3.9.2021.

RAT AS A WOUND HEALING MODEL

Wounding and wound healing takes place in all tissues and organs of the body. Many of these
repair processes are common to all tissues. Essentially it is a process through which all damaged
tissues attempt to establish structural integrity and normal functioning. Although the process of
wound healing is continuous it is arbitrarily divided into these four overlapping phases: hemostasis,
inflammatory phase, proliferative phase, and maturation. In the first phase, the main process is
coagulation which aims to protect the vasculer system, keeping it intact, so that the function of the
vital organs remains unharmed despite the injury. The humoral and cellular inflammatory phase
follows next, with the aim of establishing an immune barrier against invading micro-organisms. It is
divided into two separate phases, an early inflammatory phase and a late inflammatory phase. When
ongoing injury has ceased, haemostasis has been achived and an immune response successfully set in
place, the acute wound shifts toward tissue repair. The proliferative phase starts on the third day after
wounding and lasts for about 2 weeks thereafter. It is characterized by fibroblast migration and
deposition of newly synthesized extracellular matrix, acting as a replacement for the provisional
network composed of fibrin and fibronectin. Macroscopically can be seeb as an abundant formation of
granulation tissue. As the final phase of wound healing the remodeling phase is responsible for the
development of new epithelium and final scar tissue formation. Animal testing, also known as animal
experimentation, animal research and in vivo testing, is the use of non-human animals in experiments
that seek to control the variables that affect the behavior or biological system under study. Animal
models are important biological tools which have been developed to study the complex cellular
and biochemical processes of wound repair and to evaluate the efficacy and safety of potential
therapeutic agents. There are several factors that can influence the procces and the result of healing.
These include aging, infection, medications, nutrition, venous insufficiency, peripheral arterial
disease, obesity and diabetes. Variety of animal species are used in the study of wound healing such
as mouse, rat, rabbit, pig, and zebrafish. Many factors should be considered when choosing an animal
species for wound experiments. These include cost, availability, ease of handling, investigator
familiarity, and similarity to humans. The use of small animals has a cost advantage. There are,
however, several limitations, including limits to wound size, skin thinness relative to humans,
follicular pattern, and hair growth cycle that differs from humans. Wounds in areas with higher hair
density heal faster than those in less hairy or nonhairy areas. Rats similar to mice have independent
movement of the skin compared to deeper muscles which is called „loose skin“ and therefore heal
pedominantly by contraction. Healing by contraction is more rapid than re-epithelialization because
new tissue is not formed. Unlike humans, mice and rats do not create hypertrophic scars or keloids.
The collagen produced in their wounds come from subcutaneous panniculus carnosus musclees that is
largely absent in humans.
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Rats are often selected as a wound healing model due to its many advantages:

 Small size
 Common
 Cost effecient
 Easy to handle and maintain
 They are bigger than mice, which allows far larger or more numerous wounds per animal
 Broad knowledge base on rat wound healing from years of extensive research.

On the other side, there are also limitiations of using a rat testing model such as:

 Their loose skin and high hair density do not reflect the architecture of human skin
 They heal primarily via contraction, thus minimizing the relevance of re-epithelization
and granulation unless splinting technique is used
 Use of splinting to avoid contraction introduces foreign material to the wound site
 Less genetically tractable than mice
 Relative paucity of species-specific reagents compared with mice.

Lately, a lot of attention is focused on the rat duodenocutanenous fistula research which might
alter the duodenal ulcer disease background and therapy . The fistula is a direct connection between
two normally separated tissues which , in this case, changes the circumstances of healing in order to
create new difficulties which the regular healing mechanism wouldn't be able to deal with.
Duodenocutaneous fistula is an analogous healing model which provides insight into two different
wound healing processes, one at skin and the other at duodenum, and shows that they also contribute
each other. The main characters in this research is the NO-system and a gastric pentadecapeptide BPC
157 which ,unlike other peptides, is always given alone, and therby may act directly, always using the
same dosage range. This is different from the commonly used standard growth factors(i.e., EGF)
whicg are given with carriers and require special delivery system for skin healing (but not for
gastrointestinal ulcer healing).. BPC 157 is a safe (no lethal doses were recorded) and stable (stable in
gastric juices for a period of time of over 24 hours). This anti-ulcer peptide has positive effect in the
healing of lesions and it has been successful in inflammatory bowel disease trials,
counteracting esophagitis, sphincter failure, gastrointestinal and skin ulcers, gastrocutaneous,
duodenocutaneous or colocutaneous fistulas. The main goal of this research was to examine a role od
the NO-system in the wound healing process of duodenocutaneous fistula and to determine the effect
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of BPC 157 when the NO-system is stimulated with NOS-substrate L-arginine or NOS-inhibitor L-
nitro-arginine methyl ester (L-NAME). The experiment was carried out on Wistar Albino rats with
body mass around 200 grams. Duodenocutaneous fistula-rats were treated with BPC 157 (10 µg/kg
or 10 ng/kg, intraperitoneally or perorally (in drinking water)), L-NAME (5 mg/kg intraperitoneally),
L- arginine (100 mg/kg intraperitoneally) alone and/or together, throughout 21 days. The duodenal
mucosal defect, skin defect, volume of water intragastrically, pressure in lower esophageal sphincter
and pyloric sphincter an mortality rate was evaluated. The results showed consistent counteracting
beneficial effect in all BPC 157 rats (i.e., given perorally (in drinking water) or intraperitoneally), on
its own, and with NOS-blockade (L-NAME) and/or NOS-substrate (L-arginine). The simultaneous
effect lead to succesful closure of skin, duodenum and eventually fistula. The healing occurred macro-
and microscopically, biomechanically, and functionally (larger volume of water is tolerable before the
fistuala starts to leak). The control groups, which simultaneously received an equivolume of saline
(5.0 mL/kg intraperitoneally) or water only, maintained persistent defects, continuous fistula leakage,
sphincter failure, mortality rate at 40% until the 4th day. L-NAME further worsened the
duodenocutaneous fistula-course (mortality at 70% until the 4th day) ; L-arginine had beneficial
effect (no mortality, but maximal volume instilled not before 21th day) only if it was given alone,
without L-NAME. L-NAME- worsening was counteracted to the control level with the L-arginine
effect, and vice versa, while BPC 157 annulled the L-NAME effects. The combination of the
pentadecapeptide BPC 157 and L-arginine strengthened the otherwise mild positive effect of L-
arginine. Microscopically, in the testing group with BPC 157, the edema was less pronounced, the
superficial epithelium surrounding the defect was practically intact, and the defect was closed by
crust, and after 14 days, unlike in the controls, the treated animals showed poor granulation tissue
formation and prominent regeneration of subcutaneous muscle fibers. The BPC 157-rats sustained the
advanced volume challenge before leakage, quickly progressing until the successful instillation of the
maximal volume, which was achieved already at very early intervals

BPC 157 promotes expression of early growth factors and also has a strong direct angiogenic healing
potential and provides the direct endothelium protection which partly explains its influence on the
NO-system, with a counteracted overexpression of endothelin, counteraction of the effects of NOS-
inhibitors and NO-precursor. All mentioned positive effects of the BPC 157 were achieved regardless
of the dose (micrograms or nanograms) and administration modality (intraperitonal or peroral
administration). Iti s suggested that BPC 157 could be a potential alternative in therapy of persisting
skin wounds and duodenal ulcuses. Additionally, BPC 157 was focus of many other researches and
there are indications that it could accelerate the the healing of severe burns, used topically as a cream,
or systemically and that it is an integrative mediator that integrates the adaptive bodily response to
stress (realization of Selye's stress response).
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Although animal models provide invaluable informations that can be correlated with human
wound healing, when it comes to interpretation and implementation, one must not fail to recognize
differences in each animal model. There may never be a perfect animal healing model because taking
into consideration complexity and heterogeneity of chronic wounds in humans it is impossible for
now to recreate complete wound healing model. For now we have to adopt and choose the most
sutable model, which is and probably will be for a long time a rat. The availability of animals with
well-defined health and genetic backgrounds along side with a bounty of literature documenting
biological responses and parameters for rats, allows the rat to serve as a valuable research tool in the
search for faster, stronger, and more anatomically correct wound healing. In my opinion, animal
testing and animal models are foundation for any medical and pharmaceutical progress. as long as
Three Rs are applied. I look upon animal models as necessary „evil“, in other to advance and save
more human lives.

References:

 T Velnar, T Bailey AND V Smrkolj

The Wound Healing Process: an Overview of the Cellular and Molecular Mechanisms

 Sikiric,Predrag; Seiwerth, Sven; Rucman, Rudolf; Drmic, Domagoj; Stupnisek, Mirjana;

Kokot, Antonio; Sever, Marko; Zoricic, Ivan; Zoricic, Zoran; Batelja, Lovorka et al.

Stress in Gastrointestinal Tract and Stable Gastric Pentadecapeptide BPC 157. Finally, do
we have a Solution? // Current Pharmaceutical Design, 23 (2017)

 S. Skorjanec , A. Kokot , D. Drmic , B. Radic , M. Sever , R. Klicek , D. Kolenc , A. Zenko ,

M. Lovric Bencic , Z. Belosic Halle , A. Situm , G. Zivanovic Posilovic , S. Masnec , J. Suran
, G. Aralica , S. Seiwerth , P. Sikiric
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Duodenocutaneous Fistula In Rats As A Model For “Wound Healing-Therapy” In Ulcer

Healing: The Effect Of Pentadecapeptide Bpc 157, L-Nitro-Arginine Methyl Ester And L-
Arginine
 Ayman Grada, Joshua Mervis, Vincent Falanga
Research Techniqu3.9.2021.es Made Simple: Animal Models of Wound Healing
 Šitum, Andrej (2015)
Učinak pentadekapeptida BPC 157 na cijeljenje rane u uvjetima inducirane ishemije
stražnjih nogu štakora [Pentadecapeptide BPC 157 and wound healing after hind limb
ischemia], PhD thesis, Sveučilište u Zagrebu
 Vedran Cesarec, Tomislav Becejac, Marija Misic, Zeljko Djakovic, Danijela Olujic, Domagoj
Drmic, Luka Brcic, Dinko Stancic Rokotov, Sven Seiwerth, Predrag Sikiric
Pentadecapeptide BPC 157 and the esophagocutaneous fistula healing therapy
 Dorsett-Martin W.A., Wysocki A.B. (2008)
Rat Models of Skin Wound Healing. In: Conn P.M. (eds) Sourcebook of Models for
Biomedical Research. Humana Press
 Škorjanec, Sandra
Uloga NO-sustava i pentadekapeptida BPC 157 u cijeljenju duodenokutane fistule u štakora,
2011., doktorska disertacija, Prirodoslovno-matematički, Zagreb