THE EFFECT OF SURGICAL TRAUMA ON RAT TUNICA ALBUGINEA

T THE JOURNAL OF UROLOCY Copyright Q 1998 by AMERICAN UROUXICAL ASS~CIATION, INC.

Vol. 159, 1700-1707,May 1998 Printed in U.S.A.

THE EFFECT OF SURGICAL TRAUMA ON RAT TUNICA ALBUGINEA AHMED I. EL-SAKKA, CATHERINE A. SELPH, T. S . B. YEN, RAJVIR DAHIYA,

AND

TOM F. LUE*

From the Department of Urology, University of California School of Medicine, and the Department of Pathology, VA Medical Center, San Francisco, California

ABSTRACT

Purpose: Increased TGF-P1 protein expression in tunica albuginea has been found to be associated with Peyronie's disease. The present study is designed to investigate whether surgical trauma induces TGF-P up-regulation and histological changes in rat penis. Materials and Methods: Thirty-two adult male Sprague-Dawley rats were divided into two groups. The first group (n = 24) underwent incision and suture repair of the tunica albuginea of the penis. The second group (n = 8) received sham surgery (incision of the penile skin and underlying fascia) as the control group. The trauma-induced group was divided into four subgroups in which the rats were euthanized a t 6 hours (n = 6),1day (n = 6),3 days (n = 61,and 8 weeks (n = 6). Two sham-operated (control) animals were also euthanized a t each of the above time points. All tunical tissues from the trauma-induced and sham-operated rats were collected and examined histologically using Trichrome and Hart elastic fiber stain. Electron microscopy was used to study the ultrastructural changes of both trauma induced and control specimens. Western blotting technique was performed to study TGF-P protein expression in both experimental and sham-operated groups. Results: Tissue edema and hemorrhage between collagen bundles are noted in the experimental groups after 6 hours, 1 day and 3 days. At 8 weeks the most prominent changes observed were inflammatory cellular infiltration and disorganization of the collagen bundles. In the control group the tunica albuginea retains normal wavy regular appearance in all rats. This histological analysis is similar to the reported description of histological features of the acute phase of Peyronie's disease. Electron microscopy showed packed collagen bundles in the trauma-induced group with normal appearing elastic fibers.No abnormal change was detected in the control group. Immunoblot results revealed remarkable TGF-P1 protein expression in 1, 5, 3, and 0 rats of trauma induced subgroups after 6 hours, 1day, 3 days, and 8 weeks respectively. No TGF-P1 protein expression in any rats in the control group was detected. No significant TGF-P, or TGF-P, protein expression was observed either in the trauma induced group or in the control group. Conclusion: Trauma can induce histological changes similar to the acute phase of Peyronie's disease but not the overt picture of the chronic phase of Peyronie's disease. It can also result in an early but transient up-regulation of TGF-B1 protein expression in the rat penis. We conclude that surgical incisional trauma does not result in Peyronie's disease-like changes in the tunica. KCI WORDS:Peyronie's disease, tunica albuginea, trauma, growth factors, TGF-P

Peyronie's disease is a localized connective tissue disorder that primarily affects the tunica albuginea of the penis.' Although many tentative theories have been proposed to explain the cause of Peyronie's disease, the true pathogenesis of Peyronie's disease remains an enigma. TOdate, vitamin E deficiency: the use of beta blocking agents: increased blood level of serotonin in carcinoid syndrome, and autoimmune responses to vascular trauma4 have been implicated in the pathogenesis of Peyronie's disease. attributes the The most accepted current histological findings and symptoms t o the effects of penile t r a ~ m aMinor .~ sexual trauma with delamination of the tunica albuginea leads to bleeding into the intralaminal space and perivascular 'ymphocflic and plasmacflic cellular infiltration in the tunica albuginea.' After several months, tissue fibrosis and deposition of nonpolarized collagen with eventual scar formation occurs.' active ~ ~ ~ lpeptides ~ such ~ as platelet ~ ~derived l growth l factor (PDGF), epidermal growth factor (EGF), and transforminggrowth factorbeta ( T G F - ~have ) been implicated in

collagen synthesis and Among them, TGF-P has been proven to be involved in numerous vital processes including inflammation, stimulation of extracellular matrix production and wound healing.g TGF-P has also gained considerable attention % a factor implicated k the cause of chronic conditions such a pulmonary fibrosis rats and mice10 and hepatic fibrosis k irradiated rats.'' TGF-p may also play a sc~erosis.13 role k fibmtic liver diseae12 and The exact mechanism(s) by which TGF-P acts is not fully understood. However it has been shown to enhance the expression of specific genes'4 and to influence the metabolism of extracellular components~~5 Though many cells have been shown to produce the molecule of TGF-P, it is usually present in a latent, biologically inactive form. Latent high molecular weight TGF-P can be converted into active TGF-P by proteolytic enzymes or by the removal of carbohyTGF-P may be acti~ drates from the latent m~lecule.'~''~. vated during tissue reaction to trauma, and the molecule profoundly influences cellular processes associated with wound healing and fibrosis.15 Primary mechanical trauma with its subsequent cascade of Accepted for publication December 2, 1997. * Re uests for reprints: Department ofurology, U-575,University Cellular and molecular events has been reported to have a of Calijornia, San Francisco, CA 94143-0738. remarkable role in the induction of fibrosis in different or1700

EFFECT OF SURGICAL TRAUMA ON RAT TUNICA ALBUGINEA

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FIG. 1. A, Rat tunica albuginea 1 day after trauma (%chrome stain). Notice edema and hemorrhage in site of tunical trauma (arrows)and normal side ( m o w head). Magnification x25. B,rat tunica albuginea 8 weeks after trauma (Hart's stain). Notice regular elongated elastic fibers (arrows). Magnification ~ 1 0 0 C, . rat tunica albuginea, 8 week control group (Hart's strun). Notlce normal regular elongated elastic fibers (arrows). Magnification X 100.

gans. Among growth factors, TGF-p is considered the key factor responsible for this pathological condition.'s-21 Recently, TGF-p protein expression has been found to be associated with Peyronie's disease." The present study was designed to investigate the effects of surgical trauma on TGF-p production as well as histologicd changes in the tunica albuginea. MATERIALS AND METHODS

Experimental animals. Thirty-two adult male SpragueDawley rats were divided into 2 groups. The first group (n =

24) underwent induced trauma of the tunica albuginea of the rat penis (experimental group). The second group (n = 8) underwent incision of the penile skin and fascia only (control group). Skin incision extended from the glans to the pubic bone with dissection and opening of the penile fascia. Incision of the tunica was extended on the whole length of the penile shaft using a surgical blade and closed using 8/0 Nylon sutures under 1OX magnification. The tunica trauma group was divided into four subgroups of 6 rats each, in which the rats were sacrificed at 6 hours, 1day, 3 days, and 8 weeks. We did not exped significant changes in morphology or molecular

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EFFECT OF SURGICAL TRAUMA ON RAT TUNICA ALBUGINEA

FIG.2. A, rat tunica albu 'nea 8 weeks after trauma (Hart's stain). Notice disorganization of the collagen bundles with loss of their regular wavy appearance in area o k a u m a (arrows) and around sutures (arrow head). Magnification x100. B, rat tunica albu 'nea 8 weeks after trauma (Trichrome stain). Notice disor anization of collagen bundles with loss of their regular wavy appearance, especialg in area of trauma (arrow) and around suture (arrowheafi). Magnification X 100. C, rat tunica albuginea 8 weeks control group (Hart's stain). Notice normal regular wavy appearance of tunica with two longitudinal layers (arrows) and one transverse layer (arrow-head). Magnification x 100. biology after 8 weeks. Two control rats were sacrificed for each subgroup. All the incised tunical tissues and similar areas from the control group were collected and examined histologically using trichrome and Hart elastic fiber stain. Electron microscopy was used to study the ultrastructural changes of both trauma induced and control specimens. Western blotting technique was used to study TGF-P protein expression in all rats. Transmission electron microscopy. The samples were

immersion-fixed with 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.15 M. sodium cacodylate buffer (pH 7.4). After post-fixation in 2% osmium tetroxide, the tissue was dehydrated in graded ethanol and propylene oxide, and subsequently embedded in Epon 812. Thick sections (1Fm.) were cut on a Sorvall MT2-B ultra-microtome and stained with 1% toluidine blue. Thin sections (approximately 900 A")were mounted on 200 mesh copper grid, and stained with uranyl acetate and lead citrate as a contrasting agent. Ultrastruc-

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FIG. 3. A, rat tunica albuginea 8 weeks control group. Notice normal distribution of collagen bundles (arrows).Magnification X60,OOO. B, rat tunica albuginea 8 weeks a h r trauma. Notice increased density and clumping of collagen bundles with loss of spaces between bundles (arrows).Magnification X60,OOO .

ture examination was performed with a Philips 400 transmission electron microscope Western Blotting. The protein expression of growth factors TGF-p,, TGF-p,, and TGF-p, was studied using Western Blot technique. b i c a l tissue from each rat was homogenized in phosphate buffer (10 mM. phosphate buffer, pH 7.4; 150 mM. NaC1; 1 mM. phenylmethylsulfonylfluoride,25 pgJ ml. aprotinin, and 25 pgJml. leupeptin). The homogenate was then centrifuged in Eppendorf tubes at 10,000 RPM for

20 minutes at 4C. The protein concentration in the supernatant was determined by the Lowry micro method. Each sample was denatured for 5 minutes at lOOC in Laemmli sample buffer. Equal amounts of protein were loaded for each sample and electrophoretically separated on 16.5% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 0.2% casein and 0.1% tween-20 in 20 mM. Tris saline buffer at pH 7.2. Growth factors TGF-p,, pz, and &, were detected using specific rabbit

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EFFECT OF SURGICAL TRAUMA ON RAT TUNICA ALBUGINEA

FIG. 4. A, rat tunica albuginea 8 weeks control group. Notice normal shape and distribution of elastic bundles (arrow). Magnification

X60,OOO. B , rat tunica albuginea 8 weeks after trauma. Note packed collagen bundles and normal shape of elastic fibers (arrow). Magnification X50,OOO.

primary antibodies (1:500 dilution) to these growth factors and alkaline phosphatase conjugated goat anti-rabbit secondary antibodies (1:20,000 dilution). The membrane was processed and treated with chemiluminescence reagents supplied by Tropix (Medford, MA). The bands were visualized on an x-ray film exposed to the membrane to detect chemiluminescence signals. Statistical analysis. Statistical analyses were performed using the ?-test. (primer biostatistics, version 3.01, Macintosh version by Bret Peterson, McGraw Hill, 1992)

RESULTS

Histopathology. In the trauma-induced group, after 6 hours the prominent change was hemorrhaging between collagen bundles with the preservation of the entire architecture of the tunica. In the one-day group, edema and a dramatic influx of inflammatory cellular infiltration were the commonplace findings (fig. 1, A). ARer 3 days edema subsided; polymorphs, lymphocytes and macrophages were universally spread between collagen bundles; however, the col-

EFFECT OF SURGICAL TRAUMA ON RAT TUNICA ALBUGINEA

lagen and the normal elastic fiber lattice framework were slightly disturbed. In the group of 8 weeks the residual changes were disorganization and loss of wavy appearance of the collagen bundles (fig. 2, A and B ) . The elastic fibers remained normal (fig. 1, B and C ) . In the control group no histological changes of the tunica albuginea were noted in any rat of the different subgroups, eliminating the possibility that sham surgery may affect the tunica (fig. 2, C ) . Ultrastructural changes. In the trauma-induced group, the changes observed after 8 weeks were an increase in density and packing of collagen bundles in comparison to the control group (fig. 3, A and B ) . However, the elastic fibers appeared normal (fig. 4, A and B ) . Immunoblot. The results of Western Blotting showed statistically significant up-regulation of TGF-P1 protein expression in the experimental group that was sacrificed after one day (5/6) (fig. 5, B). Although TGF-P1 was up-regulated in V6 and 316 of the experimental groups sacrificed after 6 hours and three days, the difference between the experimental and control groups was statistically insignificant (table 1, fig. 5,A

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and C ) .There was no TGF-P1 protein expression in either the control or experimental groups after 8 weeks (0/6) (fig. 5,D ) . TGF-P2 and TGF-P3 were not significantly expressed in either the experimental or the control group (table 1). DISCUSSION

The pathogenesis of Peyronie's disease is not well understood. The most widely accepted theory attributes the symptoms and histological findings to the effect of trauma." Minor sexual trauma with delamination of the tunica albuginea may lead to bleeding into the intralaminal space followed by perivascular lymphocytic and plasmacytic inflammatory cellular infiltration in the tunica albuginea.' Primary mechanical trauma with its subsequent cascade of cellular and molecular events has been reported to have a remarkable role in the induction of fibrosis in different organs. Growth factors, particularly TGF-P are considered the key factors responsible for this pathological Multiple events involving TGF-P take place in tissue repair after

1:TGF-p, Standard ZTrauma 3:Trauma 4:Control 5:Trauma 6:Trauma 7:control &Trauma 9:Trauma FIG, 5. A, immunoblot &wed TGF-B1 protein expression in experimentaland control group after 6 hours. B , immpnoblot showed TGF-B1 p m h n expression in experimental and control group after one day. C, immunoblot showed TGF-B1 protein expression in expenmental and control group after three days. D,immunoblot showed TGF-B1 protein expression in experimental and control group after 8 weeks.

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From this study, we conclude that surgical trauma of the injury and the induction of fibrosis. Platelets contain high concentrations of TGF-p and upon degradation at a site of tunica albuginea of the rat penis produces a transient ininjury, release TGF-p into the surrounding tissue.” TGF-0 crease in TGF-p and minor tissue alteration. Although a then initiates a complex sequence of events that promotes blunt trauma to the tunica albuginea may produce changes healing, including chemoattraction of monocytes and leuko- similar to Peyronie’s disease, a clean surgical incision and c y t e ~induction ,~~ of angiogenesis? and control of the pro- suture repair does not produce long term TGF-p elevation duction of cytokines and other inflammatory mediat01-s.~~and severely distorted histological changes as seen in human Moreover, TGF-p stimulates the synthesis of matrix compo- Peyronie’s disease. The results of these experiments seem to nents including fibronectin, collagens, and proteogly- indicate that “trapping“ of the inflammatory process in a cans,z6*z7 while simultaneously blocking matrix degradation dense multi-layered structure leads to over-production of by decreasing the synthesis of proteases and by increasing TGF-p, excessive fibrosis and destruction of elastic fibers, the levels of protease inhibitors.” TGF-p increases the ex- thus resulting in the formation of Peyronie’s plaque. pression of integrin and changes their relative proportions on the surface of cells in a manner that can facilitate adhesion to REFERENCES the matrix.29 All of these events can be beneficial in tissue 1. Smith, B. H.: Peyronie’s disease. Am. J . Clin. Pathol., 45: 670, repair. However, TGF-p-induced deposition of extracellular 1966. matrix at a site of tissue injury can also lead to scarring and 2. Scardico, P. L. and Scott, W. 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