The Laryngoscope
Lippincott Williams & Wilkins, Inc.
© 2006 The American Laryngological,
Rhinological and Otological Society, Inc.
Vocal Fold Healing after Laser Cordectomy
with Adjuvant Cryotherapy
P. Daniel Knott, MD; Michael C. Byrd, MD; David G. Hicks, MD; Marshall Strome, MD, MS
Objectives: To understand the effect of adjuvant
cryotherapy on glottic wound healing after endo-
scopic CO2 laser cordectomy. Study Design: Canine
acute injury model with videolaryngostroboscopic
and histopathologic outcomes analysis. Methods:
Twelve adult male dogs underwent bilateral endo-
scopic CO2 laser transmuscular cordectomy followed
by randomized unilateral endoscopic glottic cryo-
therapy. The animals were randomly divided into
four groups and underwent videolaryngostroboscopy
followed by sacrifice at 0, 2, 6, and 12 weeks postop-
eratively. Three untreated male dogs served as con-
trols. Histopathologic sections were prepared with
Alcian blue, Giemsa, hematoxylin-eosin, movat’s, Mas-
son’s trichrome, and picrosirius stains. Results: Vid-
eostroboscopy demonstrated an earlier restoration of
glottic volume and a return of mucosal waves among
vocal folds treated with combined therapy by 6 weeks
posttreatment. The mean depth of inflammatory re-
action in the vocal cords treated with combined ther-
apy was 1.07 mm versus 1.15 mm in vocal cords
treated with CO2 laser therapy alone. At 2 and 6
weeks postoperatively, combined treatment was asso-
ciated with a decreased volume of collagen. At 12
weeks postoperatively, combined treatment was asso-
ciated with greater collagen organization, normal-
ized collagen histoarchitecture, and decreased kera-
tinization. Conclusions: Adjuvant cryotherapy appears
to alter glottis-specific wound healing, leading to de-
creased and more organized collagen formation and
decreased keratinization with a resultant improve-
ment in glottic function, when compared with CO2
laser surgery alone, in an acute canine injury model.
Studies in humans are ongoing to further evaluate
the clinical potential of cryotherapy on glottic wound
healing. Key Words: Endoscopic laser surgery, cordec-
From the Head and Neck Institute (P.D.K., M.C.B., M.S.), Cleveland
Clinic Foundation, Cleveland, Ohio, U.S.A., and the Department of Pathol-
ogy (D.G.H.), Roswell Park Cancer Institute, Buffalo, New York, U.S.A.
This paper was presented at the Middle Section of the Triological
Society Meeting. San Diego, California, U.S.A., February 3–5, 2006.
Editor’s Note: This Manuscript was accepted for publication May 25,
2006.
Send correspondence to Dr. Marshall Strome, Chairman, Head and
Neck Institute, A-71, Cleveland Clinic Foundation, 9500 Euclid Avenue,
Cleveland, OH 44195. E-mail: stromem@ccf.org.
DOI: 10.1097/01.mlg.0000231738.80952.7c
Laryngoscope 116: September 2006
1580
tomy, voice quality, cryosurgery, videostroboscopy.
Laryngoscope, 116:1580 –1584, 2006
INTRODUCTION
Recent improvements in endoscopic technology have
improved local control of early and advanced glottic ma-
lignancies. Nevertheless, speech outcomes after endoscopic
laser therapy for glottic carcinoma appear equal to or
marginally worse than the speech quality afforded pa-
tients undergoing primary external beam radiotherapy.1,2
These suboptimal vocal outcomes are likely related to the
mechanism of tissue injury. Laser surgery usually results
in some degree of thermal damage to tissue adjacent to the
ablation crater, resulting in scarring and tethering of the
epithelium, which inhibits optimal posttreatment phona-
tion.3,4 Preservation or restoration of the unique multilay-
ered structure of the true vocal folds after endoscopic
therapy is therefore important for high-quality posttreat-
ment voice production.
Cryotherapy is a well-studied technology, which has
been widely applied as an effective minimally invasive
technique for selective tumor ablation.5 This therapy has
been shown to modify wound healing at a cellular level,
reducing collagen deposition and altering the function of
fibroblasts.6,7 The senior author began to use adjuvant
cryotherapy for potential additional margin control among
patients undergoing endoscopic laser surgery for laryn-
geal cancer. Anecdotally, patients receiving adjuvant cryo-
therapy appeared to enjoy higher quality postoperative
voices compared with patients undergoing laser surgery
alone, with videostroboscopy demonstrating the restora-
tion of a vibratory phonatory surface in a limited series of
patients undergoing combined treatment.8 To character-
ize the potential effect of cryotherapy on laryngeal wound
healing, a canine study was proposed.
MATERIALS AND METHODS
All procedures and actions were approved and monitored by
the institutional animal care and use committee. Twelve male
mongrel dogs underwent suspension microlaryngoscopy with bi-
lateral transmuscular cordectomy, with one vocal fold, chosen
randomly, receiving additional treatment with cryotherapy. The
animals were divided into four groups according to time of sacri-
fice after surgery: immediately, 2 weeks, 6 weeks, or 12 weeks.
The animals all underwent laryngeal videostroboscopy (LVS) for
functional evaluation just before sacrifice. An additional 3 male
Knott et al.: Glottic Wound Healing with Cryotherapy
mongrel dogs that did not undergo cordectomy served as controls. which experienced delayed healing because of granulation
The larynges were harvested for histologic examination after tissue.
painless sacrifice of the animal.
For each procedure, the animal was placed supine on an Alcian Blue Stain
operating table and anesthetized with intramuscular atropine
These sections demonstrated greater hyaluronic acid
(0.04 mg/kg) and intravenous thiopental (20 mg/kg) and allowed
to breathe spontaneously. Each animal was given preoperative
at the base of the wounds in the animals treated with
and postoperative cephalexin (25 mg/kg). Postoperative pain was adjuvant cryotherapy at all of the time points (Fig. 1).
treated with intramuscular buprenex (0.01–.03 mg/kg).
Suspension microlaryngoscopy was performed with a Dedo Trichrome Stain
laryngoscope (Teleflex Medical, Research Triangle Park, NC) and Sections treated with adjuvant cryotherapy demon-
with a rigid telescope (Karl Storz, Tuttlingen, Germany) posi- strated a smaller volume of collagen in the 6 and 12 week
tioned above the true vocal cords (TVCs), permitting complete time points and more loosely arranged collagen at the 12
inspection of the larynx. A 14 gauge needle, passed through the week time point (Fig. 2).
trachea at approximately the third tracheal interspace, permitted
transglottic air ventilation with the application of gentle external
Picrosirius-Polarization Stain
manual compression of the larynx to cause vocal fold vibration.
LVS was performed with the Nagashima (Nagashima, Tokyo, In the control vocal cords, collagen type I was the
Japan) system to verify the normal vibratory dynamics of the predominant collagen subtype in two layers of the lamina
unoperated vocal cords. propria, one immediately below the epithelium and an-
With the animal still breathing spontaneously, bilateral other superficial to the vocal muscle. Collagen type III was
transmuscular cordectomies were performed with an operating the predominant collagen subtype in the intermediate
microscope (Leica Microsystems, Wetzlar, Germany) equipped
with a Sharplan 1055 S CO2 laser (Sharplan, Tel Aviv, Israel)
with superpulse delivery in continuous mode (5 W) coupled with
an Acuspot 712 micromanipulator (271 ␮m spot size, Lumenus,
Santa Clara, CA). Approximately 50% of the medial-most surface
of both TVCs were resected. The animals received randomized
cryosurgical treatment to one of the remnant TVCs. The cryosur-
gical probe (Frigitronics, CooperSurgical, Trumbull, CT) was
passed transorally under microscopic visualization. The unit was
activated until the tissue in a 3 mm radius around the probe tip
was visibly frozen and the tip temperature reached a nadir of
⫺40°C. Overlapping treatments were performed along the entire
length of the cordal remnant.
Just before sacrifice, each animal underwent repeat LVS. A
tracheotomy was performed between the sixth or seventh tra-
cheal rings. One endotracheal tube was positioned above the
carina for ventilation, and another tube was directed superiorly,
with its tip located approximately 1 cm below the TVCs. Humid-
ified air was then directed through this tube, and gentle external
compression of the larynx was then applied until a standing wave
form was obtained and recorded. The animals were then killed
with beuthanasia (1 mL/5 kg), and the vocal cords were excised,
divided in half in the coronal plane, with one half being fixed in
formalin and the other half being frozen. Five micron thick sec-
tions of the vocal cords were prepared and stored.

Staining Method
Alcian blue staining was used to detect hyaluronic acid,
Masson’s trichrome staining was used to detect collagen,
hematoxylin-eosin (H&E) staining was used to detect general
morphology, Giemsa staining was used to detect inflammatory
cell infiltrate, movat’s staining was used to detect elastin fibers
and connective tissue, and picrosirius-polarization staining was
used to visualize collagen type 1 and type 3.9,10

RESULTS
H&E Stain
H&E stain demonstrated that the mean depth of the
inflammatory reaction in the TVCs treated with adjuvant
cryotherapy was 1.07 mm versus 1.15 mm in the TVCs
treated with CO2 laser therapy alone. The epithelium was
healed by 6 weeks, except in one animal in each group,
Fig. 1. Alcian blue (hyaluronic acid) staining (magnification, ⫻5) of
representative vocal folds treated with CO2 laser alone and CO2
laser with adjuvant cryotherapy as well as control tissue. Hyaluronic
acid deposition within lamina propria occurs earlier and more abun-
dantly among animals treated with adjuvant cryotherapy.

Laryngoscope 116: September 2006 Knott et al.: Glottic Wound Healing with Cryotherapy
1581
Fig. 2. Masson’s trichrome (collagen) staining (magnification, ⫻5) of
representative vocal folds treated with CO2 laser alone and CO2
laser with adjuvant cryotherapy and controls. Notice decreased
density of collagen fibers in deep and superficial layers of lamina
propria in samples treated with adjuvant cryotherapy.
layer of the lamina propria. Among the 2 week animals,
collagen type I was absent. Among the 6 week animals
treated with adjuvant cryotherapy, there was a relative
abundance of type I collagen in the deeper layers and an
abundance of collagen type III in the superficial layer.
Among the 12 week animals treated with cryotherapy,
there was a relative reconstitution of the multilayered
collagen distribution, resembling that of the control tis-
sue. Furthermore, on high-power polarization microscopy,
the collagen fiber architecture was more organized and
less dense than the fibers among the sections representing
laser treatment alone (Fig. 3). Among the animals treated
with laser therapy alone, at the 6 and 12 week time points,
there was the development of thick, relatively disorganized,
predominantly type I collagen, with no reconstitution of dis-
tinct layering of different collagen subtypes (Fig. 3).
Movat’s Stain
These sections demonstrated an absence of elastin fi-
bers at the acute and 2 week intervals. There was a repopu-
Laryngoscope 116: September 2006
1582
Fig. 3. High- and low-power views (magnification, ⫻5 and ⫻20) of
picrosirius-polarization (collagen) staining of representative vocal
folds treated with CO2 laser alone and CO2 laser with adjuvant
cryotherapy and controls. Type I collagen fibers appear yellow or
red and are strongly birefringent, whereas collagen type III appears
as green, weakly birefringent fibers. Type I collagen fibers are more
abundant and less organized among sections treated with laser
alone. Specimens treated with adjuvant cryotherapy demonstrate
greater collagen organization in both high- and low-power views
with greater parallelization of fibrils.
lation of elastin fibers in the 6 week animals, which became
more uniform among the 12 week animals in both groups.
All three animals treated with laser alone experienced hy-
perkeratinization in the healed epithelium, whereas none of
the animals treated with adjuvant cryotherapy experienced
keratinization (Fig. 4).
Giemsa Stain
The Giemsa stain demonstrated relatively large pop-
ulations of mast cells and eosinophils in the normal sub-
jects. There were large numbers of inflammatory cells
(primarily macrophages and neutrophils) in the 2 week
animals, which were dramatically reduced in the 6 and 12
week animals. There appeared to be fewer inflammatory
cells among the sections treated with adjuvant cryother-
Knott et al.: Glottic Wound Healing with Cryotherapy

apy than among those treated with laser alone at the 2
week time point.
Laryngovideostroboscopy
The control animals as well as all experimental ani-
mals demonstrated normal vocal fold pliability and muco-
sal wave propagation before the performance of cordec-
tomy. Thereafter, the acute and the 2 week animals did
not demonstrate any return of a vibratory phonatory sur-
face. The 6 week animals demonstrated re-epithelialization
of the remnant vocal cords and the return of a mucosal
wave in one TVC treated with adjuvant cryotherapy.
Among the 12 week animals, there was a return of a
mucosal wave in all of the TVCs treated with adjuvant
cryotherapy, whereas there was a wave noted in two of the
three folds treated with laser alone. The waves also ap-
peared to have greater amplitude among the animals
treated with adjuvant cryotherapy.
DISCUSSION
The advancement of minimally invasive surgery and
the development of novel cryosurgical probes that deliver
very cold temperatures (⬍⫺100°C) to very finite volumes
of tissue has facilitated the application of cryosurgery to
multiple fields such as general surgery, orthopedic sur-
gery, urology, and gynecology for the treatment of solid
tumors. Cryotherapy induces local cytonecrosis in a two-
step process. During the freeze cycle, there is a rapid
formation of extracellular matrix and intracellular ice
crystals, leading to membrane disruption, the accumula-
tion of toxic electrolyte concentrations, and cellular dis-
Laryngoscope 116: September 2006
Fig. 4. Movat’s staining of representative vocal
folds treated with CO2 laser with adjuvant cryo-
therapy (A) and CO2 laser alone (B and C).
Elastin staining demonstrates return of elastin
fibers at 6 weeks, with no differences noted
between groups. Notice hyperkeratinization
present in 12 week sections treated with laser
alone, which is absent in section treated with
adjuvant cryotherapy.
ruption. This is followed by the thaw cycle, when cryother-
apy causes vasoconstriction, endothelial damage, vascular
thrombosis, and ischemic tissue loss by way of local mi-
crocirculatory failure.5 Basic scientific and clinical re-
search has shown that cryotherapy increases the prolifer-
ation of fibroblasts in vivo and in vitro. Shepherd and
Dawber11 showed an absence of damage to collagen after
repeated freeze-thaw cycles as well as a significant thin-
ning of the epithelium 6 months after cryotherapy. Sizov
et al.12 demonstrated that burn wounds treated with cryo-
therapy tended to normalize the collagen structure when
compared with untreated wounds, and cryotherapy in-
duced the production of collagen type III. Har-Shai et al.7
showed that keloids and hypertrophic scars treated with
cryotherapy were significantly associated with rejuvena-
tion of the treated areas (parallelization of the collagen),
along with the development of a more organized collagen
architecture, when compared with untreated wounds.
Although relatively little is known regarding the
modern effectiveness of cryotherapy in the larynx, the
mechanisms of wound healing in the canine and human
larynx in response to cold steel or laser trauma have
undergone extensive investigation. Vocal fold stripping is
associated with decreased elastin, increased collagen dep-
osition in thick, disorganized bundles, with resulting in-
creased stiffness, and resistance to shear flow during os-
cillatory shear deformation.13,14 The increased stiffness
and viscosity do not appear related to the increase in
collagen volume but are rather related to the disorganized
collagen histoarchitecture. Larynges undergoing laser
treatment demonstrate abundant collagen disorganiza-
Knott et al.: Glottic Wound Healing with Cryotherapy
1583
tion and decreased mucosal pliability when compared
with TVCs treated with cold steel.15
The use of adjuvant cryotherapy in treating laser
ablated wounds of the larynx, therefore, may offer distinct
advantages in terms of wound healing and ultimately
posttreatment vocal quality. Although the precise etiology
of the modulation of collagen deposition and remodeling
remains to be elucidated, it appears that cryotherapy
leads to the recruitment of specialized fibroblasts that
restore a more organized and normal collagen architecture
to the treated vocal fold. The more normalized collagen
architecture is clearly visualized in the picrosirius-
polarization stains (Fig. 3). This stain has significantly
improved the understanding of collagen architecture and
subtype and provides many clues to the particular func-
tional activity of collagen.
The use of adjuvant cryotherapy is associated with a
decreased inflammatory infiltrate at 2 weeks, followed by
a more organized and less dense final collagen histoarchi-
tecture. The control picrosirius slides demonstrate the
finely organized “basket-weave” pattern of collagen I and
collagen III fibers. The laser treated sections demonstrate
thick, disorganized collagen fibers. The specimens having
undergone treatment with cryotherapy, however, demon-
strate greater organization, both within distinctive layers
in the low-power views and greater parallelization within
the high-power views. Furthermore, the use of cryother-
apy appears to induce the deposition of a greater volume of
hyaluronic acid both acutely and at 12 weeks.
The improvement in vocal physiology afforded by this
improvement in collagen architecture and organization is
evident in the videostroboscopic images. Although there
was no appreciable vocal fold vibration among the vocal
folds treated with laser alone or laser with adjuvant cryo-
therapy at 2 weeks, one of the vocal folds treated with
adjuvant cryotherapy had phonatory wave formation at 6
weeks, and all of the vocal folds treated with cryotherapy
at 12 weeks demonstrated phonatory oscillation, whereas
two of the folds treated with laser alone demonstrated
such movement, with waves with smaller amplitude of
oscillation. It is therefore likely that the normalized flex-
ible collagen framework stabilized the lamina propria,
thereby facilitating a return of mucosal oscillatory poten-
tial. Furthermore, the adjuvant treatment did not appear
to alter the rate of elastin formation or the density of
elastin, which appeared equal in the 12 week samples.
Adjuvant cryotherapy also appeared associated with a
relative decrease in keratinization when compared with
the TVCs treated with laser alone. It is uncertain whether
this effect would have persisted beyond the 12 week time
point, but it does indicate further normalization of the
process of wound healing in the tissue treated with adju-
vant cryotherapy.
Although this animal model studied various markers of
wound healing including posttreatment LVS, true functional
improvements in vocal quality are difficult to quantify in
animal models. Rheologic investigations would likely yield
important information on the vibratory dynamics of tissue
Laryngoscope 116: September 2006
1584
treated with cryotherapy. Further investigations are there-
fore warranted before wider clinical use.
CONCLUSIONS
CO2 laser cordectomy with adjuvant cryotherapy ap-
pears to alter glottis-specific wound healing, leading to de-
creased and more organized collagen formation and de-
creased keratinization, with a resultant improvement in
glottic function as demonstrated by laryngovideostrobos-
copy, when compared with CO2 laser surgery alone, in an
acute canine injury model. These results are consistent with
previous investigations of applied cryotherapy in other organ
systems. The potential for improvement in posttreatment
voice production warrants further investigation.
Acknowledgments
The authors acknowledge the superb assistance pro-
vided by Shannon M. Tarr and Claudio Milstein, PhD, in
project organization and data analysis. Financial support
was provided by the Research Programs Council of the
Cleveland Clinic Foundation.
BIBLIOGRAPHY
1. McGuirt WF, Blalock D, Koufman JA, et al. Comparative
voice results after laser resection or irradiation of T1 vocal
cord carcinoma. Arch Otolaryngol Head Neck Surg 1994;
951–955.
2. Rydell R, Schalen L, Fex S, Elner A. Voice evaluation before
and after laser excision vs. radiotherapy of T1A glottic
carcinoma. Acta Otolaryngol 1995;560 –565.
3. Fisher SE, Frame JW, Browne RM, Tranter RM. A compar-
ative histological study of wound healing following CO2
laser and conventional surgical excision of canine buccal
mucosa. Arch Oral Biol 1983;287–291.
4. Tranter RM, Frame JW, Browne RM. The healing of CO2
laser wounds of the larynx. J Laryngol Otol 1985;895– 899.
5. Spaliviero M, Moinzadeh A, Gill IS. Laparoscopic cryotherapy
for renal tumors. Technol Cancer Res Treat 2004;177–180.
6. Dalkowski A, Fimmel S, Beutler C, Zouboulis CC. Cryother-
apy modifies synthetic activity and differentiation of keloi-
dal fibroblasts in vitro. Exp Dermatol 2003;673– 681.
7. Har-Shai Y, Amar M, Sabo E. Intralesional cryotherapy for
enhancing the involution of hypertrophic scars and keloids.
Plast Reconstr Surg 2003;1841–1852.
8. Knott PD, Milstein CF, Hicks DM, et al. Vocal outcomes after
laser resection of early glottic cancer with adjuvant cryo-
therapy. Arch Otolaryngol. In press.
9. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining
plus polarization microscopy, a specific method for collagen
detection in tissue sections. Histochem J 1979;447– 455.
10. Madruga de Melo EC, Lemos M, Aragao Ximenes Filho J, et
al. Distribution of collagen in the lamina propria of the
human vocal fold. Laryngoscope 2003;2187–2191.
11. Shepherd JP, Dawber RP. Wound healing and scarring after
cryosurgery. Cryobiology 1984;157–169.
12. Sizov VM, Pechenova TN, Volodina TG, et al. [Changes in the
structure of postburn keloid scars before and after cryo-
therapy]. Vrach Delo 1990;95–97.
13. Rousseau B, Hirano S, Scheidt TD, et al. Characterization of
vocal fold scarring in a canine model. Laryngoscope 2003;
620 – 627.
14. Thibeault SL, Gray SD, Bless DM, et al. Histologic and rheo-
logic characterization of vocal fold scarring. J Voice 2002;
96 –104.
15. Garrett CG, Reinisch L. New-generation pulsed carbon diox-
ide laser: comparative effects on vocal fold wound healing.
Ann Otol Rhinol Laryngol 2002;471– 476.
Knott et al.: Glottic Wound Healing with Cryotherapy