Megacolon: Evaluation of a Method for ExperimentalProduction of Aganglionosis *

PHILIP M. JOHNSON, M.D., MARGARET C. SWANTON, M.D.,CHARLES A. BREAM, M.D., RICHARD L. MURTLAND, M.D.

From the Departments of Radiology, Pathology, and Surgery, The School of Medicine,The University of North Carolina, Chapel Hill, North Carolina

INQUIRY into the pathophysiology of agroup of diseases related by the commonfeature of aganglionosis (congenital or ac-quired absence of ganglion cells) has beenhandicapped by inability to cause selectivedestruction of ganglion cells in the experi-mental animal. Although a remarkable in-crease in medical knowledge of esophagealachalasia, Hirschsprung's disease, mega-ileum, etc., has resulted from skilled use ofclinical material by many investigators,there can be little doubt that greater knowl-edge would accrue if such abnormalitiescould be produced and studied in labora-tory animals. Since the myenteric ganglioncells lie embedded between layers ofsmooth muscle, they are not amenable tosurgical extirpation. It would be desirableto have an agent which would, when in-jected into tissues containing ganglion cells,selectively destroy the latter without dam-age to adjacent cells and tissues.

In 1957, discovery of such an agent wasreported by Deloyers, Cordier, and Du-prez,", 2 who stated that the injection of anaqueous solution of 5 per cent "phenic acid,"or phenol (C,H,OH ),* * into the esoph-ageal wall of cats produced selective de-struction of the esophageal ganglion cellsprovided that the solution penetrated be-tween the muscle coats and did not localizesubserosally. Those cats surviving opera-tion developed esophageal achalasia or

* Submitted for publication October 14, 1959.* The identiy of "phenic acid" was obtained

by personal communication from one of theauthors.3

This investigation was supported by a grantfrom the United Medical Research Foundation ofNortlh Carolina.

"cardiospasm" characterized by stenosis atthe site of injection in the distal esophagusand dilatation of the esophagus proximally.The radiological changes of achalasia wereas well demonstrated by postmortem as byin vivo x-ray films. It was stated that afterinjection of "phenic acid," "microscopic in-vestigation did not demonstrate any kindof fibrosis or modifications of the muscularbundles." It was also stated that "examina-tion of the myenteric ganglia showed con-stant and definite lesions of the ganglioncells" of variable severity.'

In dilute solutions phenol denatures pro-teins without coagulating them. The com-pound is toxic to all tissues. When appliedlocally to the intact skin phenol penetratesto the nerve endings and exerts a local an-esthetic action. However, when it contactsexposed tissue or mucous membrane it isvery irritating initially and may cause ex-tensive tissue necrosis.4

In this country phenol has been used byveterinarians as a sterilizing agent appliedto the cut end of the intestine during in-testinal resection, and as a component ofan anesthetic syrup applied topically be-fore myringotomy.5The investigators' rationale for selection

of dilute phenol as a ganglionolytic agentwas apparently based on the fact that theagent was at one time used by Europeanveterinarians for chemical sympathectomy.No reference to this use of phenol wasfound in the American literature, nor wasthis use known to American veterinarians.5However, in 1935, Velu and coworkers 6 re-ported the employment of a 5 per cent solu-tion of phenol in periarterial sympathectomy.

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MEGACOLON Annals of SurgeryAugust 1960

TARLE 1. Summary of Experimental DataDuration of

Weight Material Survival Cause of Pathologic FindingsCat No. (kg.) Injected in Days Death Radiologic Findings at Autopsy1 1.4 Phenol 5% 6 Peritonitis No examination. Diffuse dilatation; acute peritonitis;0.7 ml. ulceration with necrotic muscularis;

massive transverse perforation.2 2.4 Phenol 5% 28 Sacrifice Day 8: Marked irregular narrow- Ring of serosal fibrosis; thinning and0.8 ml. ing of lumen at injection site. fibrosis of wall; absence of muscularis;

Day 28: Normal colon. obliteration of rugae.3 3.1 Phenol 5% 7 Sacrifice Day 7: Marked irregular narrow- Slight local constriction; localized0.8 ml. ing of lumen at injection site. organizing peritonitis; large ulcer with

necrotic muscularis; small perforation.4 1.9 Phenol 5% 13 Unknown Day 8: Moderate smooth nar- Diffuse dilatation; ring of serosal opac-1.0 ml. rowing at injection site. ity; intact wall (severe autolysis).5 1.4 Phenol 5% 8 Peritonitis No examination. Slight local constriction; local organiz-1.2 ml. ing peritonitis; early necrosis, external

muscularis.6 2.3 Phenol 5% 28 Sacrifice Day 8: Slight irregularity of lu- Slight local constriction; serosal fibrosis,

1.2 ml. men at injection site; intraperi- with barium; local thinning and fibrosistoneal extravasation of barium. of wall; absence of muscularis.Day 28: Minimal smooth nar-rowing at injection site.

7 1.6 Phenol 5% 10 Sacrifice Day 8: Moderate irregular nar- Serosal fibroblastic proliferation; lysis1.0 ml. rowing of lumen at injection site. and inflammation of muscularis; locasabsence of rugae.

8* 1.4 Sterile 10 Unknown Day 8: Normal colon. Microscopic focal serosal fibrosis;water normal muscularis.2.0 ml.

9 2.5 Phenol 5% 35 Sacrifice Day 35: Shallow thin annular Ring of serosal fibrosis; slight con-2.0 ml. stricture at injection site. striction; thinning and fibrosis of wall;absence of muscularis.

10 2.2 Phenol 5% 35 Sacrifice Day 35: Marked stenosis due to Ring of serosal fibrosis; constriction;2.0 ml. smooth, thick annular stricture. thickening and fibrosis of wall; absenceof muscularis; pyogenic granuloma pro-truding into lumen.

* Control.

In this procedure, the major artery of thelimb was exposed and surrounded by gauzepacking. The solution of phenol was thenpoured onto the adventitia of the vessel, thepacking removed and the incision closed.Postoperatively the limb exhibited hypere-mia which appeared on the second-sixthpostoperative day and lasted about sevenweeks. According to Velu et al., when gauzepacking was not used to protect the exposednonarterial tissues, a more intense inflamma-tory reaction developed, characterized earlyby necrosis and refractory suppuration, andlater by cicatrization and soft tissue deform-ity after healing by secondary intention. Al-though this report predated the use of anti-biotics, the postoperative inflammation canbe attributed primarily to the direct chem-ical action of phenol on soft tissue.

In view of the potential value of the workof Deloyers et al., and of the contradictorystatements concerning the effect of phenol

on soft tissue, it was decided that an in-dependent evaluation of dilute phenol as aselective ganglionolytic agent should beattempted.

Materials and MethodsTen adult mongrel cats provided the ex-

perimental material. A sterile 5 per centaqueous solution of phenol was preparedand was tinted by adding a few drops ofmethylene blue. The latter, by allowing bet-ter visualization of the injected portion ofthe colon, insured that no "skip areas" oc-curred. Methylene blue in this concentra-tion has no effect on tissue.

It was decided to select the colon as theinjection site to avoid both the technicaldifficulties of thoracotomy and, if possible,the high postoperative mortality (11 deathswithin 24 hours of operation in 19 cats) en-countered by Deloyers et al.Under intraperitoneal pentobarbital an-

314

Volume 152 JOHNSON, SWANTON, BREAM AND MURTLAND 315Number 2esthesia and using aseptic technic, the ab-dominal cavity of each cat was openedthrough a paramedian incision. A loop ofdescending colon was brought out throughthe incision and surrounded by moist gauzesponges; the mesentery and the blood sup-ply to this loop were left intact. Using asterile hypodermic syringe with No. 25needle, 1-2 milliliters of the 5 per cent solu-tion of phenol were injected between themuscle coats of the colon around the entirecircumference, producing an edematousring-like strip at least 1 centimeter in width.A wire clip or suture was then placed in anavascular portion of the colonic mesenteryimmediately adjacent to the injection site.The abdomen was closed in layers. No post-operative treatment was given; each animalwas fed a standard cat ration. As a controlfor the surgical procedure, one cat was sub-jected to the identical operation with injec-

FIG. 2. Cat 2. On the 8th postoperative daythe colon is markedly contracted at the injectionsite with irregularity of its wall and intramuralfistulae on its posterior aspect. Arrow indicatesmetallic suture placed in mesocolon to identifyinjection site. Dark smooth shadow below injectionsite is caused by air within the balloon of thecatheter through which barium was introduced.

tion of 2.0 cc. of sterile water tinted withmethylene blue.When possible, barium enema was per-

formed on each cat on the eighth andtwenty-eighth postoperative days, duringanesthesia with intraperitoneal pentobar-bital.Autopsy was performed in each animal

with removal of the entire colon togetherwith a portion of the rectum and ileum.The colon was opened longitudinally,cleaned, and fixed in 10 per cent formalin

FIG. 1. Cat 8 (Control). Examination on the it.th postoperaitve day sows no abnoality of pe o i ecthe distal large intestine. The identifying ietallic the colon were made at, above, and belowsiture is hidden befind the aritum-fillcd colon hiethis vidf. the viecwon .e, ident, either belowt

t

316 MEGACOLONmural changes or by the adjacent metallicclip or wire.

ResultsTable 1 summarizes the experimental

procedure and the pertinent radiologic andpathologic findings. There was no immedi-ate postoperative mortality, but four ani-mals died unexpectedly within six to 13days of operation. In two of these the causeof death was peritonitis (Cats 1 and 5); inthe others (Cats 4 and 8) the cause ofdeath was not disclosed by gross examina-

FIG. 3. Cat 3. Bariumil enema immedeiately be-fore sacrifice oii the 7tlh postoperative day showsan extentsive inflanmmiatory reaction whichl severelydistorts the colon. Bariumll lies within the colonicwall lbt is not seen intraperitoneally. The identify-ing metallic sutuire lies letween the spine and theposterior colonic wall.

Annals of SurgeryAugust 1960

tion at autopsy but presumably resultedfrom an intercurrent acute infection. Thesurviving animals were examined radio-logically and were sacrificed at intervalsfrom seven to 35 days; in addition, radio-logic studies were done on two of the ani-mals which died unexpectedly (Cats 4, 8).

Radiologic FindingsSix animals were examined by barium

enema on the seventh or eighth postoperativedays. At this time, the colon of the controlcat (No. 8) was normal (Fig. 1). The fiveremaining animals exhibited changes at theinjection site which ranged from slightsmooth constriction to marked irregularstenosis with evidence of intramural fistulae(Fig. 2, 3) and extravasation of bariuminto the peritoneal cavity. Unfortunatelyneither of the animals dying from general-ized peritonitis was examined radiologically.Four animals were examined 28 to 35

days postoperatively. Three exhibited asmooth ring-like stenosis at the injectionsite; the degree of stenosis was marked inonly one animal (Fig. 4). One animalwhich had had localized perforation of thecolon on the eighth postoperative dayshowed no abnormality of the colon at fourweeks. In a second, which had presentedmarked irregular narrowing of the lumenon the eighth day, the colon was normal onthe twenty-eighth day.No significant proximal dilatation of the

colon was observed in any animal.Pathologic Findings

Of the five cats autopsied six to 13 dayspostoperatively, three showed almost com-plete lysis of both layers of the colonicmuscularis, with destruction of the myen-teric plexus around most or all of the cir-cumference. In one there was total disrup-tion and acute inflammation of all layersof the wall arouind two-thirds of the cir-etumference; in the secon(l a small perfora-tion lhial occturred at one end of a trans-versely situated ulcer (Fig. 5). In the thirdcat, intact but nonruigose muicosa overlay

JOHNSON, SWANTON, BREAM AND MURTLAND

the area of muiscle lysis. All three had somedegree of inflammatory reaction and fibro-blastic proliferation in the serosa at thelevel of the injection (Fig. 6, 7). Of theremaining two cats, one showed chieflyserosal inflammatory and reparative reac-tion which extended irregularly into thesubjacent outer muscle layer; the latterexhibited early superficial necrosis. Themyenteric plexus contained normal num-bers of ganglion cells but early autolysisprevented more detailed evaluation. Thefifth animal had such severe autolysis ofthe colon that it could be determined onlythat all mural layers were present.

FIG. 4. Cat 10. Thereis a marked stenosis ofthe colon 35 days afteroperation. No distentionhas occurred proximal toWthis narrowing. Black ar-row identifies metallicsuture, visible in the orig-inal film.

Of the fouir cats atutopsied on the twenty-eighth to thirty-fifth postoperative days,three had some degree of constriction ofthe colon at the level of an encircling fibrousserosal band. Their colons, however, wereno more dilated above this level than belowit. Longitudinal sections of all four speci-mens through the injection site revealed acircumferential zone in which musculariswas absent (Fig. 8). In three of these, eachof the two layers was replaced by a thinlayer of young fibrous tissue merging intoand oriented in the same direction as thecorresponding muscle layer. The wall waslocally thinned, and alteration of the inner

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318 MEGACC

FIG. 5. Colon of Cat 3, sacrificed at 7 days.Colon is constricted at the site of a ring of non-rugose mucosa with a central ulcer that had per-forated at one end. See Figures 6 and 7.

layers thereof varied from lack of rugae tominimal distortion of the rugal patternwith intact mucosa (Fig. 9, 10). In thefourth specimen, the mucosa was ulceratedover a broad-based polypoid mass of chronicgranulation tissue originating both from thefibrous tissue replacing the muscularis andfrom the submucosa. This protruded intothe lumen. One of the cats sacrificed on thetwenty-eighth day had obviously had asmall perforation at the time of the firstbarium enema performed on the eighthpostoperative day. Small masses of barium-filled macrophages were imbedded withinthe thick serosa beneath an intact meso-

FIG. 6. Cat 3. The base of the ulcer is formedby adherent mesentery showing peritonitis. Ab-sence of muscularis, the 2 dark staining layers toleft, is evident in the center of the field. (Massonstain. x 15.)

)LON Annals of SurgeryAugust 1960

theliuim. The perforation and the inevitableaccompanying peritonitis had healed.The colon of the cat used as a control for

the operative procedure showed only a fewfocal microscopic areas of mild chronic in-flammation and fibroblastic proliferation inthe serosa. The muscularis was undamaged.

DiscussionThe experimental results demonstrate

that 5 per cent phenol injected into thecolonic muscularis almost invariably pro-duces necrosis of smooth muscle, leading to

FIG. 7. Cat. 3. Termination of dark stainingmuscle layers seen in Figure 6 is viewed underhigher magnification. Inner layer is necrotic (rightcenter), and outer layer is lysed and undergoingearly organization (left lower). Acute inflamma-tion in lower right corner is adjacent to ulcer.(Malsson stain. X 50.)lysis and beginning fibrous tissue replace-ment as early as the sixth day. In no casewas selective destruction of ganglion cellsin the myenteric plexuses observed. Themaximal damage observed was massive cir-cumferential perforation of the colon inone animal. Two others had small perfora-tions, one with fatal peritonitis, the otherhealing spontaneously. Of the remaining sixanimals, four showed almost complete de-struction of both layers of the muscularis,with its myenteric plexus, and considerableserosal reaction around the circumferenceof the colon, without perforation. Limita-

Volume 152 JOHNSON, SWANTON, BREAM AND MURTLAND 319Number 2

tion of damage to the serosa and the superfi-cial part of the external muscular layer inone animal of this group suggests that thephenol was predominantly injected into thesubserosa. The one animal without grossmuscular damage was unfortunately alsothe one in which autolysis was too markedfor detailed histologic study.

Summary and ConclusionsAn investigation was undertaken to eval-

uate the experimental observation that di-lute phenol is a selective ganglionolyticagent when injected into tissues containingganglion cells. It was found that, under

'.

FIG. 8. Colon of Cat 9, sacrificed at 35 days,shows slight constriction and mild rugal patternalteration. Darker colored muscularis terminates atupper and lower ends of stricture; arrows indicatethis at lower end of stricture (right).

the conditions of this study, the interstitialinjection of dilute phenol results in destruc-tion not only of ganglion cells but also ofall tissues which it contacts. The material isunsuitable for selective aganglionosis pro-duction in the laboratory animal.

References1. Deloyers, L., R. Cordier and A. Duprez: A New

Approach to the Physiology of So-Called "Car-diospasm." Experimental Production of "Car-diospasm" in Cats after Destruietion of Anier-baclh's Plextis. AIiii. Stirg., 146:167, 1957.

2. I)eloyers, L., R. Cordier and A. I)uprez: Re-cherches stur la Physiopathologie du Cardio-spasme. I. Creation de Cardiospasme Experi-mental Chez le Chat, par Lesion des Plexus

FIG. 9. Cat 2, sacrificed at 28 days. Each layerof dark staining muscularis is continuous centrallywith a much thinner, paler layer; a few musclefibers persist in the external one. Submucosa isthin, serosa thick at this level. See Figure 10. (Mas-son stain. x 15. )

d'Auerbach. Bull. Acad. Roy. de Mled., 22:47,1957.

3. Deloyers, L.: Personal Communication.4. Goodman, L. S. and A. Gilman: The Pharmaco-

logical Basis of Therapeutics. New York, Mac-millan, 2nd Ed., 1078-1080, 1955.

5. Guard, W. F.: Personal Communication.6. Velu, H., H. Weyland and L. Comby: Observa-

tions sur la Sympathectomie Chimique Chezles Equides. Bull. Acad. Vet. de France, 8:62, 1935.

1_!yS~~~~~~~~~~.

FIG. 10. Cat 2. Higher magnification of area toright in Figure 9 shows merging of each darkstaining muscle layer with a layer of delicatefibrous tissue. Serosal thickening is due to in-creased fibrous tissue. (Masson stain. x 50.)