great Dane, German shepherd, Pug and terrier

Thomson (2007) reported that brachycephalic breeds with pushed-in faces such
as the Shih Tzu, Bulldog, French bulldog, Lhasa Apso, Pekingese, and Pug were
more susceptible to ulcerative keratitis.

Kim et al. (2009) observed high incidence of corneal ulcers in the Shih-Tzu
(50%), Pekingese (25%) and Yorkshire terrier (16%) in the study of 32 dogs. The
Maltese Terrier, Pomeranian and Golden Retriever were at low risk.

Ramani et al. (2013) found a higher incidence of corneal ulcers in Pug (33.3
percent), Labrador (16.6 percent), Spitz (12.5 percent), Doberman (8.3 percent),
Mastiff (8.3 percent) and Boxer (8.3 percent).

Dawson and Sanchez (2015) investigated the prevalence of corneal surface

disorders in 100 dogs (199 eyes) in a prospective research. In this study, Seven of the
26 dogs with corneal surface disease were brachycephalic breeds, including two
French Bulldogs, one English bulldog, one Pug, one Cavalier King Charles Spaniel,
one Lhasa Apso, and one Chihuahua.

9
2.3 Grading and Classification of Corneal Ulcers

Mandell (2000) classified corneal defects according to their depth, claiming

that the treatment method to be used was determined by the depth of the ulcer.

Moore (2003) classified corneal ulcers as superficial, deep, and

descemetocele in terms of depth, as well as complex, uncomplicated, refractory, and
progressive in terms of healing ease.

Kecova et al. (2004) observed that, infectious and non-infectious keratitis

were two kinds of keratitis in humans whereas ulcerative and non-ulcerative keratitis
was typically defined in veterinary medicine.

2.3.1 Superficial Ulcers.

Mandell (2000) mentioned in his study that superficial ulcers are frequently
more painful because they involve a more innervated region of the epithelium.

Moore (2003) found that superficial corneal ulcers are comprised of corneal
epithelium and basement membrane, with or without stroma, and called as refractory
if they did not heal within 5-7 days and complicated if they grew in size or depth.

Ollivier (2003) observed that superficial wounds that healed in a few days
were typically not infected; however, antibiotic medication was advised to avoid the
emergence of secondary, opportunistic bacteria since epithelial integrity had been
compromised.

Kim et al. (2009) in their retrospective investigation of ulcerative keratitis of

dogs reported that when medical therapy was offered, superficial ulcers healed
without complications in 5-13 days.

2.3.2 Deep Stromal Ulcers

Ollivier (2003) stated that stromal ulcers are commonly infected ulcers and
are of two types i.e., progressive and non-progressive. Progressive corneal ulcer may
progress to involve deep layers.

10
 Mandell and Holt (2005) stated that if ulcer involves more than a third
of the corneal thickness, they are classified as stromal ulcers, which usually requires
required vascularization for healing and took three weeks to heal. A corneal ulcer is
present when corneal epithelium and a variable amount of stroma are missing. Small
acute ulcers heal rapidly, but chronic lesion may heal slowly if at all.
Maggs (2008) described stromal ulcers as rapidly progressive, complicated
ulcers that have areas of stromal melting, stromal loss or marked cellular infiltration
and assume to be infected.
Williams and Pinard (2013) defined stromal ulcers as complex corneal
ulcers that involve the deeper layers of corneal stroma along with the presence of
bacterial or fungal infection.
Gelatt (2014) stated that ulcerative keratitis extending to the corneal stroma
that usually involves a secondary microbial infection and some degree of proteinase
activity that initiates the stromal destruction are known as stromal ulcers
Williams et al. (2017) described stromal ulcers as ulcers that often fail to
follow a benign self-healing course and can become a sight-threatening emergency
with corneal scarring, thinning and even perforation.

2.3.3 Melting Ulcers

Whitley's (2000) stated that effective management of melting ulcers includes

infection control and minimizing the effect of collagenase and other proteases on the
cornea.

Ollivier (2003) concluded that complex corneal ulcers can be graded as

melting ulcers. When proteases, which are generated by bacteria, inflammatory cells,
corneal epithelial cells, and fibroblasts overpowered their inhibitors.

Goulle (2012) defined melting ulcers as gelatinized and liquefied corneal

stroma caused by severe keratectomy debridement of necrotic and collagenolytic
corneal tissue.

11
2.3.4 Indolent Ulcers

Gelatt and Samuelson (1982) studied histology of the epithelium of indolent

ulcers and found degeneration of basal epithelial cells, thick irregular basement
membrane and a decrease in the number of hemidesmosomes, all of which prevented
epithelial cells adherence to the stroma and resurfacing of corneal epithelium.

Morgan and Abrams (1994) defined that a non-healing (indolent) corneal

ulcer is a superficial epithelial defect of the cornea surrounded by non-adherent
epithelium.

Willeford et al. (1998) examined the levels of proteolysis in the lacrimal fluid
of dogs with normal corneas and those with persistent corneal ulcerations and found
that higher levels of proteolytic activity might be the cause of the problem.

Whitley and Gigler (1999) investigated indolent ulcers, refractory epithelial

erosions, recurrent corneal erosions syndrome and rodent ulcers, which were
superficial corneal ulcers that healed slowly and were characterized by an
overlapping lip of non-adherent epithelium around the ulcer's edge that stained
positive to fluorescein stain and usually occurred bilaterally.

Moore (2003) stated that refractory ulcers were often non-progressive due to
anterior epithelium failing to attach to stroma, which was visible as a "epithelial lip"
at the edges, highlighted by fluorescein stain.

Samuelson and Brooks (2011) stated that indolent corneal ulcers in

brachycephalic breeds could be either primary or secondary to eyelash or eyelid
abnormalities, infection, tear film abnormalities or corneal oedema.

2.3.5 Descemetocele
Wilkie and Whittaker (1997) stated that a deep corneal ulcer which did not
retain fluorescein stain in the central portion was a descemetocele.

12
 Hamilton (1999) defined descemetocele as a protrusion of Descemet's
membrane through the floor of an ulcer caused by the pressure of aqueous humour
behind it.
Featherstone and Stanley (2002) stated that descemetocele is a deep corneal
lesion characterized by complete destruction of the corneal epithelium and stroma,
leaving a lesion surrounded exclusively by descemet’s membrane and corneal
endothelium.
Ledbetter and Gilger (2015) pointed out that Descemetocele are a surgical
urgency due to the risk of infection and intraocular inflammatory injury.
Mitchell (2011) observed that a clear zone at the base of a deep hole in the
cornea following fluorescein dye instillation indicated a descemetocele because the
descemet's membrane did not pick up the lipophobic stain.

2.4 Causative Factors and Etiology of Corneal Ulcers.

Willeford et al. (1998) concluded that corneal sequestration, epithelial

dystrophy, excessive enzymatic degradation and inappropriate basement membrane
development were the reasons of epithelial adhesion failure to the underlying stroma,
resulting in recurrent corneal ulcers.

Moore and Nasisse (1999) stated that ulcerative keratitis was caused by
corneal micro-traumas, tear film irregularities, immune system disorders (immune
deficiencies, autoimmune illnesses), bacterial infection and corneal surgical treatments.

Moore (2001) observed that the position, colour, shape, and pattern of corneal
lesions will aid in determining the underlying diseases. In terms of colour, red lesions
are associated with corneal vascularization, whereas brown lesions are associated
with pigmentary keratitis. Lesions in the nasal or temporal corneal area were caused
by medial or lateral adnexal diseases, respectively, whereas a linear or geographic
pattern of corneal ulceration indicated an underlying origin. The third eyelid and its
bulbar surface must be examined if the ventromedial portion of the cornea is irritated.

13
 Mandell and Holt (2005) cited probable causes of ulcer in dogs as: Infection,
foreign bodies, trauma, topical irritants, Keratoconjunctivitis Sicca, and entropion.
Another probable cause of corneal ulcer in lagophthalmic breeds noted was exposure
keratitis.

Gilger et al. (2007) studied different etiologies for corneal ulcer and
categorized into congenital origin, infectious cause, allergic origin, trichiasis,
distichiasis, ectopic cilia, entropion, trauma, foreign body in eye and lack of tear
productions.

Kim et al. (2009) mentioned that Keratoconjunctivitis Sicca (KCS) was the
most prevalent cause of ulcerative keratitis (31%), followed by lagophthalmos (28%),
bacterial infection (11%), nasal fold trichiasis (11%) and corneal trauma (11%).

Dawson and Sanchez (2015) conducted a prospective research on 100 dogs

(199 eyes) to assess the frequency of ocular surface disorders under general
anaesthesia using preventive topical lubrication. Before and after general anaesthesia,
Schirmer's tear test 1 (STT-1), slit-lamp biomicroscopy, and fluorescein staining were
conducted. Dogs with pre-existing ocular illness were excluded from the trial. The
incidence of ocular surface disease was studied across the various lubrication groups.
Even with topical lubrication, 0.50 percent of patients' eyes developed a superficial
corneal ulcer and 18.60 percent of patients' eyes experienced corneal erosion under
general anaesthesia.

2.5 Clinical Signs Associated with Corneal Ulcers

Wilkie and Whittaker (1997) noted clinical symptoms of corneal disorders

as epiphora, blepharospasms, photophobia, pawing, and opacification

Morreale (2003) stated that the loss of corneal clarity is the most common
symptom of corneal disorder and arises as a result of non-cellular infiltration buildup,
pigmentation, fibrosis and oedema.

14
 Mandell and Holt (2005) clinically noticed relatively clear ocular discharge,
corneal oedema, conjunctival hyperemia, blepharospasms, and miosis. Yellow- white
stromal infiltration was noticed in infected ulcers. The increased ocular discharge was
attributed to expression of opportunistic bacteria and increase in bacterial load in
cornea following trauma

Hvenegaard et al. (2011) found Blepharospasm (69.72 percent), red eyes

(conjunctival hyperemia and/or congestion) (69.01 percent) and ocular discharge
(64.79 percent) in boxer dogs with indolent ulcers. When compared to the left eye
(45.77 percent), the right eye had a higher incidence of indolent ulcers (54.23
percent). The majority of ulcers (n=130) were unilateral, with just six eyes (n=6)
reporting bilateral ulcers.

Thomas (2012) studied blepharospasm, severe uveitis, corneal oedema, and

exudates of red blood cells (hyphema) and white blood cells (hypopyon) within the
anterior chamber of the eye in a 9-year-old male pug with descemetocele.

Singh et al. (2016) noticed that lacrimation, blepharospasm, and periocular

edema were the most prevalent clinical symptoms linked with corneal ulcers in dogs.

2.5.1 Corneal Edema

Wilkie and Whittaker (1997) observed that the corneal epithelium and
endothelium keep the cornea dehydrated. Corneal oedema develops from any breach
in these barriers. The authors also mentioned that corneal opacity is caused by
changes in collagen type or disturbance of the usual lamellar organization of the
stroma.

Moore (2003) stated that anterior or posterior epithelial dysfunction causes

secondary corneal oedema and can contribute to chronic corneal epithelial
abnormalities.

Myrna et al. (2009) mentioned that fibroblasts and/or myofibroblasts replace

stromal keratocytes, altering corneal transparency and eventually leading to corneal
opacity.

15
 Feizi et al. (2017) stated that corneal neovascularization is a generalized
response to corneal pathologies such as congenital diseases, contact lens-related
hypoxia, inflammatory disorders, chemical burns, limbal stem cell deficiency,
allergy, trauma, infectious keratitis, autoimmune diseases and corneal graft rejection.

Singh et al. (2018) placed on record that during the corneal wound healing
process, keratocytes develop into myofibroblasts, resulting in fibrosis. Corneal haze
and reduced corneal transparency are caused by the non-orthogonal organization of
type I and III fibrillar collagen and random scattering of light by myofibroblasts.

2.5.2 Corneal Vascularization

Renwick (1996) found that superficial corneal vasculature blood vessels

cross the limbus and branch across the corneal surface but deep vasculature blood
vessels are straighter than arborising and create a 'brush border' impression without
crossing the limbus.

Wilkie and Whittaker (1997) stated that, deep corneal vascularization was
depending on the size of the ulcer. The vessels normally form after three to five days
and progress at a rate of less than one mm per day.

Moore (2001) stated that, depending on the kind of keratitis, the layout of
corneal blood vessels varies. In superficial ulcerative or non-ulcerative keratitis, long
branching vessels are common. In deep keratitis, corneal neovascularization looks
thin and non-branched, but in intraocular illness, it forms a 3600 perilimbal pattern.

Feizi et al. (2017) him corneal neovascularization is a typical response to

corneal pathologies such as congenital diseases, contact lens-related hypoxia,
inflammatory disorders, chemical burns, limbal stem cell deficiency, allergy, trauma,
infectious keratitis, autoimmune diseases, and corneal graft rejection.

2.5.3 Corneal Pigmentation

Renwick (1996) stated that brachycephalic breeds are more prone to

pigmentary keratitis. He determined the progression of corneal pigmentation from the

16
medio-ventral quadrant to the axial portion of the cornea has been documented as a
marker of chronicity.

Slatter and Dietrich (2003) noticed that epithelial and stromal pigmentation
is more prevalent in chronic corneal disorders owing to vascularization, prolonged
exposure, or nasal fold trichiasis and distichiasis. In addition, the authors indicated
that persistent exposure may cause corneal epithelium to return to a skin pattern with
thickening, rete peg formation, keratinization, and pigmentation.

Bouhanna et al. (2008) observed that in dogs, ulcerative keratitis induces

melanin pigmentation of the anterior stroma, and a persistent inflammatory response
in the cornea leads to melanin pigment migration and progression from the periphery
to the central portion of the cornea.

Gilger et al. (2008) stated that persistent corneal inflammation may cause
migration and deposition of melanocytic cells from the limbal and perilimbal tissues
into the corneal basal epithelial cells and anterior stromal tissue, resulting in corneal
pigmentation.

Azouly (2014) documented pigmentary keratitis in dogs ranging in age from 3

to 14 years, with a mean age of 7 years. According to the author, melanin pigment
deposition in the corneal epithelium or stroma leads to superficial corneal
pigmentation. Melanocytic cells are derived from the limbal conjunctiva and deposit
in the cornea via neovascularization. The author also mentioned that melanin
deposition is induced by macrophages and fibroblasts.

Anoop et al. (2016) noticed gradual progression of peripheral pigmentation

from the limbus to the centre portion of the dog cornea. Furthermore, the authors
indicated that during stromal wound healing, keratocytes changed into disordered
fibroblast and collagen, resulting in scar formation. During healing,
neovascularization transports melanin pigment from the limbal and peri-limbal areas
and deposits it around the core scar, resulting in pigmentation.

17
2.6 Diagnostic Techniques for Corneal Ulcers

2.6.1. Ophthalmic Examination

Miller and Crenshaw (1988) observed that direct diffuse lighting can be used
to assess corneal clarity, contour and symmetry and contact between the eyelid
margin and the cornea, whereas direct focused illumination can be utilized to detect
the localization and extent of the lesions.

Renwick (1996) stated that essential prerequisites for ocular examination was
a brilliant focused source of light, a transilluminator and a mechanism of
magnification.

Ollivier (2003) pointed out that, cornea should be inspected for loss of
transparency, opacity, pigmentation, vascularization, growths, lacerations, the
presence of foreign bodies, shape alterations and ulceration.

Hazra et al. (2008) evaluated the effect of local retrobulbar nerve block for
routine cataract surgery in dogs. He reported that the exposure of the globe in all
the dogs was adequate, the desired central fixation of the eye was obtained
and surgery could be performed uneventfully.
Myrna et al. (2010) conducted a study to assess the efficacy of a retrobulbar
bupivacaine nerve block for postoperative analgesia following eye surgery in dogs. It
was observed that retrobulbar administration in conjunction with traditional
premedication prior to eye surgery was an effective form of adjunctive analgesia and
reduced the need for additional postoperative analgesics.

Gilger et al. (2014) mentioned that increased water content in cornea results
in corneal oedema, increased thickness and scattering of light, leading to reduced
transparency and loss of vision.

18
2.6.2 Visual Function Tests

2.6.2.1 Ocular Reflexes

Moore (2003) stated that the cornea is innervated by the ophthalmic branch of
the trigeminal nerve, whose proper function may be determined by touching the
peripheral cornea with a cotton-tip applicator or a wisp of cotton and eliciting a
blinking response.

Irby (2011) has observed that a complete neuro-ophthalmologic assessment is

a component of ophthalmic and neurologic examination. He emphasized that
evaluation of the cranial nerves II, III, IV, V, VI, VII, and VIII and a portion of
autonomic innervation should be done by general examination, assessment of vision,
observing ocular reflexes, movement, position, and touch response of globe and lids.
Mitchell (2011) emphasized that since ocular ailments such as blindness may
have a neurological component, a comprehensive neurological assessment is required.

2.6.2.2 Menace Response

Martin (2001) stated that threatening, quick movement near the eye causes a
blinking reflex known as the menace response. Air currents are avoided by presenting
fingers or using a transparent shield to prevent eliciting a false positive response, and
the contralateral eye should be closed to ensure that the stimulus did not elicit a
binocular response.

Moore (2001) defined visual status and eyelid function as the two
components of the threat response. A clean optic medium, a working retina, and
healthy optic and facial nerves were necessary for a good threat response.

Venugopal (2013a) studied the management of corneal ulcer in pugs of

various ages and sex. In this study on ophthalmological examination, menace reflex
was found to be sluggish whereas, corneal and palpebral reflexes were found to be
normal.

2.6.1.4 Dazzle Reflex

19
 Martin (2001) stated that bilateral narrowing of palpebral fissure occurs when
the retina is stimulated with a very bright light, which is known as dazzle reflex. This
reflex is a subcortical response and requires retina, optic nerve, optic tract, optic
chiasm and probably the supraoptic nuclei and rostral colliculi.
Mitchell (2011) summarised that if a strong light is shown into eye, elicits a
normal involuntary avoidance response which includes a blink or partial blink and
head aversion. A positive reflex suggests the function of visual pathway and a
negative reflex is a poor prognostic indicator of vision. Dazzle reflex is used to
ensure potential vision in cases with opacity of ocular media.
Featherstone and Heinrich (2014) stated that sufficient brightness of the
light source is fundamental for eliciting a reliable dazzle reflex.

2.6.2.4 Pupillary Light Reflex (PLR)

Felchle and Urbanz (2001) supported that the PLR test effectively assessed
the function of the retina, optic nerve, and iris sphincter muscle.

Maggs (2008) revealed that PLR was reduced or absent in cases of iris
atrophy, physical blockage of the pupil, iris ischemia prior to administration of a
dilating medication, or high quantities of circulating adrenaline in scared animals.

Mitchell (2011) concluded that PLR was not a function of vision since blind
animals had normal PLR, as in cataracts or occipital brain lesions, and an absence of
PLR could be seen in animals with normal vision, as in iris atrophy.

2.6.3 Direct ophthalmoscopy

Stenstorm (1966) observed that opacities in the ocular media like corneal
opacities, cloudiness of the aqueous as well as lens and vitreous opacities increases
veil and these make ophthalmoscopy impossible.
Makhdoomi and Sheikh (2001) carried out direct ophthalmoscopic
examination done using a battery ophthalmoscope .The examination was done in a
direction that the eyes of the observer and patient were brought closer such that rays

20
from retina of the patient could enter the pupil of the observer to increase the field of
vision.
Maggs (2008) described use of a direct ophthalmoscope to visualize the
ocular fundus of the canine eye after dilatation of pupil using mydriatics.

2.6.4 Schirmer Tear Test.

Munro (2001) revealed that sedation, general anaesthesia, topical

anaesthesia, and the administration of parasymphatholytic drugs all dramatically
lowered STT readings and recommended that STT be conducted prior to sedation and
administration of topical medication .

Chandler et al. (2003) observed that tear film inadequacies were of two
types: quantitative and qualitative, resulting from insufficient formation of the
aqueous layer, Mucin, and/or lipid layer. STT-1 examined the amount of tears in the
lacrimal lake, basal secretion, and trigeminal-facial reflex tearing, whereas STT-2
simply measured basal tear secretion. Tear production in dogs was unaffected by
differences in age, breed, or gender. It was noticed that for every additional year of
age, tear production decreased by 0.4mm.

Morreale (2003) noticed that the normal value of Schirmer’s tear test in a dog
ranged from 14 to 25 millimetres per minute. A Schirmer tear test result of less than
15 millimetres per minute is suspicious while that less than 10 millimetres per minute
is indicative of keratoconjunctivitis sicca.
Williams (2005) nhoticed that first quick spike in STT reading was caused by
pre-existing tearlake, while the latter gradual increase in STT value was caused by
tears formed de novo from lacrimal tissue.

Beranek and Vit (2007) observed that because the STT strips were sterile,
they had no detrimental impact on bacteriological or fungal tests.

Featherstone and Heinrich (2014) stated that, tear production should be

measured, using Schrimer tear test, before all other neuro ophthalmic examinations to

21
avoid the false negative high values due to excessive manipulation of eye or eyelids
and due to any topical agents like mydriatics, anaethetics and stains. He further
mentioned that normal values for STT vary from 18.64 ± 4.47 mm/min to 23.90 ±
5.12 mm/min.

Anoop et al. (2015) employed the Schirmer tears test (STT) to diagnose
Keratoconjunctivitis Sicca (KCS) and reported a mean value of 10.31+0.58 that was
below the normal range and was indicative of Kerato-conjunctivitis Sicca (KCS).

Williams et al. (2017) Studied changes in lacrimation and intraocular

pressure (IOP) in dogs with unilateral corneal ulceration using the Schirmer tear test
(STT) and rebound (TonoVet®) tonometry.The mean ± standard deviation for STT
values in the ulcerated and control eyes were 20.2±4.6 mm/min and 16.7±3.5
mm/min respectively. The higher tear production in ulcerated eyes was noted.
2.6.5 Fluorescein Dye test

Slatter (1990) employed Fluorescein to identify corneal abnormalities,

conjunctival epithelial defects, and pre-corneal tear-film inadequacies. The
hydrophilic fluorescein dye does not stain healthy corneal epithelium or Descemet's
membrane. When the corneal integrity is compromised, dye enters the water-soluble
corneal stroma and stains only the intracellular compartments.

Renwick (1996) explained how a fluorescein dye impregnated strip might be

used to confirm corneal ulcers.

Moore (2001) observed that cobalt blue light improved the visibility of
fluorescein stain,that was used to assess the integrity of the corneal epithelium.

Morreale (2003) pointed out that mucus stains with fluorescein dye, hence
the cornea should be free of mucus to avoid a false-positive dye test result.

Ollivier (2003) mentioned that use of a moistened paper strip impregnated

with fluorescein dye was ideal for usage in tiny animals and the moistened strip was
pressed

22
to the dorsal bulbar conjunctiva rather than directly to the cornea to avoid
misunderstanding of a positive result.

Beranek and Vit (2007) noticed that corneal abnormalities looked green
when seen using an ultraviolet light (Woods lamp) or a cobalt filter.

Janssens (2007) reported that in the case of indolent ulcers, fluorescein dye
not only marked the ulcerated region but also migrated under the loose flaps of the
epithelium and coloured the adjacent anterior stroma.

Mitchell (2011) stated that surplus dye was flushed away during the dye test
to avoid it being misinterpreted as positive dye absorption due to its pooling in
uneven surfaces of the cornea.

Kumar and Thirumalesh (2013) revealed that fluorescein, a water-soluble

dye, permeated through intercellular gaps, increased the intensity of staining in the
instance of cellular degeneration or death and staining occurred when cell-to-cell
connections allowed more penetration of the dye in case of cellular degeneration or
death.

Singh et al. (2016) observed that, external ophthalmic stain (fluorescein

sodium) was an excellent diagnostic technique for detecting corneal ulcers and
conjunctival abnormalities. Fluorescein sodium stain was taken up by exposed
corneal stroma, identifying the corneal ulcer borders as green.

2.6.6 Corneal Cytology

Hamor (2001) carried out cytological analysis of exudate and/or corneal
scrapings and found that it was a good tool for determining the origin of ocular
pathology and for recommending further treatment options.
Ollivier (2003) recorded that cotton and Dacron swabs caused the least stress
during exfoliative cytology of the cornea. He found that most prevalent cell types and
cell material during cytology of healthy cornea were non-cornified corneal epithelial
cells, lymphocytes, polymorphonuclear cells, nuclei, keratin debris, and bacteria.

23
 Kern (2004) stated that culture and cytology of ocular discharge and aqueous
humor or vitreous should be done for effective antibacterial therapy in cases of
resistant or serious ocular infections.
Anoop et al. (2015) studied impression cytology of canine corneas and
observed the infiltration of neutrophils, degenerative changes, presence of necrotic
debris and squamous epithelial cells in samples

2.7 Hematology
Chinchu (2010) observed non-significant changes in haematological
parameters in a research on the effectiveness of collagen sheets in the treatment of
ocular ulcers in dogs.
Antonia (2014) investigated the occurrence and therapy of ocular disorders in
dogs and recorded that all ophthalmic affections had normal haematological
parameters.
Nair and Vasanth (2007) studied the haematological changes in ocular surgery
.They found that there was no significant change in haemoglobin and total
erythrocyte count. Slight neutrophilia was noticed on first postoperative day due to
changes occurring in corneal healing process and there were no changes in monocytes
and eosinophils.
Venugopal (2013b) observed that all the hematological parameters are
normal in management of corneal injury.

2.8 Medical Management of Corneal Ulcers

Roberts et al. (1986) stated that a 1:50 dilution of povidone iodine was
effective as an ocular surface disinfectant for pre-surgical usage since it removed
bacterial contamination and did not cause corneal oedema. The author also mentioned
that ocular oedema was detected in instances that were disinfected with 1:20
povidone iodine.

24
 Bedford (1987) advocated the use of topical and/or systemic antibiotics in
conjunction with mydriatic and cycloplegic treatment to reduce the risk of subsequent
infection and uveitis in dogs with corneal ulcers.

Gilger and Allen (1998) noticed that cyclosporine acted on the lacrimal
acinar tissue and suppressed immune-mediated inflammation, thereby enhanced tear
production.

Peiffer (1993) advised that the ocular and surrounding periocular area
should be flushed by using of 0.5% povidone iodine solution.
Champagne (2001) observed that ciprofloxacin was a great choice for deep
or melting ulcers, descemetocele, corneal perforations, and refractory ulcers. He also
noticed that corticosteroids slowed wound healing, increased infection risk, and
increased collagenase activity. He further stated prolonged use of nonsteroidal anti-
inflammatory drugs (NSAIDs) resulted in decreased vascularisation, potentiation of
stromal necrosis, and melting.

Varges et al. (2009) studied the antimicrobial susceptibility of Staphylococci

isolated from naturally occurring canine ocular diseases and reported that they are
most sensitive to enrofloxacin fluroquinolone (25% resistance), followed by
ciprofloxacin (30% resistance)to the presence of Staphylococcus spp. in naturally
occurring extra- ocular canine ocular disease and the emergence of resistant strains to
common antimicrobial drugs and also highlighted the need for bacterial culture with
species identification and susceptibility testing in order to select the suitable
antimicrobial therapy.
Shaheen et al. (2014) described how neurotrophic corneal diseases were
treated by avoiding anti-inflammatory drugs and instead, employing neuro-
regenerative, neuro-protective, and concealing strategies by understanding the
interwoven relationship between neuro-regenerative and neuro-protective
mechanisms.

Ratnu et al. (2020) used topical instillation of antibiotics (Moxifloxacin),

NSAIDs (Flubriprofen), and cycloplegics (Atropine) to treat superficial ulcers. They

25
found that most superficial corneal ulcers healed rapidly and without complications;
simply topical antibiotics, mydriatics/cycloplegic medicines, and artificial tears were
sufficient to prevent or eradicate infection, ease pain, and promote healing. They also
noted that success rates and healing times varied and in both groups, the defect in the
surface ulcer healed within 10 to 14 days, while the reported period of recovery
ranged from 20% in 14 days to 84 percent in an average of 23 days.

2.9 Surgical Management of Corneal Ulcers

Hollingsworth (2003) proposed several surgical procedures for corneal

surgery, including corneal laceration suturing, conjunctival pedicle graft,
transconjunctival island graft, advancement graft, total conjunctival graft, frozen
tectonic corneal graft, porcine small intestine submucosa graft and superficial
keratectomy.

Gomes et al. (2005) investigated the biochemical composition of amniotic

membrane and concluded that it is a biologic tissue that may be employed as a graft
for corneal and conjunctival repair in ocular surface diseases. They further studied
that it is also avascular and has antiangiogenic, anti-scarring, and anti-inflammatory
effects, according to the researchers. They believed that the amniotic membrane
should be employed as a medium for cells to migrate and regenerate, resulting in the
formation of new and healthy tissue, rather than as a replacement for other ocular
reconstruction approaches.

Kim et al. (2009) investigated several treatment methods in dogs with corneal
ulcers. They observed that superficial corneal ulcers treated with medicine healed in
5.1 to 13.4 days on an average, but deep corneal ulcers treated with conjunctival flap
creation healed in 28.4 to 40 days.

Vongsakul et al. (2009) studied canine amniotic membrane (AM) transplan-

tation with and without a third eyelid flap to enhance healing of deep corneal ulcers.
The average time to complete corneal epithelialization in eyes having AM
transplantation in combination with the third eyelid flap was 7.33+0.21 days, which

26
was considerably lower than the average time seen in eyes getting only the third
eyelid flap of 9.17+0.31 days. They concluded that canine AM promotes fast
epithelialization without neovascularization or scarring of the cornea, which is of
paramount importance for corneal repair.

Dorbandt et al. (2015) examined the success rate of a conjunctival pedicle

flap (CPF) alone with a CPF combined with an acellular submucosa implant for the
healing of deep or perforating corneal lesions in dogs. The combined success rate for
all corneal wounds was 93%, while the rates for corneal perforations, descemetoceles,
and deep stromal wounds were 89, 95, and 100 percent, respectively. The total
success rate did not differ across groups. Deep or perforating corneal wounds
stabilised with a CPF alone compared to a CPF + acellular submucosa which had a
comparable success rate.

Cirman et al. (2014) described that amniotic membrane is the innermost layer
of the placenta. It acts as scaffold for the regeneration of new cells and promotes
epithelialization. It has antimicrobial properties and decreases inflammation, fibrosis
and neovascularization. It can be used for the reconstruction of conjunctival and
corneal defects and for the treatment of corneal ulcers.

2.10 Human Amniotic Membrane Graft for Healing of Corneal Ulcers.

Akle et al. (1981) observed that after transplanting human amniotic epithelial
cells into subcutaneous compartments, the acute immunological rejection was absent
in human volunteers. The lack of expression of HLA-A, B, C, and DR antigens or 2
macroglobulin on the surface of human amniotic epithelial cells was cited as the
cause of their low immunogenicity to the cell transplant.

Lee and Tseng (1997) investigated the use of transplanted preserved human
amniotic membrane for the treatment of persistent epithelial defects with ulceration in
humans and concluded that it could be used as an alternative method for treating
persistent epithelial defects and sterile ulceration that were not responding to
conventional treatment.

27
 Prabhasawat et al. (1997) reported that recurrence of pterygium was less
with human amniotic membrane transplantation as compared with primary closure
and concluded that human amniotic membrane can be an alternative for the primary
closure.
Fukuda et al. (1999) compared the distributions of type IV collagen and
laminin subchains in human amniotic membrane to those in the cornea and
conjunctiva, and found that the basement membrane of human amniotic membrane
contains collagen IV and VII, both of them are components of corneal epithelial
basement membrane. The scientists also noted that type IV collagen and laminin in
the basement membrane play a crucial role in epithelial cells migration, proliferation
and differentiation.

Tseng and Li (1999) observed that amniotic membrane transplantation on

human eyes, significantly aided epithelialization, decreased angiogenesis,
inflammation, and scarring, and preserved a normal epithelial phenotype. The
scientists also investigated the effects of amniotic membrane matrix on cultured
human corneal and limbal fibroblasts, finding anti-fibroblastic activity, reduction of
transforming growth factor beta isoforms and myofibroblast differentiation.

Hao et al. (2000) used immunohistochemistry and RT-PCR to demonstrate

antiangiogenic factors such as tissue inhibitors of metalloproteinase (TIMP)-1, 2, and
4 and anti-inflammatory factors such as IL-Ira, IL-10 in fresh and cryopreserved
human amniotic membrane. They found that anti-inflammatory IL-10 and IL-Ira
inhibit angiogenesis by boosting TIMP-1 production and decreasing the activity of
matrix metalloproteinases -2 and -9.

Kim et al. (2000) demonstrated suppression of proteinase activity and

infiltration of inflammatory cells after using human amniotic membrane as a
temporary patching for produced alkali burn in the rabbit cornea.

Lee et al. (2000) recorded that stromal matrix of the human amniotic
membrane contains a high concentration of foetal hyaluronic acid, which
suppresses TGF

28
signalling, proliferation and myofibroblastic differentiation of normal corneal and
limbal fibroblasts as well as pterygium and conjunctival fibroblasts.

Meller et al. (2002) found that transplanting intact human amniotic

membrane over acute chemical and thermal burns in the eye reduced longterm
scarring consequences by lowering inflammation and facilitating re-epithelialization.

Sridhar et al. (2001) carried out surgical debridement followed by human

amniotic membrane transplantation to successfully treat shield ulcers in people

Sippel et al. (2001) observed that amniotic membrane serves to provide a

basement membrane substrate when used in an inlay fashion. They further clarified
that basement membrane, in general serves to facilitate the migration of epithelial
cells, reinforce the adhesion of the basal epithelium, promote cellular differentiation
and prevent cellular apoptosis.
Burman et al. (2007) found that processed and maintained human amniotic
membrane can be utilised to replace the damaged ocular surface stromal matrix,
either as a graft or as a patch to avoid the inflammatory response on the damaged
ocular surface.

Nakamura et al. (2004) found that freeze-dried human amniotic membranes

transplanted into a rabbit model adapted well to the host corneal stroma, with no
indications of subepithelial cell infiltration or stromal oedema. The authors found no
fluorescein staining 48 hours after surgery. There was no vascularization on the
corneal surface and there was a significant increase in clarity.
Fernandes et al. (2005) stated that human amniotic membrane possesses
antibacterial characteristics and thereby reduces the incidence of postoperative
infection. The scientists also noted that the amniotic membrane functions as a
basement membrane, allowing epithelial cells to migrate. They further noticed that It
increases corneal sensitivity and tear film stability by strengthening basal epithelial
cell adhesion, promoting epithelial differentiation, preventing epithelial apoptosis,
and preventing epithelial apoptosis.

29
 Wichayacoop et al. (2005) employed preserved human amniotic membrane
to heal generated corneal stromal lesions in dogs and noticed full corneal
epithelialization in seven days with subsequent eye sight restoration.

Ollivier et al. (2006) subjectively compared outcome of amniotic membrane

transplantation (n=9 eyes) and bulbar conjunctival grafting following keratectomy in
equines after excision of corneo-limbal squamous cell carcinoma (scc). Strontium-90
irradiation or cryotherapy was also used in 3 eyes. They concluded that amniotic
membrane transplantation (n=9 eyes) received eyes showed a minimal level of
scarring in a cornea and regained a greater transparency in comparison to bulbar
conjunctival graft treated eyes.
Seitz (2007) stated the therapy for persistent corneal epithelial defects. He
explained that with persistent corneal ulcers AMT should be considered early, to
avoid keratoplasty à chaud or even a conjunctival flap. He noted that differentiated
microsurgical technique results in different integration patterns of AM into the human
cornea and the sandwich technique seems to have advantages in contrast to the
solitary graft or patch techniques with respect to primary success and recurrence rate.
Corneal infections were extremely rare after AMT, but may happen. Postoperatively,
phosphate-containing eye drops should not be used to avoid corneal calcification.
AMT had a great variety of merits before, instead of, during, or even after
keratoplasty. The consequent systemic therapy of underlying diseases was absolutely
indispensable for durable success of AMT.
Riau et al. (2010) recorded that human amniotic membrane contains a variety
of growth factors including epidermal growth factor (EGF), transforming growth
factor (TGF)-a, -b1, -b2, and -b3, keratinocyte growth factor (KGF), KGF receptor
(KGFR), hepatocyte growth factor (HGF), HGF receptor (HGFR), basic fibroblast
growth factor (bFGF) and vascular endothelial growth factor (PDGF). According to
the scientists, EGF is a strong stimulator for epithelial cell proliferation and its high
level of expression might explain the promotion of ocular surface wound healing
following transplantation.

30
 Gholipourmalekabadi et al. (2015) found that even after decellularization of
typical human amniotic membrane tissue, revealed the presence of human collagen
types I, III, and IV in basement membrane proteins.

Gurpreet Singh (2017) applied dry human amniotic membrane overlay graft
in four eyes. Two layered human amniotic membrane graft was placed over ulcer
without suturing or any tissue adhesive. It was followed by partial temporary
tarsorrhaphy. Human amniotic membrane transplantation was applied as double layer
membrane transplant. All animals treated by overlay amniotic membrane graft
showed healed corneal ulcer within 11.25±2.50 days. Success rate of technique was
evaluated in terms of restoration of vision and resumption of normal corneal
transparency. Vision was restored in all 4 eyes and normal corneal transparency was
resumed on 45th post- operative day. A 100% success rate was recorded following
amniotic membrane transplantation technique. Complications in terms of
neovascularization and corneal scarring were minimum in amniotic membrane
grafting group as compared to bulbar cunjuctival grafting group.

Jing et al. (2018) noted that amniotic membrane (AM) has been widely used
as a temporary or permanent graft in the treatment of various ocular surface diseases.
In this study, they compared the epithelial wound healing and tissue remodeling after
ocular surface reconstruction with intact amniotic membrane (iAM) or denuded
amniotic membrane (dAM). Partial limbal and bulbar conjunctival removal was
performed on New Zealand rabbits followed by transplantation of cryo-preserved
human iAM or dAM. In vivo observation showed that the epithelial ingrowth was
faster on dAM compared to iAM after AM transplantation. Histological observations
showed prominent epithelial stratification and increased goblet cell number on dAM
after 2 weeks of follow up. Collagen VII degraded in dAM within 2 weeks, while
remained in iAM even after 3 weeks. The number of macrophages and α-SMA
positive cells in the stroma of remodelized conjunctiva in the dAM transplantation
group was considerably less. In conclusion, dAM facilitates epithelial
repopulation and goblet cell

31
differentiation and further reduces inflammation and scar formation during
conjunctival and corneal limbal reconstruction.
Costa et al. (2019) found epitheliotropic properties in human amniotic
membrane and used the negative absorption of fluorescein dye to measure corneal
wound healing in dogs. The authors found that cryopreserved human amniotic
membrane transplantation was extremely successful for surgically managing the
severe corneal ulcers in dogs, providing 99.1% anatomical and 97.4% visual results
with very few complications.

Korittum et al. (2019) conducted an experimental investigation to assess the

effectiveness of human freeze dried amniotic membrane transplantation on canine
corneal wound healing. The authors utilised 8-0 polygalcatin 910 to secure the human
amniotic membrane graft over the corneal defect, adjacent to the healthy cornea using
a simple interrupted suture pattern and found that the amniotic membrane improved
corneal epithelial wound healing.

2.11 Complications Associated with Corneal Ulcers

Bussieres et al. (2004) found that the most common problems identified
following corneal surgeries are aqueous leakage, conjunctival flap dehiscence,
synechia, cataract and fibrin in the anterior chamber. Fundus examination revealed
evidence of vitreal degeneration. Retinal scarring consisted of several linear streaks
that were assumed to be caused by a prior retinal detachment, because the retina was
normal when evaluated prior to surgery, panuveitis was suggested as the origin of
these lesions.

Dorbandt et al. (2015) identified corneal fibrosis at the site of damage,

corneal vascularization, corneal pigmentation, and recurrent corneal ulceration as
significant postoperative sequelae. However, none of these issues had a substantial
impact on the visual result or the success rate. The research found no evidence of
glaucoma as a post- surgical consequence.

32
Materials & Methods
 CHAPTER III

MATERIALS AND METHODS

The present research work entitled “Clinical Evaluation of Amniotic

Membrane Graft for Management of Corneal Ulcer in Dogs” was carried out in the
clinical cases of dogs suffering with corneal ulceration which were referred to
Veterinary Clinical Complex (VCC), KNP College of Veterinary Science Shirwal.
Also, cases referred to government veterinary polyclinics, mini-veterinary
polyclinics, veterinary dispensaries and private pet clinics around Satara, Pune, and
Mumbai and nearby areas were included in the present study.
Design of research work
Clinical cases suffering with various eye affections were screened for
confirmatory diagnosis of corneal ulcer. On the day of presentation, signalment and
anamnesis, general condition and physiological parameters of the patient were
recorded. Further, all dogs underwent detailed ophthalmic examination that included
gross examination of eye, tests for vision viz; menace response, dazzle reflex,
pupillary light reflex and direct ophthalmoscopy. The ancillary tests such as corneal
cytology, hematology, schirmer tear test along with confirmatory test i.e.
fluorescein dye test were carried out.
After through screening, dogs suffering with various types of corneal ulcers
were initially subjected to instillation of Ciprofloxacin 0.5% eye drops QID for 5
days,1% Atropin eye drop for 3 days and Meloxicam@ 0.5 mg/kg body weight PO
BID for 3 days. Dogs which did not respond to above medical treatment, were treated
surgically.
Total 12 dogs with non-healing corneal ulcers were randomly divided into two
groups based on the surgical technique adopted: In group I (1-6), six dogs were
subjected to transplantation of dry human amniotic membrane graft, whereas in group
II (7-12), six dogs were subjected to transplantation of wet human amniotic
membrane graft.

34
3.1 Signalment and anamnesis
History pertaining to age, breed, sex, symptoms, duration of the corneal ulcer,
concurrent illness if any and any previous illness with medications or treatment were
recorded in all animals during preliminary general examination. The general
condition of all the animals was visually assessed on the day of presentation and also
on the day of surgery and it was categorized as excellent, good, fair or poor.

3.2 Physiological Parameters

The physiological parameters in all cases i.e. rectal temperature (˚F), pulse
rate (per minute), respiration rate (per minute) and color of conjunctival mucous
membrane were recorded at the time of preliminary examination.

3.3 Ophthalmological examination

Pre-operatively, all animals were subjected to detailed ophthalmic
examination using following tests:
3.3.1 Gross Ophthalmological examination
Preliminary gross examination of ocular and surrounding structures was
carried out in routine manner. Examination of all dogs from a distance included
ocular symmetry along with the size, shape, gaze, movement of the eyes and ocular
conformation. The eyes of all the dogs were observed for lacrimation and nature of
ocular discharge like watery, mucoid, mucopurulent or purulent and relevant laboratory
investigation was carried out to rule out the ocular infection if any.
The closer examination of the eye was carried out which included inspection
of each visible structures such as bulbar conjunctiva, palpebral conjunctiva and the
eyelids for presence of any disorder initiating from external to internal structures.

3.3.2 Tests for vision

Following tests were performed in dogs with corneal ulcers, to rule out the
extent of blindness.

35
3.3.2.1 Menace response
Menace response test was conducted by making a sudden gesture to provoke
a blink response. Care was taken not to create air currents or touch facial hairs. The
reflex included blinking so as to protect the eyes from potential damage. This also
included turning the neck, head and even trunk away from the potential stimulus and
it was graded as normal (++), sluggish (+) and absent (-).

3.3.2.2 Dazzle reflex

The torch light was directed at the eye to observe the blink reflex. The reflex
included involuntary blinking of eyelids as a response to a precipitant bright light. It
was graded as normal (++), sluggish (+) and absent (-).

3.3.2.3 Pupillary light reflex (PLR)

In a semi-dark room, a torch light was directed to the eye spontaneously to
observe the pupils response. It was recorded as normal (++), sluggish (+) and absent
(-).
3.3.3 Direct ophthalmoscopy
The iris, pupil, cornea and associated structures were examined with the help
of direct ophthalmic microscope by setting it at 0 diopter from a distance of 25 cm.
Followed by close examination of the patient's eye, various structures of the anterior
and posterior segment were observed at respective diopters as described by Bowersox
and Criox (2001), by using ophthalmoscope. Simultaneously corneal clarity, corneal
edema and type and extent of corneal ulcer was also recorded with the help of
ophthalmoscope.
3.3.4 Schirmer tear test (STT)
The Schirmer tear test was carried out to assess the quantitative production of
tears. In this test the, Schirmer tear test strips1 with printed millimeter scale on it were

1.
Tear Touch, Madhu Instruments Pvt. Ltd, New Delhi.

36
placed in the lower eyelids of the dog for the time period of one minute and the
quantity of tear production was checked as the distance of wetness on strip measured
immediately and compared against the normal range of 15-25 mm/min (Gelatt, 1991).

3.3.5 Fluorescein dye test

Fluorescein dye test was performed using Fluro touch sterile strips2. The
extent of corneal ulcers were assessed on the basis of pooling of stain and findings
were recorded.
The tip of strip was held against the bulbar conjunctiva and two drops of
sterile normal saline was instilled on it for the dye to elute and fall on the eye. The
animal was allowed to blink the eye to distribute the dye across the ocular surface.
The excess stain was flushed out with sterile normal saline. The eye was then
examined under cobalt blue light and observation was noted as staining (+) or no
staining (-).

3.3.6 Corneal Cytology

Corneal cytology was conducted in dogs showing signs of ocular discharge,
conjunctivitis, keratoconjunctivitis Sicca, corneal edema and corneal pigmentation.
The eyelids were gently retracted and the sides of sterile ocular swabs were rolled
over the ulcerated part of cornea without touching eyelid margin or eyelashes.
These samples were smeared on the glass slide and stained with Giemsa stain for
microscopic examination. The corneal cytology was conducted preoperatively as well
as on 12th postoperative day.

3.4 Hematology
The Blood samples were collected aseptically from the cephalic vein,
from every case in the Ethylene Diamine Tetra Acetic Acid (EDTA @ 1mg/1ml)
vials and were subjected for complete blood count.

2.
Fluro Touch, Madhu Instruments Pvt. Ltd, New Delhi

37
 The parameters viz; Hemoglobin (Hb gm/dl), packed cell volumes (PCV %),
total erythrocyte count (TEC x106 /µl), total leukocyte count (TLC x103/µl)), mean
corpuscular vale (MCV fl), mean corpuscular hemoglobin concentration (MCHC
gm/dl) and platelets count (x106 /µl), were estimated using automated hematology
analyzer3.The differential leukocyte count (DLC) was also measured manually on
blood smears stained with Giemsa staine. The blood analysis was carried out
preoperatively as well as on 12th postoperative day and was compared statistically.

3.5 Instrumentation.
3.5.1 Direct ophthalmoscope
The direct ophthalmoscope4 is a diagnostic instrument which has several
lenses that magnify up to 15 times. It was used to examine anterior chamber lens, iris,
optic disc, fundus etc. by directing artificial light beam into the eye.

3.5.2 Ophthalmic surgical instruments

In present study, special ophthalmic instruments like operating ophthalmic
microscope5, wire speculum, colibri forceps, castroviejo corneal scissors, jaffe tying
forceps and barraquer needle holder were used throughout the procedures. Vicryl
no.10-0 was used to secure the graft with cornea whereas, Vicryl 5-0 was used for
tarsorrhaphy.

3.5.3.1 Preservation of Amniotic membrane grafts.

The procured grafts were preserved as per the instructions of the manufacturer
i.e. the dry amniotic membrane graft6 were preserved at room temperature. Whereas
wet amniotic membrane graft7 were preserved in a deep freezer at -700C.

3.
Abacus Jr.Vet 5, Diatron Hungary.
4.
Direct Opthalmoscope, Heine K180, Made in Germany.
5.
Appasamy Brilliant Advent FS-9, Chennai, Tamil Nadu.
6.
Amnio cornea GraftTM, Reea Carewell Products, Nizamuddin West, New Delhi.
7.
CryobankTM, Cryobank fertility research center, Jalna, Maharashtra.

38
3.6 Preparation of patient and anesthetic protocol
3.6.1 Preoperative medication
In both the groups, preoperative medication included use of Moxifloxacin8 eye
drop 0.5% (one drop 60 minutes prior to surgery) and Proparacain HCL9 0.5% eye
drop (instilled 15 minutes prior to surgery) as topical anesthetic, systemic antibiotic
i.e. inj. Amoxycillin & Sulbactum Sodium10 @ 7 mg/kg body weight and inj.
Meloxicam11 @ 0.2mg/kg body weight by intramuscular route (30 minutes prior to
surgery).
3.6.2 Anesthetic protocol:
In present study for both the groups, inj. Butorphanol Tartrate12 @ 0.3 mg per kg
body weight intramuscularly and inj. Triflupromazine HCL13 @ 1.2 mg per kg body
weight intravenously was used as a sedative. Whereas, induction was achieved with
14
the help of inj. Propofol @ 4-6 mg/kg body weight intravenously. Anesthesia was
15
maintained by using 2-3% Isoflurane as gaseous general anesthetic. To avoid
movement of the eyeball during the procedure, retro bulbar nerve block was also
performed by using Lignocaine HCl 2% 16.

3.6.3 Positioning and Preparation of patient

All the dogs were positioned in lateral recumbency with affected eye in an
upward position. Head was kept in an elevated position using soft bedding material
for proper visualization under the operating ophthalmic microscope. The eyelashes of
affected eye were clipped and the ocular discharge was cleaned with wet cotton swab.

8.
Moxicip eye drop 0.5%, Cipla Ltd., Haridwar, Uttarakhand.
9.
Paracaine0.5%, Sunways, India Pvt.Ltd. Mumbai.
10.
Amoxirum Forte, Virbac, Bhivandi, Maharashtra.
11.
Melonex, Intas Pharmaceuticals Ltd., Ahmadabad, Gujrat.
12.
Butodol-2, Neon Laboratories Ltd., Thane, Maharashtra.
13.
Siquil, Sarabhai Zydus Animal Health Ltd. Ahmadabad, Gujrat.
14.
Neorof, Neon Laboratories Ltd., Andheri East Mumbai Maharashtra.
15.
Sosrane, Neon Laboratories Ltd., Andheri East Mumbai Maharashtra.
16.
Lox* 2%, Neon Laboratories Ltd., Andheri East Mumbai Maharashtra.

39
 The affected eye was thoroughly cleaned with 0.5% povidone iodine solution
and was covered with sterile eye drape (Plate. 3.1).

3.7 Surgical procedure for corneal grafting

3.7.1 Preparation of ulcer bed and placement of Amniotic membrane Graft
Eyelids were kept in a retracted position using self-retaining eye speculum.
Stay sutures were applied on the eyelids for proper exposure of the eye (Plate 3.2).
Loose epithelium from the lesion was debrided (up to 2 mm surrounding area) by
using sterile cotton swab. (Plate No. 3.3)
This was followed by preparation of graft site by using sterile b.p blade no.15
The remaining debris were removed by frequently flushing the eyeball with isotonic
saline solution.
After preparation of ulcer bed, the commercially available sterile dry and wet
human amniotic membrane grafts (greater than the size of the ulcer) were placed on
prepared ulcer bed in the animals from the respective groups i.e. group I and group II.

These grafts were then secured with adjacent cornea by using 10-0 Vicryl
with simple interrupted suture pattern at four different points. After the grafting
procedure, the tarsorrhaphy was performed by using 5-0 vicryl so as to prevent self-
mutilation and to promote healing process. During tarsorrhaphy, a minute slit was
kept at the lateral canthus to facilitate instillation of topical eye drops.
3.8 Postoperative Care
Topical medication by using Moxifloxacin8 0.5% eye drops one drop TID for
5 days.
1% atropine17 eye drop one drop TID for 3 days and Carboxy methyl cellulose
sodium 1% 18 eye drops one drop QID for 12 days was carried out . All dogs received
inj. Amoxycillin & Sulbactum Sodium10 @ 7 mg per kg body weight i/m and inj.
Meloxicam11 @ 0.2 mg per kg body weight i/m for 5 days. Tarsorrhaphy sutures were
removed on the 4th postoperative day.
17.
Atropine eye drops 1%, Jawa Pharmaceuticals (India) Pvt. Ltd., Jaipur, Rajasthan.
18.
Just tears Liquigel, Sunways, India Pvt.Ltd. Mumbai.

40
 All the dog owners were advised to keep Elizabethan collar to avoid
scratching or other self-mutilation.
3.9 Parameters for evaluation of post-surgical outcomes
3.9.1 Tests for vision
All dogs were subjected to assessment of vision after corneal grafting
included menace test, dazzle reflexes and pupillary light reflexes on 4th, 8th and 12th
post-surgical day.
3.9.2 Schirmer tear test (STT)
The Schirmer tear test was done to assess the quantitative production of tears.
STT was carried out on 4th, 8th and 12th post-operative day and observations were
compared statistically.
3.9.3 Fluorescein dye test
Fluorescein dye test was recorded on 4th, 8th and 12th day.
3.9.4 Corneal cytology
Was performed on 12th post-operative day
3.9.5 Hematological parameter
Hematology was conducted on 12th post-operative day and were compared
statistically with preoperative findings.
3.9.6 Appearance of grafted ulcer
All dogs were kept under observation on 4th, 8th and 12th postoperative days
and they were monitored for healing of cornea, corneal edema and corneal scarring.
3.9.6.1 Corneal Edema
The corneal edema was assessed and graded as Grade 3(severe edema),
Grade 2 (Moderate edema), Grade 1(Mild edema) and Grade 0(No edema).
3.9.6.2 Corneal opacity
The corneal opacity was assessed and assigned scores from complete opacity
to no opacity and was graded as Grade 3+ (severe), Grade 2+ (Moderate opacity),
Grade 1+ (Mild opacity), Grade 0+ (No Opacity).
3.9.6.3 Corneal healing
Corneal healing was observed on 4th, 8th and 12th postoperative days.

41
Plate no. 3.1 Lateral positioning of dog under operating microscope.

Plate no. 3.2 Placing of eye speculum along with stay suture.

A
Plate no. 3.3 Debridement of loose epithelium with the
help of sterile cotton bud.

B
3.9.7 Complication if any
Complication if any were recorded during present investigation.
3.9.8 Stastical Analysis
The present clinical data was analyzed with the help of SSP by using t-test and
ANOVA.

42
Results & Discussion
 CHAPTER IV

RESULTS AND DISCUSSION

In the present study, a total of 180 dogs with various eye affections were
screened. Out of these 25 cases (13.88%) were diagnosed with various types of
corneal ulcers and were subjected to medical management. Out of 25 dogs, 12 dogs
(48.00%) did not respond to said treatment protocol and hence they were subjected to
amniotic membrane graft.
These 12 dogs were randomly divided into two groups viz; group I (1-6) and
group II (7-12). The non-healing corneal ulcers of dogs from group I were treated by
using dry human amniotic membrane graft technique whereas, in group II, wet human
amniotic membrane graft technique was advocated.
4.1 Signalment and anamnesis
The age of dogs from both the groups, ranged from 4 months to 11.4 years
(4.12±3.70 years).The present finding is in accordance with Sood and Mohindroo
(2005) who observed that dogs with a mean age of 5.46± 1.03 years were more prone
to corneal affections. Whereas, Sale et al. 2013 found that ocular diseases were
common in all age groups of dogs. In all the twelve dogs the average duration of
corneal ulcer was 11.08± 3.54 days.
The breed wise distribution of corneal ulcers included Shih Tzu (33.33%)
(Plate4.1) followed by Doberman (16.66%), Rottweiler (16.66%) (Plate 4.2),
Nondescript (16.33%), Labrador (8.33%) and Pitbull (8.33%) which is in accordance
with the findings of Holmberg, (1981) who reported highest incidence of corneal
ulcers in Shih Tzu (73%) followed by Boxer (20%). In the present study, the highest
incidence of corneal ulcers reported in Shih Tzu breed might be due to exophthalmic
nature of eyes which is highly prone to trauma. Similar findings were recorded by
(Wolfer and Grahn 1994).
Thomson, (2007) and Kim et al., 2009 who found that brachycephalic breeds
with pushed-in faces, such as the Shih Tzu, Bulldog, French bulldog, Lhasa Apso,
Pekingese, and Pug were more susceptible to ulcerative keratitis.

43
 The incidence of corneal ulcers was higher in males (n=9; 75%) than in
females (n=3; 25%). This finding is in agreement with the findings of Turner and
Blogg (1997) who reported highest incidence of corneal ulcers in males (80%) than
females (20%). Ramani et al., 2013 and Moore, (2003) also recorded higher
incidence of corneal ulcers in males (67%) than in females (33%). In contrast,
Murphy et al., (2001) reported that there was no sex predilection as far as occurrence
of corneal ulcers in dogs is concerned.
All the dogs from both the groups had unilateral involvement of the eye
amongst which, irrespective of groups 50% of dogs had their right eye affected and
50
% of dogs had their left eye affected. There was no predilection on ulcers for one
particular eye (Nagashree, 2019).
As assessed visually on the day of presentation and on the day of surgery the
dogs from both the groups were having good health with normal physiological
parameters, indicating traumatic origin of ulcer. Similar findings were recorded by
Wolfer and Grahn (1994) and Mandell and Holt (2005) who observed that the dogs
with corneal ulcers were having good health and reported that most of the corneal
ulcers in dogs is due to their playful nature.
The results of signalment and anamnesis are depicted in (Table no. 4.1).
4.1.1 Classification of corneal ulcers
Total 12 eyes with corneal ulcers were classified into five groups on the basis
of location of ulcers on corneal surface (Table no.4.1). This revealed that, most of the
corneal ulcers were located centrally (n=5) (Plate no. 4.3.a and Plate no. 4.3.b)
followed by ventro-nasal (n=3) (Plate no. 4.4), ventro temporal (n=2) (Plate no. 4.5)
dorso-nasal (n=1) (Plate no. 4.6) and dorso-temporal (n=1) (Plate no. 4.7).
The results of classification based on location of corneal ulcers are depicted in
(Table no. 4.1).
Similar finding is corroborated with Dorbandt et al., (2015) who found high
percentage of centrally located corneal ulcers in dogs.

44
Table no. 4.1 Signalment and anamnesis of dogs from group-I and group-II (n=12).
Duration
Age Eye
Group Dog no. Breed Sex of ulcer Type and extent of ulcer
(Years) affected
(days)
1 Doberman M 2.5 10 Left Ventro temporal, stromal
2 Rottweiler M 4.3 7 Right Central stromal
3 Shih Tzu M 0.9 10 Right Ventro nasal(melting)
4 Labrador F 5.4 8 Right Dorso nasal stromal
I
5 Shih Tzu M 0.8 12 Left Ventro nasal stromal
6 Nondescript F 10.4 15 Left Ventro nasal superficial
7 Rottweiler M 2 .9 17 Right Darso temporal
superficial
8 Pitbull M 6.2 8 Right Central decemetoceal
9 Shih Tzu M 0.8 11 Left Central superficial
10 Shih Tzu F 2.3 8 Right Central deep stromal
II
11 Nondescript M 11.4 9 Left Central superficial
12 Doberman M 0.4 18 Left Ventro temporal deep
stromal
Mean ±SE 4.12± 11.08 ±
3.70 3.54
 Plate no. 4.1 Corneal ulcer in Shih Tzu breed of dog.

Plate no. 4.2 Corneal ulcer in Rottweiler breed of dog.

C
Plate no. 4.3 (a) Centrally located corneal ulcer in Dog no. 2.

Plate no. 4.3 (b) Centrally located corneal ulcer in Dog no. 8.

Plate no. 4.4 Ventro nasally located corneal ulcer in Dog no. 5.

D
Plate no. 4.5 Ventro temporally located corneal ulcer in Dog no. 12.

Plate no. 4.6 Dorso nasally located corneal ulcer in Dog no. 4.

Plate no. 4.7 Dorso temporally located corneal ulcer in Dog no. 7.

E
4.2 Physiological parameters
Felchle and Urbanz (2001) suggested that many a times ocular involvement
indicates some systemic diseases therefore, a general physical examination should
precede the ophthalmic examination.
In the present study, the physiological parameters of all the animals from both
the groups were within normal range, which included body temperature
(100.50±0.670F), heart rate (72.7±8.48 /min) and respiration rate (29.75±3.67/min).
The results of physiological parameters are depicted in (Table no. 4.2)
4.3 Ophthalmic examination
Pre-operatively, detailed ophthalmic examination of all animals was carried
out by adapting following tests.
4.3.1 Gross examination
Gross examination of all eyes showed normal eye symmetry and did not show
structural deformity in all cases.
The ocular discharge was grossly noted on the day of presentation from
animals of both the groups. The ocular discharge was watery (n=4), mucoid (n=5) and
mucopurulent (n=3).The increased ocular discharge was attributed to expression of
opportunistic bacteria and increase in bacterial load in cornea following trauma
(Whitley, 2000; Mandell and Holt 2005).
The discharge gradually changed from mucopurulent to mucoid and from
mucoid to watery and then gradually became normal up to 12th postoperative day
following grafting and postoperative treatment i.e. Moxifloxacin 0.5% eye drops one
drop TID for 5 days, 1% atropine eye drop one drop TID for 3 days and
Carboxymethylcellulose sodium 1% eye drops one drop QID for 12 days.
Simultaneously, all dogs received inj. Amoxycillin & Sulbactum Sodium @ 7 mg per
kg body weight i/m and inj. Meloxicam @ 0.2 mg per kg body weight i/m for 5 days.
The postsurgical observation revealed that on 12th postoperative day, all the
dogs from both the groups showed watery to normal ocular discharge indicating
decrease in the bacterial load and inflammatory responses which could be attributed
to the antibacterial property of the grafted human amniotic membrane Fernandes et
al.

45
(2005) as well as topical Moxifloxacin antibiotic eye drops. Varges et al., (2009) also
used fluoroquinolone group of eye drops and found to be most effective in naturally
occurring canine ocular infections.

4.3.2 Vision test (preoperative)

Preoperatively, i.e on day 0 all dogs underwent routine vision tests to
diagnose concurrent blindness along with corneal opacity.

Nine dogs showed absence of menace response and three dogs showed
sluggish menace response which was attributed to loss of corneal transparency that
obscured visual axis. This finding is in accordance with Gilger et al., (2014) who
described that increased water content in cornea results in corneal oedema, increased
thickness and scattering of light, leading to reduced transparency and loss of vision

Dazzle response was sluggish in the majority of dogs (n=11) and was normal
in one dog. This was due to severe oedema and disturbed corneal transparency which
diminished the intensity of a bright focal light reaching the retina. This is in
accordance with Featherstone and Heinrich (2014) who stated that sufficient
brightness of the light source is fundamental for performing dazzle reflex test and this
bright focal light should reach retina.

In present investigation in all dogs pupillary light reflexes could not be

accessed due to deep and large sized epithelial defect created extensive corneal opacity
and corneal oedema which prevents the light to pass through pupil and thereby prevent
the pupil constriction. Therefore, all dogs showed absence of pupillary light reflex
(Cullen and Grahn 2005). These findings in present study are in accordance with the
observations of Felchle and Urbanz (2001) who used pupillary light reflex for
confirmation of visual function and corneal transparency.

The results of preoperative vision tests are depicted in (Table no. 4.3).

4.3.3 Direct ophthalmoscopy

Direct ophthalmoscopic examination of all the dogs from group I and groupII
revealed that ten dogs were having severe edema (grade 3) and remaining two dogs
had moderate edema (grade 2) (Table no.4.3 ).This was due to breach in epithelium
and

46
 Table no. 4.2 Mean (±S. E) of physiological parameters recorded
in dogs from group-I and group-II (n=12).

Dog Body Temp. Heart rate Respiration rate

Group
No. (˚F) (per minute) (per minute)
1 100.5 78 28
2 101.1 63 25
3 100.2 86 35
4 99.8 68 27
I
5 100.6 79 29
6 101.7 62 26
7 100.9 69 32
8 99.8 66 30
9 101.3 84 36
II 10 99.8 80 34
11 99.9 64 27
12 100.5 74 28
Mean±SE 100.50 ±0.67 72.75 ± 8.48 29.75 ± 3.67
Table no. 4.3 Preoperative evaluation of vision tests viz: Menace response (M), Dazzle
reflex (D), Pupillary light reflex (PLR), corneal edema and corneal
opacity in dogs from group-I and group-II (n=12).

Group Dog M D PLR Edema Opacity

No.
1 + + Grade 3 Grade 2+

2 - + Grade 2 Grade 2+
I
3 - + Grade 3 Grade 3+

4 - + Grade 3 Grade 3+
Could
5 - + not be Grade 3 Grade 3+
assessed
6 - + Grade 3 Grade 3+

7 - + Grade 2 Grade 2+

II 8 + ++ Grade 3 Grade 2+

9 + + Grade 3 Grade 3+

10 - + Grade 3 Grade 3+

11 - + Grade 3 Grade 3 +

12 - + Grade 3 Grade 3 +

Normal (++) Sluggish (+) absent (-)

endothelium which lead to accumulation of infiltrates resulting into oedema (Wilkie
and Whittaker, 1997; Moore 2003).
Out of twelve dogs from group-I and group-II, eight dogs showed grade 3+
i.e. severe corneal opacity and remaining four dogs were having grade 2+ i.e
moderate corneal opacity along with ulceration (Table no.4.3). Corneal opacity
occurred due to changes in collagen type or disturbance of the usual lamellar
organization of the stroma (Wilkie and Whittaker 1997).
Corneal ulcers were observed by using direct ophthalmoscope and classified
on the basis of depth as described by Moore, (2003). Six dogs (50 %) showed
stromal/deep stromal ulcers, four dogs (33.33%) were having superficial ulcers, and
one dog each was having melting corneal ulcer (8.33%) and descemetocele (8.33%)
(Table no. 4.4).
The results of direct ophthalmoscopy are shown in (Plate no. 4.8 A & B).

4.3 Preparation of patient and anesthetic protocol

4.3.1 Preoperative medication
In the present study, topical eye drops of 0.5% Moxifloxacin was used to
control the ocular infection in both the groups. Varges et al., (2009) also used
fluoroquinolone group of eye drops and found to be most effective in naturally
occurring canine ocular infections.
Proparacain hydrochloride 0.5 % eye drop was instilled prior to surgery as a
topical anesthetic. Willams, (2005), Silva et al. (2010) and D Kumar, (2012) also
found 0.5% Proparacaine hydrochloride as effective topical anaesthesia for cornea.
Ganesan and Ramani (2018) also used 0.5% proparacaine hydrochloride as topical
anaesthetic.

4.3.2 Anesthetic protocol

In all dogs, balanced anesthesia was achieved using combination of inj.

Butorphanol Tartrate @ 0.3 mg per kg body weight intramuscularly and inj.
Triflupromazine HCL @ 1.2 mg per kg body weight intravenously as sedatives.
General anesthesia was induced by inj. Propofol @ 4-6 mg/kg body weight
47
intravenously and was maintained by using 2-3% Isoflurane. Retrobulbar nerve block
was also performed by administrating 2 ml of 2 % Lignocaine hydrochloride (Plate
No. 4.9). This protocol provided smooth induction and recovery of anesthesia .All
dogs were properly anaesthetized and no anesthetic complications were observed
during the study. These findings are in consonance with (Sabnis S. A. 2018).

In the present study, retrobulbar nerve helped to achieve sufficient proptosis

along with excellent and long-lasting analgesia. Bhadsavle, (2006) also reported that
proptosis of the eyeball is a pre-requisite for comfortable access to the eye during
ophthalmic surgeries.
Similar findings were also recorded by Hazra et al. (2008) and Myrna (2010)
in their study and found that retrobulbar administration in conjunction with traditional
premedication prior to eye surgery was an effective form of adjunctive analgesia and
reduced the need for additional postoperative analgesics.

4.3.3 Positioning and Preparation of patient

All the dogs were positioned in lateral recumbency with affected eye in
upward position. Head was kept in an elevated position using soft bedding material
for proper visualization under the operating ophthalmic microscope.
The eyelashes of the affected eye were clipped and the ocular discharge was
cleaned with a cotton swab. The affected eye was thoroughly cleaned with 0.5%
povidone iodine solution and was covered with sterile eye drape as prescribed by
perifer (1993).

4.5 Surgical procedure for corneal grafting

The dogs from both groups were subjected to debridement of epithelial tissue
debris accumulated around the ulcerated part of cornea by using B.P blade no. 15
(Plate no. 4.10) so as to prepare ulcer bed prior to grafting (Plate
no.4.11).Debridement was found to be more effective for healing of corneal ulcer
(Sridhar et al., 2001). The commercially available dry human amniotic membrane
graft of the (size of 2 cm x 2

48
 Table No.4.4 Classification of Ulcers on the basis of depth in
dogs from group-I and group-II (n=12).
Sr. No Type of ulcers No. of dogs Percentage
affected
1 Stromal/deep stromal 6 50%
2 Superficial 4 33.33%
3 Melting 1 8.33 %
4 Descemetocele 1 8.33 %
Total 12 100%
Plate no. 4. 8 (A) Corneal edema and opacity noticed by direct
ophthalmoscopy on day 0 prior to membrane grafting.

Plate no. 4.8 (a) Corneal ulcer with grade 2 edema

and grade 3+ opacity in Dog no. 1.

Plate no. 4.8 (b) Melting ulcer with grade 3 edema

and grade 3+ opacity in Dog no. 3.

A
Plate no. 4. 8 (B) Corneal edema and opacity noticed by direct
ophthalmoscopy on day 0 prior to membrane grafting.

Plate 4.8 (c) Corneal ulcer with grade 2 edema

and grade 2+ opacity in Dog no. 7.

Plate 4.8 (d) Corneal ulcer with grade 3 edema

and grade 3+ opacity in Dog no. 12.

B
 Plate no. 4. 9 Site of retrobulbar nerve block.

Plate no. 4.10 Preparation of ulcer bed for grafting.

Plate no. 4.11 Ulcer bed prepared for grafting.

C
Plate no. 4.12 Grafting of cornea with dry
human amniotic membrane.

Plate no. 4.13 Grafting of cornea with wet

human amniotic membrane.

Plate no. 4.14 Tarsorrhaphy suture.

D
cm) and wet human amniotic membrane graft of (2cm x 2cm size) (2mm greater than
size of ulcer) was procured in advance and was preserved. After debridement, these
grafts were placed on the corneal defect in group-I and group II respectively (Plate
no.4.12 and Plate no. 4.13). The grafts were sutured with surrounding cornea by
simple interrupted suture using Vicryl 10-0 suture material so that, neighbouring
recipient epithelial cells can migrate onto the amniotic membrane and the wound
begins to close as per Sippel et al. (2001) and Seitz (2007).In their study, they
observed that debridement of corneal ulcer and suturing the graft with cornea allows
migration of recipient cells to the amniotic membrane graft for effective healing.
In all the dogs tarsorrhaphy (Plate no. 4.14) was performed by using Vicryl 5-
0 suture material so as to prevent self-mutilation and to promote healing process.
Gurpreet Singh (2017) found that in case of amniotic membrane transplantation, rate
of healing can be significantly improved by using tarsorrhaphy as protective measure.

The dry and wet amniotic membrane grafts were used for treating persistent
epithelial defects by (Lee et al. (1997) and Sridhar et al., 2001) in their study. In
another study, Wichayacoop et al., (2005) used preserved human amniotic membrane
grafts in dogs for healing of induced stromal lesions.
4.6 Post surgical evaluation
The dogs from group I and group II were kept under observation for 12 days post
operatively and following parameters were recorded.
Vision tests, STT, fluorescein dye test, corneal cytology, hematology,
appearance of grafted ulcer viz; corneal edema and corneal opacity and corneal
healing.
The results of postperative vision tests are depicted in (Table no. 4.5).

4.6.1 Assessment of vision

Assessment of vision was carried out on 4 th, 8th and 12th postoperative days
after corneal grafting. The tests used were menace response test, dazzle reflex test and
pupillary light reflex test.

49
4.6.1.1 Menace response test
On 4th day five dogs from group I showed absence of menace response and
one dog showed sluggish menace response. whereas from group II, one dog showed
absence of menace response and remaining five dogs showed sluggish menace
response.
On 8th day from group I, two dogs showed absence of menace response, three
dogs showed sluggish menace response and one dog had normal menace response.
From group II, three dogs showed sluggish menace response and three dogs showed
normal menace response
On 12th day from group I, one dog showed sluggish menace response and five
dogs showed normal menace response. From group II, all dogs showed normal
menace response.
Absent to sluggish response on day 4 th and 8th was attributed to the location
respective ulcers, corneal opacity and severe oedema which directly obscured the
visual axis.
On 12th day all dogs showed blinking response with eyelid movement i.e.
normal menace response that indicated regaining of normal vision.
.
4.6.1.2 Dazzle reflex test
On day 4, all dogs from both the groups had sluggish dazzle reflex. On 8th
post- operative day, two dogs from group I, showed sluggish dazzle reflex and four
dogs showed normal dazzle reflex. From group II, one dog had sluggish dazzle reflex
whereas remaining five dogs showed normal dazzle reflex .On 12 th day all the dogs
from both the groups showed normal dazzle reflex.
The normal dazzle reflexes recorded in terms of blinking of eye lids, globe
retraction, eyelid protrusion and head movement are in accordance with Moore
(2001) and Mitchell (2011) who reported that proper eyelid closure before triggering
a palpebral reflex and a normal blink response to the dazzle test indicates regaining
of vision.

50
Table no. 4.5 Postoperative evaluation of vision tests viz: Menace Response (M),
Dazzle Reflex (D) and Pupillary Light Reflex (PLR) on day 4, 8, and 12
in dogs group-I and group-II (n=12).

Group Dog 4th day 8th day 12th day

No.
M D PLR M D PLR M D PLR

1 + + ++ ++ + ++ ++ ++
I 2 - + + ++ + ++ ++ ++

3 - + + ++ + ++ ++ ++

4 - + Could - + + ++ ++ ++
not be
5 - + assess + ++ ++ ++ ++ ++
ed
6 - + - + + + ++ +

7 + + + ++ + ++ ++ ++

8 + ++ ++ ++ ++ ++ ++ ++
II 9 + + ++ ++ ++ ++ ++ ++

10 + + + + + ++ ++ ++

11 + + + ++ + ++ ++ ++

12 - + ++ ++ + ++ ++ ++

Normal (++) Sluggish (+) absent (-)

4.6.1.3 Pupillary Light Reflex test
On 4th post-operative day the pupillary light reflex test was performed
however the reflexes could not be assessed in all dogs from both groups, which might
be due to the corneal opacity and edema that obscures the light rays to fall on the
retina.
On 8th postoperative day five dogs from group I showed sluggish PLR reflex
and one dog showed normal PLR reflex. From group II, four dogs showed sluggish
PLR reflex and two dogs showed normal PLR reflex.
On 12th day from group I, one dog showed sluggish PLR reflex and five dogs
showed normal PLR reflex. Whereas all dogs from group II showed normal PLR
reflex.
Normal vision can be attributed to the reduction in edema and improvement in
clarity of the cornea which resulted due to the anti-inflammatory effects of the
decellularised human amniotic membrane graft (Tseng and Li, 1999; Meller et al.,
2000; Wichayacoop et al., (2005); Riau et al., 2010 and Korittum et al., 2019).
Costa et al., (2019) reported that cryopreserved human amniotic membrane
transplantation was very efficient for surgical management of complicated corneal
ulcers in dogs, which provided 99.1% good anatomical and 97.4% of visual outcomes
with minimal complications in dogs.

4.6.2 Schirmer tear test (STT)

The dogs from both the groups showed fluctuating but non-significant
differences in values of STT (Plate no.4.15). The mean vales of STT was 25.11 ±
4.76 mm/min , 23 ±4.2 mm/min, 22 ±4.02 mm/min and 19.5 ± 1.9 mm/min on day
0, 4th
,8th and 12th day respectively (Table no. 4.6). Lacrimation resulting from ocular
irritation or pain due to corneal ulceration might be responsible for fluctuating STT
values throughout the investigation Williams and Burg (2017), but these values were
within the normal range. The normal values for STT vary from 18.64 ± 4.47 mm/min
to 23.90 ± 5.12 mm/min (Featherstone and Heinrich 2014).
The present findings of STT are in consonance with Williams and Burg
(2017) who recorded higher range of STT in dogs with corneal ulceration. The higher
values

51
of STT were recorded in the initial days but later this values decreased which might
be due to anti-inflammatory effects of human amniotic membrane (Tseng and Lee,
1999).

4.6.3 Fluorescein Dye Test

Fluorescein dye test was used for the confirmation of corneal ulceration
Renwick, (1996).
Fluorescein dye test was carried out in all cases from both the groups on day 0
except in dog. No. 8, where in, due to descemetocele only margins of the ulcers took
the stain because the ulcer was so deep that the corneal stroma was abraded resulting
in protrusion of descemet’s membrane (Plate No. 16).Kern, (1990) also observed that
only the ends of abrasive cornea stained greenish in cases of descemetocele as the
exposed descemet’s membrane in the centre does not stain.
In the present study, fluorescein stain aided in diagnosis of corneal ulcers and
the dye not only stained the ulcerated area but also migrated under the loose flaps of
epithelium and stained the surrounding anterior stroma which made the ulcer appear
larger than the actual size (Plate No.4.17) (Janssens 2007; Kumar and Thirumalesh
2013).
On 4th, 8th and 12th day of observation in all the dogs, fluorescein dye test was
found to be negative due to epithelialization and covering of ulcer with the graft.
Negative Fluorescein dye test was considered as an indication of healthy
cornea or complete corneal healing Absence of staining could be attributed to the re-
epithelialization of the lost corneal epithelium by the sterilized, processed human
amniotic membrane graft. Similar findings were observed by Costa et al., (2019) who
studied epitheliotropic properties of human amniotic membrane and assessed corneal
healing by the negative uptake of fluorescein stain. Wichayacoop et al.(2009)
observed completely epithelialized cornea within four days after transplantation of
human amniotic membrane.
Ledbetter et al. (2014) stated that epithelial migration starts in one hour and it
gets completed by 48-72 hours.

52
Table no. 4.6 Evaluation of Schirmer Tear Test on day 0, 4, 8 and 12
in dogs from group-I and group-II (n=12)
Group Dog no. Day 0 Day 4 Day 8 Day 12
1 28 25 24 20

2 25 23 23 18

3 33 31 29 23
I 4 31 29 28 22

5 21 20 22 20

6 23 20 19 18

7 22 19 19 20

8 17 18 17 18

9 26 23 20 19
II 10 24 22 19 17

11 28 25 24 20

12 25 23 23 18

Mean±SE 25.11 23.00 22.00 19.50

± 4.76 ±4.2 ±4.02 ± 1.90
 Plate no. 4.15 Schirmer tear test.

Plate no. 4.16 Clear patch at the center in case of descemetocele.

Plate no. 4.17 Staining of ulcer patch by using fluorescein dye.

E
 The observations of fluorescein dye test of all the twelve cases are depicted in
(Table no.4.7)

4.6.4 Corneal Cytology

Corneal cytology is a diagnostic tool used for diagnosis and assessment of
treatment regimen for ocular conditions that does not respond to empirical therapy
Slatter, (1990). It is a quick and non- invasive method of detecting the corneal
pathology based on which treatment can be followed.
In present study, corneal cytology was performed preoperatively i.e. on day 0
and on 12th day post-operatively.
In all the dogs, the corneal cytology performed on day 0 showed sloughed
squamous epithelial cells from different layers of anterior epithelium of the cornea
and varying number of inflammatory cells mainly neutrophils along with fibrin and
keratin debris (Plate no. 4.18 (a) –Plate no.4.21 (a)).
Anoop et al., (2015) carried out impression smear cytology of canine cornea
and observed infiltration of neutrophils, squamous epithelial cells as major cells,
presence of necrotic debris and degenerative changes in the impression smear.
Gerding et al., (1988) carried out corneal cytology of a normal eye. He observed non
cornified superficial corneal epithelial cells, intermediate corneal epithelial cells,
lymphocytes and polymorphonuclear neutrophils as normal corneal cells. He further
observed that in bacterial infection, polymorphonuclear neutrophils, keratin debris,
mucus, and bacterial clumps were predominant cells in corneal cytology.
The cytological samples collected on 12th post-operative day revealed very
few squamous epithelial and very few to none inflammatory cells. (Plate no.4.18 (b) -
Plate no.21 (b)) indicating improvement in inflammatory condition. This could be due
the complete re-epthialization of cornea and anti-inflammatory role of amniotic
membrane graft along with the post-operative medication (Ledbetter et al., (2014).

53
4.6.5 Hematological findings

On day 0, the mean values of Hb, PCV, TEC, TLC, MCV, MCHC, platelets
and DLC count viz: Neutrophils, Lymphocyte, Monocytes, Eosinophil and Basophil
of six dogs from group-I (dry amniotic membrane graft) were 12.81 ±0.82
gm/dl,37.78 ±
2.5 %, 5.83 ± 0.56 x106/µl,13.00 ± 0.67 x103/µl, 63.83 ±1.99 fl, 34.98 ± 1.10 gm/dl,
1.96± 0.34x106/µl, 71.16±1.60%, 23.66±1.90%,3.16±0.79%, 2.00± 0.36%, and 0.33±
0.33%, respectively. Whereas the mean value of six dogs from group II (wet amniotic
membrane graft) were14.18 ±0.48 gm/dl,41.26 ± 1.70 %, 6.09 ± 0.44 x106/µl,11.34 ±
0.46 x103/µl, 64.33 ±2.24 fl, 34.83 ± 0.72 gm/dl, 2.43± 0.28x106/µl, 65.16±1.22%,
29.50±2.29%,2.50±0.56%,1.16±0.16%, and 0.00 respectively.

The mean values of Hb, PCV, TEC, TLC, MCV, MCHC, platelets and DLC
count viz: Neutrophils, Lymphocyte, Monocytes, Eosinophil and Basophil on12th
day post operatively for group-I (dry amniotic membrane graft) were 12.67 ±1.75
gm/dl,37.53 ± 5.42 %, 6.09 ± 0.44 x106/µl,10.30 ± 0.46 x103/µl, 58.00 ±4.50 fl, 34.76
± 0.48 gm/dl, 3.04 ± 0.64 x106/µl, 70.83±2.3%, 24.50±1.92%,2.50±0.61%, 2.16±
0.47%, and 0.00, respectively whereas the mean value for group II (wet amniotic
membrane graft) were13.00 ±1.67 gm/dl,38.6 ± 4.50 %, 6.88 ± 0.49 x106/µl,9.60 ±
1.60 x103/µl, 60.00 ±2.68 fl, 35.05 ± 0.54 gm/dl, 3.40± 0.64x106/µl, 65.66±2.20%,
30.00±2.47%,2.66±0.61%,1.33±0.21%, and 0.00 respectively.

The results of hematological findings are depicted in (Table no. 4.8).

There was non-significant difference in the values of all hematological

parameters of group I (dry amniotic membrane graft) between day 0 and on 12th post-
operative day.

Similarly, the values of all hematological parameters of the dogs from group
II (wet amniotic membrane graft) between day 0 and on 12th post-operative day did
not differ significantly.

So also, there was non-significant difference between the values of

hematological parameters of group I and group II.

54
Table no. 4.7 Evaluation of Fluorescein Dye Test on day 0, 4, 8 and
12 in dogs from group-I and group-II (n=12).
Group Dog no. Day 0 Day 4 Day 8 Day 12

1 + - - -

2 + - - -

I 3 + - - -

4 + - - -

5 + - - -

6 + - - -

7 + - - -

8 - - - -

II 9 + - - -

10 + - - -

11 + - - -

12 + - - -

Staining (+) No Staining (-).

 Table no. 4.8 Mean (±S.E) of Hematological parameters on preoperative day and on 12th post- operative
day in dogs from group-I and group-II (n=12).

TEC TLC PLT LY- MO-

HB PCV MCV MCHC NEU EOS BASO
(x106/ (x103/ (x106/ MH NO
(gm/dl) (%) (fl) (gm/dl (%)
(%) (%)
(%) (%)
µl) µl) µl)
Group
I
Pre-
12.81 37.78 5.83 13.00 63.83 34.98 1.96 71.16 23.66 3.16 2.00 0.33
operati
±0.82 ± 2.5 ±0.56 ±0.67 ±1.99 ±1.1 ±0.34 ±1.6 ±1.90 ±0.79 ±0.36 ±0.33
ve
12th 37.53
Post- 12.67 6.50 10.30 58.00 34.76 3.04 70.83 24.50 2.50 2.16
±5.4 00
operati ± 1.75 ±0.70 ±1.89 ±4.50 ±0.48 ±0.64 ±2.3 ±1.92 ±0.61 ±0.47
2
ve day
Group
II
Pre- 41.26
14.18 6.09 11.34 64.33 34.83 2.43 65.16 29.50 2.50 1.16
operati ± 00
± 0.48 ±0.44 ±0.46 ± 2.24 ± 0.72 ±0.28 ±1.22 ±2.29 ±0.56 ±0.16
ve 1.70
12th 38.6 3.40
Post- 13.00 6.88 9.60 60.00 35.05 65.66 30.00 2.66 1.33
±4.5 ±`` 00
operati ±1.67 ± 0.49 ±1.60 ±2.68 ±0.54 ±2.20 ±2.47 ±0.61 ±0.21
0 0.64
ve day
Plate no. 4.18 (a)
Neutrophils as predominant
cell type along with few
epithelial cells on day 12, in
Dog no. 2 (Giemsa stain
1000x).
Plate no. 4.19 (a) Neutrophils
as predominant cell type
along with few epithelial
cells on day 12, in Dog no. 5
(Giemsa stain 1000x).
F
Plate no. 4.18(b) Showing
only epithelial cells on day
0, in Dog no. 2 (Giemsa
stain 1000x).
Plate no. 4.19 (b) Showing
only epithelial cells on day
0, in Dog no. 5 (Giemsa
stain 1000x).
Plate no.4.20 (a) Neutrophils
as predominant cell type
along with few epithelial
cells on day 12, in Dog no. 7
(Giemsa stain 1000x).
Plate no. 4.21 (a) Neutrophils
as predominant cell type
along with few epithelial
cells on Day 12 in Dog no. 10
(Giemsa stain 1000x).
G
Plate 4.20 (b) Showing only
epithelial cells on day 0, in Dog
no. 7 (Giemsa stain 1000x).
Plate no. 4.21 (b) Showing
only epithelial cells on day
0,in Dog no. 10 (Giemsa
stain 1000x).
 Above findings of hematological parameters related to RBCs, WBC and
platelets indicated absence of anemia and leukocytosis. This can be attributed to no
loss of blood during and after the operative procedure in both the groups.
The mean TLC and DLC values were within normal reference range Chinchu
(2010), Venugopal 2013 and Antonia (2014) and did not show statistically significant
difference in both groups before operative and on 12 th post-operative day. This
indicated absence of leukocytosis and could be due to very small area involved in
inflammation.

4.6.6 Postoperative appearance

The results of postoperative appearance are depicted in (Table no.4.9).
4.6.6.1 Corneal edema

On 4th day, four dogs from group I showed moderate edema whereas two dogs
showed mild edema. From group II, five dogs showed moderate edema and one dog
showed sever edema.

On 8th day five dogs from group I showed mild edema whereas there was no
edema in one dog. From group II, one dog showed moderate edema, four dogs
showed mild edema and one dog showed no signs of edema.

On 12th day all dogs from both the groups showed no signs of edema.

The post-operative appearance of corneal edema in dogs from group I is

shown in (Plate no. 4.23 (A) and Plate no. 4.23(B)) and from group-II, is shown in
(Plate no.4.24 (A) and Plate no. 4.24 (B)).

In present study, all the dogs from both the groups showed recovery on 12 th
postoperative day, which might be due to anti-inflammatory properties of human
amniotic membrane graft. The present clinical findings are in accordance with the
findings of Hao et al., (2000) who demonstrated anti-inflammatory properties of
amniotic membrane in corneal grafting via Immunohistochemistry and RT-PCR test.
Similarly, Meller et al. (2002) and Gurpreet Singh (2017) evaluated transplanting
intact

55
human amniotic membrane in dogs and reported drastic reduction in inflammation
and facilitation of re-epithelialization and concluded that, the restoration of vision
and resumption of normal corneal transparency was observed on 11.25±2.50
postoperative day.

4.6.6.2 Corneal opacity

On 4th day four dogs from group I showed severe opacity and three dogs
showed moderate opacity .From group II, all dogs showed moderate opacity,

On 8th day three dogs from group I showed moderate opacity and three dogs
showed mild opacity. From group II, one dog showed moderate corneal opacity
whereas four dogs showed mild corneal opacity and in one dog corneal opacity was
not observed.

On 12th day, two dogs from group I showed a hazy spot of opacity however
this opacity did not affect the vision and four dogs from this group showed no corneal
opacity. From group II, none of the dog had corneal opacity.

The post-operative appearance of corneal opacity in dogs from group I is

shown in (Plate no. 4.23 (A) and Plate no. 4.23(B)) and from group-II, is shown in
(Plate no.4.24 (A) and Plate no. 4.24 (B)).

Corneal opacity might have been observed due to disorganized stromal layer
because of the ulcer (Wilkie and Whittaker 1997).
The improvement in the corneal clarity could be attributed to the antifibrotic
effects of the decellularised human amniotic membrane as the human amniotic
membrane suppresses the transforming beta growth factor signaling which, is responsible
for fibrosis (Tseng and Li, 1999; Lee et al., 2000).
Amniotic membrane was in close adherence to cornea and the ulcer were well
surrounded by the graft. This might be the possible reason behind the greater corneal
transparency in the operated animals in both the groups. Furthermore, amniotic
membrane also possesses anti-angiogenetic effect, anti-scarring effect and anti-
inflammatory action which makes it more suitable occular graft (Gomes et al., 2005).

56
Table No.4.9 Postoperative appearance of cornea in dogs treated with dry & wet human amniotic membrane graft
on 4th , 8th and 12th post-operative day in dogs from group-I and group-II (n=12).

Group Dog No. 4th day 8th day 12th day

Edema Opacity Edema Opacity Edema Opacity
1 Grade 1 Grade 1+ Grade1 Grade1+ Grade 0 Grade 0+
2 Grade 1 Grade 2+ Grade 0 Grade1+ Grade 0 Grade 1+
3 Grade 2 Grade 2+ Grade1 Grade1+ Grade 0 Grade 0+
4 Grade 2 Grade 2+ Grade1 Grade 1+ Grade 0 Grade 0+
I
5 Grade 2 Grade 3+ Grade1 Grade 2+ Grade 0 Grade 0+
6 Grade 2 Grade 3+ Grade1 Grade 2+ Grade 0 Grade 1+
7 Grade 2 Grade 2+ Grade1 Grade 2+ Grade 0 Grade 0+
8 Grade 2 Grade 2+ Grade1 Grade 1+ Grade 0 Grade 0+
9 Grade 2 Grade 2+ Grade 0 Grade 0+ Grade 0 Grade 0+
10 Grade 2 Grade 2+ Grade 1 Grade 1+ Grade 0 Grade 0+
II 11 Grade 2 Grade 2+ Grade 1 Grade 1+ Grade 0 Grade 0+
12 Grade 3 Grade 3+ Grade 2 Grade 1+ Grade 0 Grade 0+

Grade 3=severe edema, Grade 2= Moderate edema, Grade 1 = Mild edema and Grade 0= No edema.
Grade 3+ = severe opacity, Grade 2+ = Moderate opacity, Grade 1+=Mild opacity and Grade 0+ = No Opacity.
Plate no. 4.23 (A) Post-operative appearance viz: corneal edema, corneal
opacity and corneal healing in Dog no. 1.

Day 4, Ulcer with grade 1 edema, grade 1+ opacity, and

glistening dry amniotic membrane graft.

Day 8, Ulcer with grade 1 edema and grade 1+ opacity.

Day 12, Clear transperant cornea similar to normal eye with no edema.

H
Plate no. 4.23 (B) Post-operative appearance viz: corneal edema, corneal
opacity and corneal healing in Dog no. 3.

Day 4, Ulcer with grade 2 edema, grade 2+ opacity and pink

coloration of graft with vascularization beneath the ulcer.

Day 8, Ulcer with grade 1 edema, grade 1+ opacity and glistening

appearance of graft along with regressing blood vessels.

Day 12, Ulcer with no edema and transparent cornea with

single vasculature.

I
 The present observations are in agreement with Gurpreet Singh, (2017) who
used dry human amniotic membrane overlay graft in four eyes of dogs with
tarsorrhaphy and found clear corneal transparency on 11.25±2.50 postoperative days.
Ollivier et al., (2006) found that eyes receiving amniotic membrane grafts
possesses minimal degree of corneal scarring and regained a greater transparency of
cornea.
4.6.6.3 Corneal healing and acceptance of
graft in group- I
The visual appearance of corneal graft in dog no. 1 and dog no.2 revealed that,
grafts appeared to be granulating, glistening and devoid of hemorrhage on 4 th day of
observation. On 8th day, the amniotic membrane graft was absorbed to further extent
and its appearance was hazy. The grafted area on 12th day appeared blanched and was
at par with the remaining part of cornea.
In dog no.3, the grafted ulcer appeared pink with vascularization on 4th day.
On 8th day, the graft was absorbed to further extent and appeared glistening and also
the blood vessels started regressing. On 12th day the graft appeared normal like a
transparent cornea with only single regressing blood vessel.
In one dog no.4 and dog no.5 the grafted ulcer appeared granulating like a
proud flesh with irregular surface on 4th day of observation. On 8th day, in the same
dog, the depth of ulcer was found to be reduced and the extent of granulation was also
reduced and it appeared light pink with smooth surface. On 12th day of observation,
the area appeared further pale and showed continuation of its surface with the
remaining cornea. In dog no. 6, the grafted ulcer bed appeared shallow on 4th day of
observation.
The depth appeared further reduced on 8th day along with few areas of granulation.
On 12th day, grafted area appeared healed with hazy scar at the site.
The post-operative appearance of corneal healing in dogs from group I is
shown in (Plate no. 4.23 (A) and Plate no. 4.23(B)).

In group -II
In dog no. 7, 11 and 12, graft appeared to be cloudy without vascularization on
4th day of observation. The corneal surface was smooth with good luster and reduced

57
cloudiness on 8th day, while on 12th day the cornea and grafted area appeared smooth,
moist and transparent.
In dog no. 8, the grafted ulcer had a follicular appearance like a granulating tissue
with shallow ulcer bed on 4th day of observation. The extent of granulating tissue reduced
on 8th day of observation. On 12th day, the surface appeared smooth and continuous with
remaining cornea and resembled almost similar to normal cornea.
In dog no. 9, the grafted ulcer appeared pink with extensive neovascularization
on 4th day. On 8th day the graft was absorbed to further extent, appeared glistening and
fine parallel radiating blood vessels towards the ulcer site were observed. On 12th day
the graft appeared normal like a transparent cornea with no visible blood vessels.
In dog no.10, the grafted ulcer appeared pink with two blood vessels running
parallel from the adjacent limbus and divided into fine branches of vessels on the
graft on 4th day. On 8th day the graft was absorbed to further extent and appeared
glistening and also the blood vessels started regressing. On 12 th day the graft was
absorbed to a greater extent with complete regression of blood vessels and appeared
like normal transparent cornea.
The post-operative appearance of corneal edema in dogs from group-II, is
shown in (Plate no.4.24 (A) and Plate no. 4.24 (B)).

These findings could be attributed to the healing effects of the grafted human
amniotic membrane (Fernandes et al., 2005).

Above finding suggests that human amniotic membrane contains a number of

growth factors that favors epithelial healing within a short time period Koizumi et al.
(2000). Moreover, the basement membrane of human amniotic membrane facilitates
the migration of epithelial cells that helps in re-establishment of adhesion process
between newly generated epithelial cells and underlying basement membrane
(Khodadoust et al. 1968).
There was no significant difference between appearances of wet or dry grafts
in both the groups. Jie et al., (2018) studied use of intact AM (iAM) and denuded
AM (dAM) in ocular defects in rabbits and concluded that, both the grafts
facilitates

58
Plate no. 4.24 (A) Post-operative appearance viz: corneal edema, corneal
opacity and corneal healing in Dog no. 7.

Day 4, Ulcer with grade 2 edema, grade 2+ opacity

and hazy appearance of graft.

Day 8, Ulcer with grade 1 edema, grade 1+ opacity

and smooth corneal surface with luster.

Day 12, no edema with smooth moist and transperent cornea

similar to normal cornea.

J
Plate no. 4.24 (B) Post-operative appearance viz: corneal edema, corneal
opacity and corneal healing in Dog no. 12.

Day 4, Ulcer with grade 3 edema, grade 3+ opacity

and cloudy apperence of membrane graft.

Day 8, Ulcer with grade 2 edema and grade 1+ opacity.

Day 12, apperece of normal cornea with no edema .

K
epithelial regeneration and goblet cell differentiation. It also reduces the ongoing
inflammation and scar formation at defect sites. Dry amniotic membrane grafts were
found to be more suitable for preservation as these grafts are to be preserved at room
temperature. In contrast, wet amniotic membrane grafts are to be preserved in deep
freezer at – 700c. and this specific condition of preservation at -70 0c may not be
available at all places there by limiting the wide spread use of wet amniotic
membrane grafts.

4.6.7 Complication if any

In present study, the allograft rejection was not reported because of the fact
that the amniotic membrane consisted of avascular and non-immunogenic material.
This finding was supported by the study carried out by Tseng et al., (1999) who
found that amniotic membrane was devoid of any live cells and thus it does not
initiate any immune reaction.

59
Summary &
Conclusions
 CHAPTER V

SUMMARY AND CONCLUSION

In the present study, 12 dogs were randomly divided into two groups viz;
group I (1-6) and group II (7-12). The corneal ulcers of dogs from group I were
treated by using dry human amniotic membrane graft technique whereas, in group II,
wet human amniotic membrane graft technique was advocated. The mean age of the
dogs presented in all the dogs from both groups ranged from 4 months to 11.4 years
(4.12±3.70 years) showed corneal ulcers. The breed included Shih Tzu (33.33%)
followed by Doberman (16.66%), Rottweiler (16.66%) Nondescript (16.33%),
Labrador (8.33%) and Pitbull (8.33%) The incidence of corneal ulcers was higher in
males (n=9; 75%) than in females (n=3; 25%). The duration of ulcer observed was
11.08 ± 3.54 days. Irrespective of groups, 50% of dogs had their right eye affected
and 50 % of dogs had their left eye affected.

The physiological parameters of all the animals from both the groups were
within normal range, which included body temperature (100.50±0.67 0F), heart rate
(72.7±8.48 /min) and respiration rate (29.75±3.67/min).

Total 12 eyes with corneal ulcers were classified into five groups on the basis
of location viz; central (n=5) followed by ventro-nasal (n=3) ventro temporal (n=2)
dorso-nasal (n=1) and dorso-temporal (n=1). These dogs were also classified on the
basis of depth ulcer. Six dogs (50 %) showed stromal/deep stromal ulcers, four dogs
(33.33%) were having superficial ulcers and one dog each was having melting
corneal ulcer (8.33%) and descemetocele (8.33%).

Gross examination of all eyes did not show structural deformity in any case.
The ocular discharge was grossly noted on the day of presentation and recorded as
watery (n=4), mucoid (n=5) and mucopurulent (n=3). This discharge gradually
became normal up to 12th day following grafting and postoperative treatment.

Preoperative vision assessment revealed that, Nine dogs showed absence of

menace response and three dogs showed sluggish menace response.Dazzle reflex was

60
sluggish in the majority of dogs (n=11) and was normal in one dog and in all dogs
pupillary light reflexes could not be accessed.
Direct ophthalmoscopic examination of all the dogs revealed that ten dogs
were having severe edema (grade 3) and remaining two dogs had moderate edema
(grade 2). Eight dogs showed grade 3+ i.e. severe corneal opacity and remaining four
dogs were having grade 2+ i.e moderate corneal opacity along with ulceration.
All the dogs were subjected to general anesthesia and retrobulbar nerveblock
for the surgical procedure.The surgical procedure included debridment, formation of
ulcer bed, placement of amniotic membrane graft, securing of graft with vircyl 10-0
suture and tarsorrhaphy.
Topically 0.5% moxifloxacin, 1% atropine and 1% Carboxymethylcellulose
eyedrops and systemically, Amoxicillin sulbactum and Meloxicaim was used
postoperatively.
Post operatively, all the animals were evaluated for visual functions i.e.
Schirmer tear test, fluorescein dye test corneal healing, corneal oedema and corneal
opacity on 4th, 8th and 12th post-operative days.
Initially values of Schirmer tear test were higher which later got reduced post-
operatively. Also, there was progressive reduction of corneal oedema and
improvement in corneal clarity.
In all the dogs, sloughed squamous epithelial cells from different layers of
anterior epithelium of cornea and varying number of inflammatory cells mainly
neutrophils along with fibrin and keratin debris were found preoperatively, which
later got reduced to very few squamous epithelial and very few or none inflammatory
cells. The mean TLC and DLC values of dogs from both the groups were within
normal range and there was no statistically significant difference in these values
during pre-operative and on 12th post-operative day.
The dogs from group I and group II were kept under observation for 12 days
post operatively and following parameters were recorded.
Assessment of vision was carried out on 4th, 8th and 12th postoperative days
after corneal grafting. On 4th postoperative day five dogs from group I showed
absence of

61
menace response and one dog showed sluggish menace response. whereas from group
II, one dog showed absence of menace response and remaining five dogs showed
sluggish menace response. On 8th post-operative day from group I, two dogs showed
absence of menace response, three dogs showes sluggish menace response and one
dog had normal menace response. From group II, three dogs showed sluggish menace
response and three dogs showed normal menace response.On 12 th day from group I,
one dog showed sluggish menace response and five dogs showed normal menace
response. From group II, all dogs showed normal menace response.
The dazzle reflex test showed that on day 4 all dogs from both the groups had
sluggish dazzle reflex. On 8th post-operative day, two dogs from group I showed
sluggish dazzle reflex and four dogs showed normal dazzle reflex. From group II, one
dog had sluggish dazzle reflex whereas remaining five dogs showed normal dazzle
reflex .On 12th day all the dogs from both the groups showed normal dazzle reflex.
Pupillary light reflex test was performed. However the reflexes could not be
assessed in all dogs from both groups on 4th post-operative day. On 8th postoperative
day five dogs from group I showed sluggish PLR reflex and one dog showed normal
PLR reflex. From group II, four dogs showed sluggish PLR reflex and two dogs
showed
normal PLR reflex.
On 12th day from group I, one dog showed sluggish PLR reflex and five dogs
showed normal PLR reflex. Whereas all dogs from group II showed normal PLR
reflex. The dogs from both the groups showed fluctuating but non-significant
differences in values of STT. The mean vales of STT was 25.11 ± 4.76 mm/min , 23
±4.2 mm/min,
22 ±4.02 mm/min and 19.5 ± 1.9 mm/min on day 0, 4th ,8th and 12 day respectively
Fluorescein dye test was positive from both the groups on day 0 except in dog no. 8,
where in, due to descemetocele it was found negative. On 4th, 8th and 12th day of
observation in all the dogs, fluorescein dye test was found to be negative due to
epithelialization. Preoperative cytological examination suggested inflammatory
condition with predominant neutrophils whereas, on 12th postoperative day, very few
epithelial cells and no neutrophils were seen. The Mean of hematological parameters
of both the groups were within normal range
62
 The post-operative apperence of grafted ulcer in terms of corneal edema,
opacity and healing was studied on 4th, 8th and 12th post oprative day. Till 12th day, all
dogs from both the groups showed no signs of edema. Two dogs from group I
showed a hazy spot of opacity. Four dogs from this group showed no corneal opacity.
From group II, none of the dog had corneal opacity. All the dogs from both the
groups showed recovery on 12th postoperative day. The Progressive healing was also
observed on respective days. The ulcerated cornea appeared transparent in ten dogs
and only focal haziness observed in two cases from group I. Allograft rejection was
not reported, because of the fact that the amniotic membrane consisted of avascular
and non- immunogenic material.

Conclusions:

 Fluorescein dye test was confirmatory diagnostic test for identification and
classification of corneal ulcers.

 The dry human amniotic membrane grafts and wet human amniotic membrane
grafts possesses anti-angiogenetic effect, anti-scarring effect and anti-inflammatory
action which together accelerated the healing process of corneal ulcers there by
helped to achieve early healing of ulcer.

 The dry human amniotic membrane grafts were found to be easier for use, as
it can be preserved at room temperature and easily carried at different locations, in
contrast to wet human amniotic membrane grafts which needs special preservation
conditions of -700c.

63
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 i

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Vita
 xvi

VITA

Dr. Attar Tayyab Ismail was born on, 15th August, 1994 at Aurangabad,
Maharashtra. He completed his secondary school education in 2011 from
Mount Valley English School Harsul Savangi, Aurangabad and higher
secondary education from A.U.B.H.Sc & Jr. College, Pune, Maharashtra, in
2013.
He belongs to farmer’s family and has a special bond with the
speechless animals from childhood. That is why he chose the field of
Veterinary Science. After completion of his higher secondary education, he got
in K.N.P College of Veterinary Science Shirwal, Satara, Maharashtra where he
completed his undergraduate degree in year- 2018 with distinction in the first
class.
Apart from academics, he represented his university in the National
level Digital Financial Literacy Campaign Held by HRD, GOI in 2017. He has
also represented the university at state level in Social media Mahamitra
programme and State Republic Day Parade on 26th Jan 2018. He bagged
second prize in the College level quiz competition held by Virbac Animal
Health India Pvt. Ltd. in 2017.
He worked under the renowned small animal cardiologist, Dr.Sangeeta
Vengserkar Shah for a year after completing graduation. While working with
his senior colleague Dr. KamalKumar Jiddimani, a budding ophthalmologist
who was conducting research work on cataract surgeries in dogs. Dr.Tayyab
developed a keen interest in Veterinary Ophthalmology and is looking forward
to work in the same field.
He got selected as a livestock development officer Grade -1 in the State
Department of Animal Husbandry, government of Maharashtra. He aims to use
his knowledge for the well-being of society.
Abstract
 xvii

THESIS ABSTRACT

a) Title of the thesis (In capital Letters) : CLINICAL EVALUATION OF

AMNIOTIC MEMBRANE
GRAFT FOR MANAGEMENT
OF CORNEAL ULCER IN DOGS

b) Full name of student : Attar Tayyab Ismail

c) Name and address of Major Advisor : Dr. A. H Ulemale
Associate Professor, Department
of Vety. Surgery & Radiology,
KNPCVS, Shirwal-412801.

d) Degree to be awarded : M.V.Sc.

e) Year of award of degree : 2022

f) Major subject : Veterinary Surgery

and Radiology

g) Total number of pages in the thesis : 63

h) Number of words in the abstract : 297

i) Signature of Student :

j) Signature, Name and address of : Dr.V.D.Aher, Professor &

Forwarding authority (HOD/SH) Head Dept. of Veterinary
Surgery & Radiology,

ABSTRACT

The present study titled “clinical evaluation of amniotic membrane

graft for management of corneal ulcer in dogs” was conducted on twelve
cases of corneal ulcer in dogs. After detailed signalment, physical,
 xviii

ophthalmic, cytological and hematological examinations, these twelve dogs

which were diagnosed for corneal ulcer and were divided into two groups
based on the type of graft to be used. In Group I (n=6) dry, and in Group II
(n=6) wet human amniotic membrane graft was used.
All the dogs were placed in lateral recumbency. Under general
anesthesia, ulcer bed was prepared aseptically for acceptance of the grafts by
using cotton bud and 15 no. bp blade. The dry and wet human amniotic
membrane graft was then placed on prepared ulcer bed as per respective
groups and secured in place by suturing with 10-0 Vicryl. Tarsorraphy was
performed to promote healing and was removed on 4th postoperative day.
Topically 0.5% moxifloxacin, 1% atropine and 1%
Carboxymethylcellulose eyedrops and systemically, Amoxicillin sulbactum
and Meloxicaim was used postoperatively.
All the dogs were evaluated on, 4th, 8th and 12th postoperative day. In
dogs from both the groups vision improved gradually and showed normal
vision tests viz menace response, dazzle reflex and PLR on 12th postoperative
day.
Values of STT were higher in initial days of observation, which later
reduced to normal. Preoperative cytological findings suggested inflammatory
condition with predominant neutrophils whereas, on 12 th postoperative day,
very few epithelial cells and no neutrophils were seen.The Mean of
hematological parameters of both the groups was within normal range.
Progressive healing, reduction of corneal oedema and opacity was
observed on respective days. The ulcerated cornea appeared transparent in ten
dogs and only focal haziness was observed in two cases from group I. On 12th
postoperative day, edema was not observed in any case.
 xix

प्रबंध सारांश
"श्वानांमधील कॉर्न ष य ल अ ल् सर च् या
: उप चा रा सा ठी म्नी
ओर्ि
क मेम्ब्रेन
१ प्रबंधाचे र्ष
क ग्राफ्टचे नैदार्नक
मूल्ांकन"
२ र्िद्यार्थ्ाषचे : आत्तार तय्यब इस्माईल
पूर्ष नाि
डॉ. अ. र्ि. उलेमाले
सि यो र्ी प्राध्यापक,
प( शशल्र्चर्कत्सा आ र्र् क्ष र्करर् र्िभार्,
मार्षदशषकाचे नाि ि : क्रा.ना.पा.प( द् यकीय
द्यकीयशुिै
३ पत्ता
मिार्िद्यालय र्शर्रिळ , र्ि.सातारा.

४ पदिीचे नाि : एम.व्ही.एस्सी

५ पदिी घेण्याचे िर्ष: : २०२२
प( शशल्र्चर्कत्सा आर्र् आर्र् क्ष
६ मुख्य र्िर्य: : र्करर् स् त्र
स्त्रर्शा

प्रबंध मधील एकू र्

७ पृष्ांची : ६३
संख्या
मधी
प्रबंध सरां मधील लशा
८ एकू र् शब्द : २७५

९ र्िद्यार्थ्ाषची स्वाक्षरी :
१० पुढे पाठर्िर्ार् या
अर्धकार् याची स्वाक्षरी,
नाि ि पत्ता
डॉ. व्ही.डी.आिेर
: प्राध्यापक ि र्िभार्
प्रमुख
प( शशल्र्चर्कत्सा आ र्र् क्ष र्करर् र्िभार्,
क्रा.ना.पा.प( द् यकीय
द्यकीयशुिै
मिार्िद्यालय र्शर्रिळ , र्ि.सातारा.
 xx

पपपपपप पपपपपप

श्वानांमधील कॉर्नषयल अल्सरच्या उपचारासाठी ॲम्नीओर्िक मेम्ब्रेन ग्राफ्टचे नैदार्नक

मूल्ांकन" या ज्या श्वा वानांना कॉर्नषयल अल्सर आिेत अ शा बारा श्वानांमध्ये
शीर्षकाचा अभ्यास आयोर्ित करण्यात आला िोता. शारीररक, नेत्रर्िकार,
सायिॉलॉर्िकल आर्र् रक्तनमुने इत् या दी तपासण्या के ल्ा नंतर, या बारा
श्वानांमधे कॉर्नषयल अल्सरचे र्नदान करण्यात आले आर्र् त्यांना
िापरल्ा िार्ार् या ग्राफ्ट प्रकारानुसार आधाररत दोन र्िांमध्ये
र्िभार्ले र्ेले
िोते. र्ि १ मध्ये (n=६) कोरडे आर्र् र्ि २ (n=६ ) मध्ये ओले
मानिी म्नी ओर्िक मेम्ब्रेन ग्राफ्ट िापरले र्ेले.
सिष श्वानांना बािूच्या कडािर झोपिण्यात आले िो ते. श्वानांना
पुर्षता: भूल देऊन, कापसाची कळी आर्र् बी. पी. ब्लेडचा िापर करुन ग्राफ्ट
करण्यासाठी ग्राफ्ट बेड तयार करण्यात आला. कोरडे आर्र् ओले
मानिी ॲम्नीओर्िक ग्राफ्ट संबंर्धत र्िांनुसार तयार के लेल्ा अल्सर
बेडिर ठे िण्यात आले आर्र् 10-0 व्हायक्रीलने र्शिुर् ( नत्या िार्ी
सुरर्क्षत के ले र्ेले. बरे
िोण्यास प्रोत्सािन देण्यासाठी िारसोरॅ फी के ली र्ेली आर्र् ती
चौर्थ्ा ऑपरे शन नंतर चौर्थ्ा र्दि शकाढण्यात आली.
िॉर्पकली ०.५ % मोक्सीफ्लॉक्सार्सन, १ % ऍिर ोर्पन आर्र् १ %
काबोक्झझमेर्िलसेल्ुलोि आयडर ॉप्स आर्र् अमोक्क्सर्सर्लन सलबॅक्टम
आर्र् मेलॉक्क्सकॅ मचा शस्त्रर्क्रयेनंतर र्शर्रेतुन देण्यात आले.
शस्त्रर्क्रयेनंतर चिर्थ्ा, आठव्या आर्र् बाराव्या र्दि शसिष श्वानांचे मूल्मापन
करण्यात आले. दोन्ही र्िातील श्वानांमध्ये दृष्टी िळू िळू सुधारली आर्र् १२ व्या
शस्त्रर्क्रयापश्चात र्दि शसामान्य दृष्टी सा ठी च् या चाचण्या िसे र्मनेस ररस्पॉन्स, डॅ झ ल
ररफ्लेक्स आर्र् पी. एल. आर. सकारात्मक आढळु न आल्ा.
र्नरीक्षर्ाच्या सुरुिातीच्या र्दिसांमध्ये एस िी िी ची मूल्े
िास्त िोती,
िी नंतर सामान्य झाली. शस्त्रर्क्रयापूिष सायिोलॉर्िकल र्नष्कर्ाषमध्ये,
मुख्यत : न्यूिर ोर्फल्स पे शसि दािक क्थिती, तर 12 व्या शस्त्रर्क्रयापश्चात
र्दि श , फारच कमी एर् पिेर्ल
यल पे आर्र्न् यि
र ोर्फल्स पे शर्दसलया. दोन्ही
र्िांच्या िेमॅिोलॉर्िकल पॅरामीिसषचे सरासरी प्रमार् सामान्य
श्रेर्ीमध्ये िो ते. उपचारानंतर कॉर्नषयल एडेमा कमी िोर्े आर्र् पारदशषकता
पुिषपदािर आली. दिा श्वानांमध्ये कॉर्नषया पारदशषक र्दसला मात्र
पिील्ा र्िातील दोन
 xxi

श्वानांमध्ये कॉनीयाच्या अत्यंत लिान भार्ािर अपारदशषकता र्दसली. 12

व्या शस्त्रर्क्रयापश्चात र्दिशी, एकािी शिा नांमध्येसूि र्दसून आली नािी.