Experimental Hyphema in Rabbits*

EYE PAIN AND HOMONYMOUS r o w i n g of the posterior cerebral arteries. E i g h t e e n months later the patient w a s still blind and d e mented. DISCUSSION

The clinical histories of these five patients and that of Förster show that ocular pain can be significantly associated with a vascular lesion of the parieto-occipital area of the brain. In one of our patients this site was proven by the finding of an old infarct of the striate occipital cortex. The mechanism of this referred pain may be attributed to the sensory innervation of the dura mater. Recurrent branches (nervi tentorii) leave the ophthalmic division of the trigeminal nerve in the cavernous sinus and spread over the ipsilateral tentorium cerebelli, the posterior third of the falx cerebri and the outer surface of the parietal-occipital region of the cerebral hemisphere. Infarcted tissue in the parietal-occipital area could produce painful stimuli which would be referred to the eye. 2

HEMIANOPIA

1093

opic scotomas; the eye pain being on the side of the headache. H e also describes eye pain as a manifestation of tumors in occipital areas and that patients experience eye pain at the time of needle puncture when ventriculography is performed through the occipital lobe. SUMMARY

Pain in the eye may be a symptom of a vascular accident in the parieto-occipital area of the brain and is associated with homonymous hemianopia. In five of our patients and in one patient described in the literature, the pain was localized on the side of the lesion, was not severe and was noticed only after the visual field defect had become apparent. Three of these patients had an associated defect of opticokinetic nystagmus. This referred pain is presumed to be mediated through the dural branches of the ophthalmic division of the trigeminal nerve.

Walsh* states that ocular pain may accompany ophthalmic migraine with hemian-

243 Charles Street

(14).

REFERENCES

1. F ö r s t e r : U e b e r Rindenblindheit. A r c h . f. Ophth., 3 6 : 9 4 , 1890 . 2. Brash, J. C , and Jamieson, E . B . ( e d . ) : Cunningham's T e x t b o o k of A n a t o m y . London, O x f o r d , 1951, p. 999. 3. W a l s h , F . B . : Clinical N e u r o - O p h t h a l m o l o g y . Baltimore, W i l l i a m s & W i l k i n s , 1956, ed. 2, pp. 1044; 1137.

EXPERIMENTAL H Y P H E M A IN RABBITS* V.

THE

PRODUCTION OF A HEMOLYTIC AGENT A N D ITS EFFECT ON

ROBERT

M.

THE

SINSKEY,

RATE

M.D.

Los

OF

AND

Angeles,

It was noted in the hyphema studies reported in 1957 that hemolyzed red blood cells leave the anterior chamber very rapidly. This report concerns the investiga-

ABSORPTION ALICE

R.

KRICHESKY,

A.B.

California

tion of hemolytic agents and their effectiveness in lysing red blood cells in the anterior chamber of the rabbit eye.

4

METHODS AND

* F r o m the D i v i s i o n of Ophthalmology, D e p a r t m e n t of Surgery, U n i v e r s i t y o f California Medical Center. T h i s investigation w a s supported by grantin-aid B-929 f r o m the N a t i o n a l Institute of N e u r o logical D i s e a s e s and Blindness, U n i t e d S t a t e s P u b lic H e a l t h Service, Bethesda, Maryland.

CHEMICAL LYTIC

AND

MATERIALS

PHYSIOCHEMICAL

HEMO-

AGENTS

The use of chemical and physiochemical hemolytic agents to produce hemolysis of

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R O B E R T M. S I N S K E Y A N D A L I C E R.

rabbit red blood cells was investigated. In vitro studies were carried out to determine the lowest concentration of these agents which would hemolyze the red blood cells. Benzalkonium chloride (Zephiran, Winthrop-Stearns), a cationic detergent, was used in a 1:2,000 dilution. Five-tenths of a cc. of the diluted Zephiran was combined with 0.5 cc. of rabbit blood. Approximately 33-percent lysis of the cells in 60 minutes was observed and up to 50-percent lysis occurred over a period of 40 hours. Mephesen (Tolserol, Squibb), a synthetic chemical compound, was combined with 0.5 cc. of rabbit blood in the following amounts : 0.2 c c , 0.4 c c , 0.6 c c , 1.0 cc. and 1.5 cc. After one hour approximately 25-percent lysis of the cells occurred in the specimens containing 0.4 cc. through 1.5 cc. of Tolserol. After a three-hour period approximately 50-percent lysis was observed in these same specimens'. Crude cobra venom (Hynson, et al.), an agent which produces hemolysis by partially hydrolyzing the lecithin of red blood cells to lyso lecithin, was used as follows: 1:10 through 1:10,000 dilutions of the venom in 0.5-cc. amounts were combined with 0.5 cc. of rabbit blood. N o lysis occurred in any concentration. The lowest concentrations of Zephiran and Tolserol which hemolyzed the rabbit red blood cells in vitro, were injected into the anterior chamber of the rabbit eye. A toxic reaction of the eye tissues was observed within eight hours, increasing in intensity over a period of 30 hours. This toxicity precluded further use of these agents in the studies of hyphema absorption. HEMOLYTIC ANTIBODY

AGENTS

PRODUCED BY

ANTIGEN-

REACTION

Sprague-Dawley rats were used as the host animal in all the studies which were successful in producing a hemolytic antiserum (hemolysin) to rabbit whole blood. The first studies produced a hemolysin of a low titer. The reaction between the low titer antiserum and the eye tissues was de-

KRICHESKY

termined by injection of 0.05 cc. of the hemolysin into the anterior chamber of the rabbit's eye. A severe inflammatory reaction of the eye tissues resulted. Modification of the technique used in the first studies effectively resulted in the consistent production of a high titer hemolysin. The technique for production of this hemolysin was as follows: under ether anesthesia, 0.5 cc. of a 30-percent saline suspension of rabbit whole blood was injected into the saphenous vein of a 300-gm. SpragueDawley rat. Three weeks after the initial injection the rat received 0.5 cc. of a 5.0percent saline suspension of rabbit whole blood intravenously. Five days after the second injection the rat was exsanguinated by cardiac puncture. 3

The rat blood remained at room temperature for one hour and in the refrigerator at a temperature of 4°C. to 5°C. overnight. The following morning the serum was decanted from the clot and immersed in a 56° C. water bath for 30 minutes to inactivate any natural complement that may have been present. The hemolytic titer of the serum was determined by means of a doubling dilution titration. The first dilution contained 0.4 cc. of serum and 0.4 cc. of saline ( 1 : 2 dilution). Each consecutive dilution was doubled until a 1:16,384 dilution was reached. Each dilution received 0.2 cc. of a 2.0-percent saline suspension of rabbit whole blood and 0.2 cc. of complement (Lyo complement, Sharpe and Dohme). Lyo complement is a lyophilized guinea pig complement. The complement was reconstituted with 5.0 cc. of stabilizing solution per 7.0 cc. vial and diluted to a 1:30 solution with sterile saline. The titration was incubated at 37°C. for two hours. The degree of hemolysis was recorded from 0 (no hemolysis) to 4 plus ( + + ' + + ) (complete hemolysis). \ The degree of hemolysis (titer) of the hemolysin produced by this technique, in a series of four experiments on a total of 12 rats, was an average of 1 plus ( + ) hemolysis in a 1:8,192 dilution of the antiserum. Mathe1

EXPERIMENTAL HYPHEMA

matical computation of the titration established that 0.02 cc. of a 1:20 and a 1:10 dilution of the whole antiserum plus 0.01 cc. of complement were necessary to produce 2 plus ( + + ) and 4 plus ( + + + + ) hemolysis, respectively, of the experimental hyphema. N o observable damage to the eye tissues was produced subsequent to the injection of 0.05 cc. of a 1:10 dilution of this high titer antiserum into the anterior chamber of the rabbit eye. T o evaluate the effect of hemolysin on the absorption of experimental hyphema, standard animals and techniques previously established were used for labeling of red blood cells, injection into the anterior chamber and the counting of peripheral blood specimens.* T w o series of acute experiments were conducted to determine the effect of hemolysin on hyphema absorption. In the first series, hemolysin alone plus complement was used in two dilutions as follows: ( a ) hemolysin in a 1:10 dilution; ( b ) hemolysin in a 1:20 dilution. In the second series, a 1:10 dilution of hemolysin plus complement was combined with streptokinase and streptodornase (Varidase). The animals were killed at the end of each experiment and disposed of according to radiation safety specifications. Series 1: (a) Hemolysin in a 1:10 dilution; (b) hemolysin in a 1:20 dilution The

hemolysin

(hemolytic

antiserum)

* Adult albino rabbits w e r e used throughout the study. O n l y one e y e o f each rabbit w a s subjected t o experimental study. Rabbit w h o l e blood in A C D solution ( a c i d citrate d e x t r o s e ) w a s incubated at 8 0 ° F . ( p l u s or m i n u s 2 ° F . ) f o r 90-plus minutes w i t h 250 microcuries of C r ^ per 3.0 cc. of w h o l e blood. A f t e r w a s h i n g at least three times w i t h saline, the uptake o f the Cr** by the cells w a s "fixed" by the addition of 6.0 ( p l u s o r m i n u s ) m g . of ascorbic acid per 3.5 cc. of w h o l e blood. T a g g e d cells, 0.05 c c , w e r e injected into the a n terior chamber of the rabbit's eye after the removal of 0.05 cc. of aqueous humor, u s i n g a 2 7 - g a u g e , one-inch needle. Peripheral blood specimens w e r e obtained by bleeding f r o m a n ear v e i n at specified time intervals after injection o f the t a g g e d cells into the anterior chamber. A l l blood specimens w e r e counted in a deep-well scintillation counter.

IN

RABBITS

1095

was reduced to the specified dilution (a or b ) . One hour after the injection of the Cr -tagged cells into the anterior chamber of the rabbit's eye, 0.02 cc. of the diluted hemolysin and 0.01 cc. of a 1:30 dilution of Lyo complement were injected into the anterior chamber. The control animals received 0.02 cc. of saline plus 0.01 cc. of complement. Duration of experiment. Experimental observations were made over a period of 48 hours. Specimens for counting were obtained 3, 5, 7, 24 and 48 hours after induction of the hyphema. 51

Series 2: Hemolysin, 1:10 dilution, plus complement and streptokinase and streptodornase (Varidase) 156 units One hour after the induction of the hyphema in the rabbit's eye, 0.02 cc. of hemolysin, diluted 1:10, plus 0.01 cc. of Lyo complement, diluted 1:30 were injected into the anterior chamber. This injection was followed immediately by an injection of 156 units of Varidase in 0.05 cc. of saline solution. The control animals received 0.02 cc. of saline plus 0.01 cc. of complement followed immediately by an additional 0.05 cc. of saline. Duration of experiment. Experimental observations were made over a period of 48 hours. Specimens for counting were obtained 3, 5, 7, 24 and 48 hours after the hyphema induction. STANDARD

A N D PERCENT ABSORPTION

The counts per minute per 1.0 cc. of the tagged-blood injected into the anterior chamber were obtained for each experiment and known as the standard. The counts per minute for each peripheral blood specimen were reduced to a percent of its standard and designated as the percent absorption of the hyphema at the indicated time intervals. The blood dilution factor was considered to be a constant since it fell within acceptable statistical limits of variability. The percent absorption figures for all animals in each series at each time interval were subjected

1096

R O B E R T M. S I N S K E Y A N D

A L I C E R.

KRICHESKY

F i g . 1 ( S i n s k e y and K r i c h e s k y ) . T h e difference in rate of absorption between hemolysin-treated ( 1 : 1 0 dilution) animals and control animals.

0 To 7 Hrs. Hem.= 12% Faster

7 To 24 Hrs Hem. = 9% Foster

24 To 48 Hrs. Hem = 30% Faster

to the rank sum and t-test. The percent difference of absorption between the treated animals and the control animals were calculated by the Unit of Biostatistics, Department of Preventive Medicine, University of California Los Angeles Medical Center under the supervision of Wilfrid J. Dixon, Ph.D. Block graphs showing the difference in rate of absorption between the treated animals and the control animals for each time interval and the average of these for the total period of the experiment were made from this statistical analysis. RESULTS SERIES 1

a. Hemolysin in a 1:10 dilution was used in a series of 11 animals and compared with 11 control animals. Seven hours after the hyphema induction the rate of absorption of the red blood cells was 12-percent faster in the treated animals than in the control animals ; from seven to 24 hours the absorption rate was nine-percent faster in the treated animals than in the control animals; from 24 to 48 hours the absorption rate was 30percent faster in the treated animals than in the control animals. The average rate of absorption over the 48-hour period was 16percent faster in the treated animals than in the control animals (fig. 1 ) . b. Hemolysin in a 1:20 dilution was used in a series of 12 animals and compared with 11 control animals. Seven hours after the hyphema induction the rate of absorption of the hyphema was 36-percent slower in the treated animals than in the control animals ;

48 Hr. Average Hem.= 16% Faster

from seven to 24 hours the rate of absorption was 42-percent slower in the treated animals than in the control animals ; from 24 to 48 hours the absorption rate was 30percent slower in the treated animals than in the control animals ; and the average rate of absorption over the 48-hour period was 36-percent slower in the treated animals than in the control animals. SERIES

2

Hemolysin in a 1:10 dilution and 156 units of streptokinase and streptodornase (Varidase) were used in a series of 17 animals and compared with 16 control animals. Statistically there was no significant difference in the rate of absorption of the treated animals and the control animals. DISCUSSION

Chemical and physiochemical hemolytic agents cannot be used in studies of hyphema, even in low concentrations, due to their toxicity to the eye tissues. Ideally, a hemolysin produced by antigenantibody reaction is a more physiologic approach to the destruction of red blood cells in the anterior chamber of the rabbit eye. Such an agent was produced, initially, in a low titer. When the low titer antiserum proved to be toxic to the eye tissues upon injection into the anterior chamber of the rabbit eye, the production technique was modified. By means of the modified technique consistently high titer hemolysin was produced. The high titer antiserum, in a

EXPERIMENTAL HYPHEMA IN

diluted form, proved to be relatively nontoxic to the eye tissues of the rabbit when injected into the anterior chamber. High titer hemolysin, in critical dilution, did increase the absorption of hyphema. Although the results were promising, it was possible to carry out only limited studies at this time. It would be desirable to increase the titer of hemolysin by further modification of techniques and to determine minimum, maximum and optimum concentrations of both hemolysin and complement necessary to increase hyphema absorption in the rabbit. SUMMARY

1. Chemical and physiochemical hemolytic agents, such as Zephiran and cobra venom, were investigated but not used in studies

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1097

of hyphema due to their toxic effects on the eye tissues. 2. A high titer hemolysin to rabbit red blood cells was produced in the rat. 3. Dilutions of high titer hemolysin injected in the anterior chamber of the rabbit eye were nontoxic. 4. Intraocular injection of hemolysin in rabbits, in critical dilution, increased the absorption rate of hyphema in a small controlled experiment. Department of Surgery (24). ACKNOWLEDGMENTS

W e w i s h to e x p r e s s appreciation t o D r . Bradley R. S t r a a t s m a f o r support and advice during the course o f this w o r k ; to D r . W i l f r i d J. D i x o n , biostatistician, for his supervision of the statistical analysis of experimental d a t a ; and to M r . Robert J. H e n d r i c k s o n and M r . J a m e s Craig f o r assistance in the laboratory.

REFERENCES

1. Helper, Ο. E . : Manual o f Clinical Laboratory M e t h o d s . Springfield, 111, T h o m a s , 1949, pp. 6-8. 2. Ponder, E . : H e m o l y s i s and related phenomena. N e w Y o r k , Grune & Stratton, 1948, pp. 2 5 8 - 2 8 8 ; 324-335. 3. Roberts, S , A d a m s , E , and W h i t e , Α . : Influence of m o d e of immunization o n the relationship b e t w e e n the development of tissue titers and the release o f h e m o l y s i n in vitro. J. I m m u n o l , 6 2 : 1 5 5 , 1949. 4. Sinskey, R. M . : E x p e r i m e n t a l hyphema in rabbits. A m . J. O p h t h , 4 3 : 2 9 2 , 1957. 5. Sinskey, R. M . a n d K r i c h e s k y A . R.: E x p e r i m e n t a l hyphema in rabbits: I I . T h e effect o f acetazolamide ( D i a m o x ) o n the rate of absorption. A m . J. O p h t h , 5 0 : 7 4 3 , I960.

U S E

OF

PREPARED

FASCIA

LATA

ELTON

IN

YASUNA,

Worcester,

The excellent symposium on blepharoptosis presented in 1958 was introduced by Fralick with this statement: "Ptosis surgery is infrequently undertaken by the average ophthalmologist. The operations for ptosis notoriously often leave much to be desired." It would be useful to keep these thoughts in mind as this paper develops. Approximately 100 methods have been described for correcting ptosis. All these procedures can be classified basically into three general groups: ( 1 ) shorten the leva1

2

8

* F r o m the T u f t s Medical School, Boston, M a s s achusetts, and the Ophthalmological Service, W o r cester City H o s p i t a l

CORRECTION

OF

PTOSIS*

M.D.

Massachusetts

tor muscle, ( 2 ) attach the levator to the superior rectus and ( 3 ) suspend the lid from the brow. It is generally accepted that, when levator function is present in reasonable degree, the operation of choice is resection of the levator. This procedure with its variations has been well presented and discussed in the literature. It is not the final answer in many instances and, as McDonald* succinctly states (on shortening the levator) "the endresult is frequently disappointing to both the surgeon and the patient." The surgical principle of attaching the levator to the superior rectus has its advo-