870 Reports Investigative OphthalmologyNovember 1974

5. Campbell, F. W., and Michaelson, I. C :Blood-vessel formation in the cornea, Br. J.Ophthalmol. 33: 248, 1949.

6. Benezra, D., and Sachs, U.: Growth andtransplantation of organ cultured corneas, inpreparation.

7. Benezra, D., Gery, I., and Davies, A. M.:Transformation of rabbit lymphocytes by spe-cific antigens, Proc. Soc. Exp. Biol. Med. 125:1305, 1967.

8. Benezra, D., and Hochman, A.: In vitroactivation of lymphocytes from patients withmalignant diseases. I. Kinetics and differencesin magnitude of response, Isr. J. Med. Sci.7: 553, 1971.

9. Benezra, D., Gery, I., and Davies, A. M.:Specificity of blast transformation. II. Studieswith erythrocyte antigens, Int. Arch. Allergy39: 488, 1970.

Cytological changes in the conjunctiva inthe megaloblastic anemias. J. D. BROD-

RICK, I. M. STRACHAN, AND T. SPEED.

An attempt has been made to demonstrate in-creased nuclear size of the conjunctival cells inthe viegaloblastic anemias. Conjunctival scrapingswere taken, wet fixation employed, and nucleardiameters measured under the microscope. Theresulting data was subjected to statistical analysis.Two approximate tests of the null hypothesis thatthe affected group has the same nuclear size asthe control group have been performed. Both testsreject this hypothesis at the 5 to 10 per cent level.

Cytologic changes in the mucous membrane ofthe mouth and vagina are well recognized in pa-tients suffering from megaloblastic anemia. Thenature of these changes is similar irrespective ofwhether folic acid or vitamin Bi2 deficiency is theunderlying cause and it would be somewhatsurprising if similar cytologic changes did notoccur in the conjunctiva. As far as we are able toascertain, the only attempt made to find theseexpected changes led to the conclusion that therewas no alteration of the conjunctival cytology inmegaloblastic anemia.1

This communication deals with the results ofour investigations into the conjunctiva] cytologyof proved cases of megaloblastic anemia.

Material and methods. Patients were obtainedfrom the Department of Hematology, having beenreferred there for sternal marrow examination onthe basis of symptoms, clinical examination, anddemonstration of the characteristic changes in theperipheral blood film. Before treatment was com-menced, conjunctival scrapings were taken by thesame observer following instillation of 0.5 percent Ophthaine. The specimens were spread onto

Table I. Table showing high and low classificationin control and affected specimens

High Low Total

ControlAffected

Total

49

13

101121

the center of a slide, immersed immediately in50:50 absolute alcohol and ether, and fixed for24 hours. They were then air dried, taken downthrough 70 per cent and 50 per cent alcohol todistilled water, treated with hot (60° C.) N HC1for 10 minutes, washed in distilled water, andstained with 1 per cent Cresyl fast violet acetate.The stained sections were then taken through 95per cent absolute alcohol and xylol and mountedin Canada Balsam. Scrapings were taken from aseries of clinically normal control specimens andthe whole series was randomized so that the sub-sequent microscopic observations were carried out"blind."

When the affected patients were ultimatelyidentified, the hematology record was consultedfor details of the blood picture, sternal marrow,hemoglobin, serum vitamin Bi2, and serum folate.

The smears were viewed monocularly with amicroscope using the xlOO oil immersion objec-tive and an eyepiece graticule. Certain criteriawere adopted regarding the nuclei to be measured.In general, the largest, most regular nuclei in thefield were selected and those with overlapping orcontiguous borders and shrunken, irregular, orabnormal nuclei due to artifacts of preparationwere excluded.

One-hundred nuclei were selected on each slideand two measurements were made on each nucleusat 90° to each other. The measurements were madein arbitrary scale division to the nearest half-unit,so that the effect of magnification and eyepiecedistance can be discounted.

The results obtained were submitted for statisti-cal analysis.

Results. Data. The data available is in the formof measurement of areas (in arbitrary units) ofnuclear cross-sections.

A Mann-Whitney Test was performed with thenull hypothesis being that the control and affectedgroups were the same (as far as the nuclear areawas concerned) and (one-sided) alternative thatthe affected group had larger nuclear areas thanthe control group. The test statistic was significantat the 2.5 per cent level, indicating an upwardshift in the area of nuclei of affected individualsin comparison with the control group (Fig. 1).

Another more crude analysis was performedas follows: The averages were classified as high(H) or low (L) according to whether they were

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Reports 871

• Affected

o Control

20 30

OOO OO

40 50 6 0 70 8 0 9 0

Fig. 1. Distribution graph of area of affected and control nuclei showing upward shift in areaof nuclei of affected individuals in comparison with the control specimens.

greater or less than 46, respectively. This gave thefollowing 2 by 2 table (Table I) .

Under the hypothesis of no association betweenthe average (H/L) and the classification C/A(C = control, A = affected), the probability ofgetting a configuration like that above, or moreextreme, is about 0.08, so this hypothesis wouldbe rejected at about the 10 per cent level.

No correlation was found between nuclear sizeand the hemoglobin, serum vitamin B]^ or serumfolate.

Discussion. Extensive reviews of conjunctivalcytology2-4 do not mention any changes thoughtto be characteristic of the megaloblastic anemiasand we have been unable to find any referenceto such changes in a search of the literature. Thefailure of Boddington and Spriggs1 to find anychanges may have been due to their method ofdry fixation which inevitably leads to considerableshrinkage and distortion. Atypical conjunctivalcells have, however, been described in other con-ditions. Sekino5 described extensive deformation ofthe comeal epithelial cells with loss of glycogen,RNA, and DNA in rats fed on a folic acid-deficient diet.

Kimura and Thygesonfi demonstrated largemultinucleate epithelial cells which they regardedas pathognomonic of viral infection and, in addi-tion, large epithelial cells have been observed inBowen's disease.

Liotet and Iris,7 in a study of normal andpathologic conjunctival epithelium using mainlyPappenheim, Poleff, and Toluidine Blue stains,demonstrated some cells in which, although thenuclei were of normal size, the nuclear patternwas rather coarse, suggesting that the cells wereundergoing chromatolysis. In addition, they notedlarge cells with pyknotic nuclei and consideredboth these types to be separate stages in thenormal degeneration of conjunctival cells.

The nuclear chromatin in the former type andthe large cell size of the latter might be considereda source of confusion in differentiating normal cellsfrom those seen in megaloblastic anemia. In fact,however, the large nuclei seen in the conjunctivain megaloblastic anemia appear to stain less in-

Fig. 2. Nuclei of affected case showing increasein size and open lattice-like chromatin pattern.(Cresyl fast violet acetate, x2,500.)

Fig. 3. Nuclei of unaffected control. (Cresyl fastviolet acetate, x2,500.)

tensely than apparently normal ones and thechromatin pattern in these large nuclei is more"open and lattice-like" (Fig. 2). These changesappeared to be so striking to us that we felt theyalone might be indicative of an affected individualby comparison with a normal control specimen(Fig. 3).

In this study, superficial conjunctival scrapingshave demonstrated the expected changes in theconjunctiva of patients with megaloblastic anemia.

From the Department of Ophthalmology, Uni-versity of Texas Southwestern Medical School,

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872 Reports Investigative OphthalmologyNovember 1974

Dallas, and the Department of Ophthalmology,The Hallamshire Hospital, Sheffield, England.Submitted for publication March 25, 1974. Reprintrequests: Dr. J. D. Brodrick, University of TexasSouthwestern Medical School, 5323 Harry HinesBlvd., Dallas, Texas 75235.

Key words: conjunctival cytology, megaloblastic,smear, nuclear size, chromatin pattern.

R E F E R E N C E S1. Boddington, M. M., and Spriggs, A. I.: The

epithelial cells in megaloblastic anemias, J.Clin. Pathol. 12: 228, 1959.

2. Theodore, F. H.: In: Modern Trends in Oph-thalmology. London, 1955, Butterworth, p. 98.

3. Norn, S.: Cytologic examinations of conjunctivain practice using the quantitative pipette meth-od, Acta Ophthalmol. 40: 206, 1962.

4. Duke-Elder, S.: System of Ophthalmology.London, 1965, Kimpton, vol. 8, pt. I, p. 49.

5. Sekino, I.: Experimental studies on ocularmanifestations of folic acid deficiency, ActaSoc. Ophthalmol. Jap. 64: 2218, 1960.

6. Kimura, S. J., and Thygeson, P.: The cytologyof external ocular disease, Am. J. Ophthalmol.39: 137, 1955.

7. Liotet, S., and Iris, L.: Analytical study ofnormal and pathological conjunctival cytology,Arch. Ophthalmol. 23: 143, 1963.

Genesis of light-induced avian glaucoma.ADRIANNE KINNEAE, JEAN K. LAUBER, AND

T. A. S. BOYD.

Light-induced avian glaucoma is characterizedby eye enlargement, high intraocular pressure(IOP), low outflow facility (C), and reducedaqueous space volume. In this study we haveidentified several lesions occurring in the earlypathologic process, from five to 28 days of age.At seven days, comeal lactic dehydrogenase (LDH)is low and aqueous LDH is high. By 28 days,aqueous LDH is 3.5 times normal levels andcornea! LDH is reduced by 10 per cent. Bynine days, aqueous space volume is reduced, andeye enlargement is evident by three weeks. IOPand C remain normal during this period, althoughC is later impaired (at six weeks) and homeostaticcontrol of IOP breaks down at approximately16 weeks. Though the primary cause(s) for thedimensional changes in cornea and vitreous bodyhave not been identified, the difference in the timeof their appearance indicates that these two lesionsmay be independent of one another. The comealLDH change suggests early alteration in endo-thelial permeability, allowing excessive enzymeloss to the aqueous humor.

Domestic chicks reared under continuous light(24L/0D) develop severe morphologic and physi-

ologic ocular lesions which culminate in glaucomaand blindness. We have called the condition light-induced avian glaucoma. Although human open-angle glaucoma is a serious clinical problem, fewexperimental animals develop such a disease andin no others, to our knowledge, can the conditionbe precipitated at the will of the investigator.

Light-induced avian glaucoma is associated withincreased eye weight and diameter, reduced cor-neal curvature, impaired outflow facility, andelevated intraocular (IOP) pressure.1'- Eye en-largement and refractive error are detectableseveral weeks before outflow facility is impaired,and IOP is elevated still later in the disease pro-cess.8 Although the iridocorneal angle is narrow,iridectomy of chicks, subsequently reared under24L/0D, failed to alter the course of the de-veloping glaucoma, as might be expected ifpupillary block had contributed to iris bombe.1

A systemic rather than a local etiology was sug-gested by the finding that eyes of 24L/0D birds,when covered by an opaque vision occluder,showed no less enlargement than those not socovered.5

However, these positive findings have providedfew clues about the etiology of light-inducedavian glaucoma. Improvements in our techniqueshave now made it possible to monitor aqueousfluid dynamics in chicks as young as seven daysafter hatching: we here detail these early pre-glaucomatous changes. We have also measuredlactic dehydrogenase levels during the same period.This enzyme is thought to be essential for normalcorneal metabolism0 and its relative activity incornea and aqueous has been used as an indica-tion of the viability of the corneal endothe-lium.7

Materials and methods. White Rock (broilertype) chicks were reared from hatching in eithercontinuous incandescent light (24L/0D) or adiurnal photoperiod of 14 hours of light per day(14L/10D). Birds were maintained in tempera-ture-controlled environment chambers and weresupplied with food (commercial chick startercrumbles) and water ad libitum. Brooder heatwithout light was supplied by heating the inflowair of the positive-pressure ventilation system.

Eye weight, corneal height, corneal diameter,and aqueous space volume were determined aspreviously described.2. s To separate the effect ofbody growth from eye enlargement, a numberof chick pairs were selected, one bird from eachlighting treatment, but having equivalent bodyweights. Eye weights of these birds were com-pared by paired t-test. In evaluation of all otherdata, Student's t-test was applied.

Under Combuthal anesthesia,9 IOP was moni-tored by closed manometry and aqueous outflowfacility (C) was estimated after constant-rateperfusion, as previously described.3

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