effect of freezing on the ciliary body...

Effect of freezing on the ciliary body

(cyclocryotherapy)

Frank M. Polack* and Andrew de Roetth, Jr.

Freezing of several areas of the ciliary body of normal rabbit eyes was clone with a cryocautery.Tonogaphic tracings were 'performed before and from 6 to 8 iveeks after freezing. It wasobserved that the average intraocular pressure of these rabbit eyes had decreased from 18to 14 mm. Hg, the coefficient of outflow facility (C) was reduced from 0.24 to 0.17, andaqueous floto (F) was decreased from 2.26 to 1.06. Histologic observations showed an im-mediate postfreezing stage of edema, exudates, and hemorrhages, with destruction of cellularelements. The histologic structure of the frozen area regenerated rapidly, acquiring an almostnormal appearance one week after freezing. Hyalinization of connective tissue around vesselsand in the subepithelial area was observed in sections of eyes 5 to 10 weeks after freezing.It seemed apparent that these changes might be partially responsible for the decreased secre-tion of aqueous.

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.reezing of the ciliary body as an anti-glaucomatous treatment was first describedby Bietti1 in 1950. He reported tonometricand histologic studies on rabbits and alsotonometric results in several types of glau-coma in humans. Further reports on thismethod have not appeared, although it isstill used by Bietti- in some cases ofglaucoma.

The purpose of this investigation was toobtain tonographic information concerning

From the Department of Ophthalmology, ResearchCollege of Physicians and Surgeons, ColumbiaUniversity, and the Institute of Ophthalmology,Presbyterian Hospital, New York, N. Y.

This investigation was supported in part by NonrContract 266 (71), and in part by ResearchGrant B 492 (C7) from the National Instituteof Neurological Diseases and Blindness, NationalInstitutes of Health, United States Public HealthService.

"Special Fellow, National Institutes of Health(BT-819).

the changes in aqueous production andoutflow facility in rabbit eyes followinglocal freezing of the ciliary body (cyclo-cryotherapy), and to study the histologicchanges, especially in the early regenera-tion period.

Clinical observations have been madeby us, and will be reported at a later date.

MethodsTonography. Adult albino rabbits (2.5 to 3 kilo-

grams) were anesthetized with intravenous sodiumpentothal (30 mg. per kilogram) and locally withone or two drops of Ophthaine." Tonographictracings were obtained with a Miiller electronictonometer, and recorder in 30 eyes. The 1955tables of Friedenwald were used to interpret thetonographic results. To obtain a measure ofaqueous production, the formula F = (Po - Pv) xC was used, where F = rate of aqueous flow(fd per minute), Po = intraocular pressure (mm.Hg), C = outflow facility (fi\ per minute permm. Hg), and Pv = episcleral venous pressure

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Effect of freezing on ciliary body 165

(mm. Hg). Pv was assumed to be 9 throughoutthese experiments. It is generally recognized thatcalculating aqueous production from tonographicdata is inaccurate, and one cannot obtain reliabledata by this method. However, since we wereinterested only in relative changes in aqueousformation before and after freezing, this method ofmeasuring aqueous humor production seemedadequate for our purposes. Tonography was donein each eye before freezing and then at weeklyintervals thereafter.

Freezing of the ciliary body was carried outwith a copper cup filled with dry ice and alcohol(-79° C.) with a conical tip 4 mm. in diameter.The cryocautery was applied over the conjunctivafor one minute, 1 mm. from the limbus {Fig. 1).Four, 6, or 8 applications were made. The frozensurface was 5 to 5.5 mm. in diameter, and insidethe eye a ball of ice formed gradually, whichreached as far as the peripheral vitreous. Sincethe ciliary processes in the rabbit extend to theposterior surface of the iris, a complete freezing ofthese structures was probably not always obtained.The eyes were examined daily with penlight andsometimes with slit lamp during the first weekafter freezing. The tonograms obtained 6 to 8weeks after freezing were used to evaluate theeffects of cryotherapy.

Histology. The eyes of 5 albino and 6 pig-mented rabbits (2.5 to 3 kilograms) were frozenin four areas as described above. At periods rang-ing from 4 hours to 7 days, the animals werekilled with intravenous Nembutal. The eyes wereremoved and fixed in 10 per cent formalin. Inaddition, the rabbits studied tonographically werekilled, and the eyes removed for histologic studyat various time intervals ranging from 4 to 10weeks. Four control eyes were also studied. Afterfixation, the treated area of the ciliary body wasexamined macroscopically while being removedfrom the globe. Tissues were processed in paraffin.Sections were stained with hematoxylin and eosin,Masson's trichrome stain, and van Giesson's stain..

Labeling regenerating cells with tritiatedthymidine, which is incorporated in the premitoticstage of DNA synthesis, is of interest because onecan localize these cells in preparation for mitosis,and obtain a view of their regenerative activityat the time of labeling, For this purpose, in 6eyes in which cryotherapy had been applied infour areas, 2 /*c of tritiated thymidine,* dis-solved in 0.10 c.c. of Eagle's basal media, wasinjected in the vitreous cavity adjacent to thefrozen area 24 hours after freezing. These eyeswere removed 24 hours later (48 hours afterfreezing), fixed in 8 per cent formalin, embeddedin paraffin, and cut at 5 n. Radioautographs were

Fig. 1. Schema showing the cryocautery beingapplied to the rabbit eye. The circular area in-dicates the approximate size of the frozen tissueafter one minute.

Table I

Before freezing

Po C F

6 to S weeksafter freezing

Po | C F

152521112115241912181919

0.250.240.180.170.280.200.310.350.170.190.260.23

1.503.842.160.340.161.204.653.500.511.712.602.30

172113111712199

14131113

0.120.200.220.110.190.110.310.230.110.170.150.17

0.962.400.880.221.520.333.10

0.550.680.300.68

'New England Nuclear Corp., Boston, M.iss.

Average 18.25 0.24 2.26 14.2 0.17 1.06Po = intraocular pressure,C = outflow facility (/tl/min./mm. Hg).F = rate of aqueous flow (/tl/inin.),

made with Kodak AR-10 stripping film, and wereexposed for 7 days. The slides were developedin Kodak developer D-19, and stained withHarris' hematoxylin after fixation.

Results

Tonography. After thawing, the treatedarea showed a moderate amount of hy-peremia and occasionally a small conjunc-tival hemorrhage. Twenty-four hours afterfreezing there was marked ciliary conges-tion and dilatation of iris vessels. In one


166 Polack and de Roetth Investigative OphthalmologyApril 1964

case, blood was present in the anteriorchamber, and for the first 3 or 4 days afterfreezing all eyes showed a discrete aque-ous flare. One week after freezing, theexternal appearance of all the eyes was

normal, and the anterior chambers wereclear. Table I represents the tonographicobservations in 12 eyes before freezingand 6 to 8 weeks after freezing. Other tono-grams were not included because they

Fig. 2. Four hours after freezing, the ciliary processes show edema, some hemorrhages, andloss of the nonpigmented epithelium. In some areas the ciliary epithelium has not beendestroyed (arrow). Edema is also present under the pigmented layer of the iris. Fibrinousexudate occupies the posterior chamber. (Masson's trichrome. x72.)

Fig. 3. Twenty-four hours after freezing, hemorrhages predominate in the stroma of theciliary processes. Edema has formed cystic deposits between the epithelial layers. The non-pigmented epithelium has been destroyed, and the pigmented epithelium persist for the mostpart. (Hematoxylin and eosin. x72.)


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were technically inadequate. The tableshows that before freezing the average Powas 18, C was 0.24, and F was calculatedto be 2.26. Six to 8 weeks after freezing,the tonograms indicated an average Po of14, a C of 0.17, and a calculated F of 1.06.

Histology. Globes of albino rabbitswhich had been fixed in formalin 4 hours,1 and 2 days after freezing, on sectioningrevealed areas of congestion and hemor-rhages in the ciliary body. One week afterfreezing, these hemorrhages were not seenmacroscopically.

Microscopic examination of tissue sec-tions 4 hours after freezing showed swell-ing of the ciliary processes caused byedema and hemorrhages in the stroma(Fig. 2). Rupture of small capillaries anddamage of the endothelium of larger ves-sels accounted for these alterations. Thenonpigmented epithelium was absent overmost of the ciliary processes, although insome areas clusters of these cells remainedin place. The pigmented epithelium wasalso destroyed in many ciliary processes;however, in eyes of chinchilla rabbits,where the pigmented epithelium could bebetter observed, flat pigmented cells re-mained, lining the injured ciliary body inmost places. Vascular congestion andedema were present in the iris stroma, andfrequently the pigmented epithelium ofthe iris was partially detached due toedema (Fig. 2). Coagulated fibrin waspresent in the anterior and posterior cham-bers, and irregularly stained strands ofvitreous were present around the ciliarybody suggesting that this structure wasaltered by freezing. In the treated area,the sclera showed a pink staining insteadof the usual bluish color obtained with thetrichrome stain. Subconjunctival edemawas present at this level.

Twenty-four hours after freezing, theedema of the injured area had subsided.The appearance of the ciliary processeswas otherwise similar to that described.In some sections, hemorrhages predomi-nated (Fig. 3), the basal layer of the cili-ary epithelium was ruptured, and red

cells were present in the posterior cham-ber (Fig. 4). Mounds or excrescences ofpigmented cells were observed in several

Fig. 4. Part of a ciliary process 24 hours afterfreezing, showing stromal hemorrhage and rup-ture of the basement layer of the ciliary epithe-lium. (Hematoxylin and eosin, xl44.)

CB

Fig. 5. Radioautograph of a section of the ciliarybody (CB) of an albino rabbit labeled with tri-tiated thymidine 24 hours after freezing. There isheavy uptake of the isotope by regenerating cellsof the ciliary epithelium (E). Arrow points a mi-totic figure in the pigmented epithelium. Labeledconnective tissue cells are present in the stroma.{Hematoxylin. x72.)


168 Polack and de Roetth Investigative Ophthalmology

areas as well as free pigment granules inthe surrounding fibrinous exudate. Dis-crete leukocytic reaction composed of

ft

>

Fig, 6. Radioautograph of the ciliary body (CB)and peripheral retina (R) showing cells of thepigmented epithelium (p) labeled with tritium24 hours after freezing. (Hematoxylin. x72.)

Fig. 7. Eight weeks after cryotherapy, the ciliaryepithelium is of normal appearance. There is amoderate hyalinizaticm of the connective tissue inthe ciliary processes. (Masson's trichrome. x570.)

polynuclears and lymphocytes was presentin the ciliary body and also in the angleof the anterior chamber. Except in 2 eyesin which no labeling was observed, allradioautographs of the ciliary body labeled24 hours after freezing and removed 4Shours after freezing showed that cells ofboth the pigmented and nonpigmentedepithelium were heavily labeled, indicating,that regeneration of the epithelium wasmost active at this stage. Figures indicat-ing mitosis were present in the layer of thepigmented epithelium (Fig. 5), as well asclumps of labeled cells which seemed tocorrespond to pigmented cells. The cells ofthe pigmented epithelium of the peripheralretina were also labeled in a case wherethe freezing injury was more posterior(Fig. 6), demonstrating that these cellsreacted in a similar way after freezing.

Two to three days after freezing, theedema of the injured area was minimal. Aconsiderable epithelization of the ciliarybody was present, and, at one week afterfreezing, the ciliary processes showed acomplete regeneration of the epithelium;except in a few instances where the layerof nonpigmented cells was still absent, theciliary processes being covered by flat pig-mented cells. The appearance of the filtra-tion angle was normal, as well as the stain-ing characteristics of the sclera.

Five to 10 weeks after freezing, the mi-croscopic appearance of the ciliary bodyand processes in the treated area were in-distinguishable from the normal whenstudied with hematoxylin and eosin, ex-cept for a layer of homogeneous hyalinetissue around the blood vessels and in thesubepithelial layers (Fig. 7), which wasbetter observed in sections stained withMasson's trichrome stain. Occasionally,areas of hyperplasia of the pigmented epi-thelium were observed. It also appearedthat there was an increase in the amountof connective tissue in the ciliary bodywhen compared to sections of normal eyes(Fig. 8). This thickening of the stroma ofthe ciliary body was found in eyes 5 weeksor more after freezing.


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Fig. 8. Ten weeks after freezing, in most cases there is an increase in the amount of con-nective tissue of the ciliary body and areas of hyalinization, as shown in the figure. (Hema-toxylin and eosin. x72.)

Comments

These experiments indicate that freezingof the ciliary body decreases the intra-ocular tension in normal rabbit eyes. Tono-graphic studies confirm the supposition ofBietti that the hypotony was caused by adecrease in the production of aqueoushumor. Our studies, although not extensive,indicate that concomitant with the decreasein aqueous flow there is a compensatorydecrease in outflow facility, perhaps sim-ilar to that observed by Becker and Con-stant* in Diamox-treated eyes. Hemorrhageand edema are the initial lesions in theciliary body. These alterations are causedby a destruction of the endothelium ofcapillaries and arterioles and rupture ofthe wall of small vessels. Destruction ofboth epithelial layers also occurs, but thereis evidence that the pigmented layer per-sists in several injured ciliary processes. Inpreliminary experiments we have observedthat the intraocular pressure rises some-what {5 to 10 mm. Hg) shortly after freez-ing, and remains elevated for 1 or 2 daysbefore hypotony begins. This elevation of

the ocular tension is probably caused bythe hemorrhages and edema in the treatedareas.

The epithelial layers regenerate rapidly,as shown by a high uptake of tritiatedthymidine 24 hours after freezing. Thisregeneration seems more active at the levelof the pigmented epithelium, which, as hasbeen noted before, can persist in frozenareas and may show areas of hyperplasia.Cryotherapy did not produce permanentchanges in the sclera. This observationis supported by studies proving that freez-ing of the cornea did not change its col-lagen structure or transparency.4 Possibly,fibrosis of the connective tissue of the cili-ary body results from the hemorrhagescaused by freezing and by the stimulus forrepair, as may occur in other tissues and asoccurs in stomachs frozen in vivo5 inwhich hemorrhages and edema were theinitial lesions. The changes described inthe stroma of the ciliary body might bepartially responsible in decreasing thesecretion of aqueous humor. Further stud-ies are planned to elucidate this point.


170 Polack and cle Roetth Investigative OphthalmologyApril 1964

REFERENCES1. Bietti, G.: Surgical interventions on the ciliary

body, J. A. M. A. 142: 889, 1950.2. Bietti, G.: Personal communication.3. Becker, B., and Constant, M.: Experimental

tonography (the effect of carbonic anhydraseinhibitor, acetazolamide, on aqueous humor),A. M. A. Arch. Ophth. 54: 321, 1955.

4. Polack, F. M.: Isotopic labeling of cornealstromal cells prior to transplantation, Trans-plantation 1: 83, 1963.

5. Allcook, E. A., Carpenter, A., Berstein, E. F.,Peter, E. T., and Wangensteen, O. H.: Struc-tural changes following gastric freezing, Sur-gery 53: 764, 1963.


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