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Experimental Glaucoma in the Rabbit*
GENETICS AND PRIMARY OPEN-ANGLE GLAUCOMA
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Michels, V. : L'hérédité dans le glaucome pri Strehlar, E. : Zwei Stammbäume von Glaucoma maire. Thèse, Lausanne, 1957. simplex. Thèse Zurich 1938. Miller, S. J. H., and Paterson, G. D. : Studies Sveinsson, K. : Glaucoma and heredity in Ice on glaucoma relatives. Brit J. Ophth., 46:513, land. Acta Ophth., 37 :191, 1959. 1962. Tiscornia, B. : Erblichkeit und Blutsverwandt Olivier, W. : Familiäres Glaukom über zehn schaft in der Augenheilkunde. Zentralbl. Ges. Generationen. Deutsche Ophth. Ges. Heidelberg, Ophth., 38:339, 1937. 65:547, 1963. Törnquist, R. : Genetics of senile glaucoma. Acta Penzani, B. : Conbributo allô studio dell'eredità nel Ophth., 32 :513. 1954. glaucoma giovanile tardivo. Giorn. Ital. OftaL, 8: Waardenburg, P. J. : Ueber das Familiäre Vor 53, 1955. kommen und den Erbgang des praesenilen und Pimentel, P. C. : Consanguinity and morbid he senilen Glaukoms. Genetica, The Hague, 25:79, redity. Ophthalmos, 2:329, 1941. 1950. Politzer, M. : The gonioscopic picture of he Waardenburg, P. J., Franceschetti, A., and reditary glaucoma. (In Czech.) Csl. oftal., 18:446, Klein, D. : Genetics and Ophthalmology (Glau 1962. coma). Assen, Von Gorcum. 1961, v. 1, pp. 601Posner, A., and Sohlossman, A. : Role of in 626. heritance in glaucoma. Arch. Ophth., 41:125, 1949. Weekers, R., Gougnard-Rion, C, and Gougnard, Probert, L. A. : A survey of hereditary glau L. : Considérations cliniques sur l'hérédité des coma. Canad. M. A. J.. 66:563, 1952 glaucomes. Bull. Soc. Belge Opht, 110:255, 1955. Rossi, V. : Glaucoma primario e constituzione. Westerlund, E. : Clinical and genetic studies on Arch. Ottal., 39:1, 1932. the primary glaucoma diseases. Vol. XII, opera ex Sedan, J., and Sédan-Bauby, S. : Dépistage d'une Domo biologiae Heredit. Hum. Univ. Hafmensis. "glaucomatose" latente chez deux jeunes gens Copenhague, Munksgaard, 1947. ayant une hérédité de trois et quatre générations Woerner, E. : Primäres Glaukom und Konstitu pour cette affection. Bull. Soc. Ophtal. France, tion. Thèse Tübingen, 1936. 1949, pp. 29-36. Wolf söhn- Jaffe, E.: Ueber 9 Fälle von hereditä Stankovic, L. : Ein Beitrag zur Kenntnis der rem Glaukom der Erwachsenen in 3 Generationen. Vererbung des Pigmentglaukoms. Klin. Mbl. Klin. Mbl. Augenh, 94:622, 1935. Augenh., 139:165, 1961. Zorab, A. : Glaucoma simplex familialis. Tr. Stokes, W. H. : Hereditary primary glaucoma : Ophth. Soc. U. Kingdom, 52:446, 1932. A pedigree with five generations. Arch. Ophth., 24:885, 1940.
E X P E R I M E N T A L GLAUCOMA IN T H E RABBIT* MAURICE H. LUNTZ, F.R.C.S. Johannesburg, South Africa Our knowledge and understanding of primary simple glaucoma in man has in re cent years been enriched by a vast amount of research undertaken on a world-wide basis. Nevertheless, there are still many un solved problems, especially as regards etiolo gy and natural history. Progress has been hampered because the disease cannot be sat isfactorily reproduced in animals for exper imental study. Sir George Pickering (1955) wrote : "A time-honoured method of investi gating disease is to try and reproduce it in animals. This method has proved of the ut* From the Department of Ophthalmology, University of the Witwatersrand Medical School.
most value and without it the advances that have taken place all over the front of medi cine would not have occurred." This state ment will no doubt prove as true of glauco ma as of other diseases. One essential in any attempt to reproduce disease experimentally is that it should re semble the malady as it occurs in man. Pre vious workers, attempting to reproduce glau coma and bearing this in mind, have adapt ed the current theories for the etiology of primary simple glaucoma in man to their techniques for reproducing it in the experi mental animal. Some managed to produce raised intraocular pressures over a period as long as six months, but always at the price
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of gross structural and functional damage to the eye. However, affected eyes in man characteristically appear to be clinically and histologically normal apart from the effects of prolonged ocular hypertension, for exam ple, cupping of the optic disc. What is re quired therefore is a technique which pro duces ocular hypertension in an experimen tal eye that is maintained over some months or longer but which does not cause any ap parent structural or functional damage to the eye. Such damage may occur later secon dary to the ocular hypertension and would be permissible and indeed expected. It is the purpose of this paper to describe such a technique and to discuss the results ob tained.
where it was absorbed into the general cir culation. Aqueous drainage from the anterior chamber is considered to take place predom inantly at the angle of the anterior chamber of the eye via Schlemm's canal. The aque ous gains access to Schlemm's canal by per colating through the trabecular tissue and leaves Schlemm's canal mainly through col lector channels reaching the episcleral ve nous plexus and the venous circulation by way of the deep scierai and intrascleral plexuses. A limited proportion of this drain age from Schlemm's canal reaches the epis cleral venous plexus directly via aqueous veins which bypass the deep scierai and in trascleral plexuses.
The cause of primary simple glaucoma in man is now widely accepted as being an in creased resistance to the outflow of aqueous humor at the anterior chamber angle. This concept has assumed pride of place since Leber (1873) described the angle of the an terior chamber and postulated that aqueous drainage took place mainly at this site. This was confirmed by Priestley Smith (1891) in a brilliant series of perfusion experiments on enucleated eyes. He went further and stressed that obstruction at the angle of the anterior chamber was primarily responsible for the raised ocular tension. Gonioscopy has shown that acute closed-angle glaucoma oc curs when the iris obstructs the entry of aqueous into the angle.
Considering the anatomy of the anterior chamber angle in the light of present-day knowledge, it is evident that obstruction to the aqueous outflow in primary simple glau coma in man could occur at one of three sites :
Investigations have made it clear that the aqueous humor from Schlemm's canal drained into the episcleral venous plexus by a series of intercommunicating plexuses in the sciera and episclera, eventually reaching the superficial vessels of the episclera (Ashton 1951, 1952). Approximately 30 external collector channels, irregularly distributed and varying in size and shape, conveyed aqueous from Schlemm's canal to the deep scierai plexus which in turn anastomosed with the intrascleral plexus, the aqueous finally reaching the episcleral venous plexus
a. The trabecular tissue, immediately proximal to Schlemm's canal. b. At Schlemm's canal (rare). c. The outflow channels from Schlemm's canal of which the epsicleral venous plexus forms the ultimate stage. The purpose of this paper is to describe a technique for producing chronic simple glaucoma in rabbits by injecting phenol into the episcleral tissue. PRESENT EXPERIMENTS MATERIALS
Twelve gray female rabbits were used be cause they are more docile and easier to handle than males. Six were bred from the stock at the Cape Town University Medical School, which are not a pure breed and are closest to pure-bred chinchillas. A second series of six were chinchilla rabbits obtained at Oxford University. Rabbits are docile animals, have relatively large eyes and cor-
EXPERIMENTAL GLAUCOMA IN THE RABBIT neas and submit uncomplainingly to tonometry. Histologically the rabbit anterior cham ber angle has structural differences from those of man. These were well described by Davis (1929). Essentially these differences occur in the meshwork which is better developed in the rabbit. The collagenous fibers are much finer and not enclosed by elastic fibers, as in man. They run as isolated fibers. Schlemm's canal is not a single channel, as in man, but rather a series of channels forming a plex us. In spite of these anatomic differences Ruskell (1961) showed that the rabbit has a close functional similarity to man, which justifies its use as an experimental animal. Using the Neoprene cast technique, as de scribed by Ashton (1951), he showed that in rabbits the major outflow of aqueous is to the episcleral vessels, as in man. Ruskell's results also supported the idea of a secondary route for aqueous otflow as ad vocated by Nemetz (1956) from the filtra tion angle through the intrascleral plexus to the choroid. These connections had also been demonstrated in man by Ashton (1955). There is considerable disagreement about the extent to which this route could be used in rabbits under physiologic and patho logic conditions. The fact that prolonged ocular hypertension can be produced by blocking the episcleral outflow strongly im plies this route is of minor importance as an alternative outflow channel in the breed of rabbit used in these experiments. It is gen erally agreed that this is also the case in man. The rabbit, then, has an aqueous drainage system which is similar to that in man, apart from some minor anatomic dif ferences. Monkeys have an angle very similar to that in man but, unfortunately, it is impossible to do tonometries on them with local anesthetics. Another argument in favor of using rab bits was that normal rabbits produce fairly constant intraocular pressure readings with
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repeated indentation tonometries, using local anesthetics. The average tension in 28 rabbit eyes was found by Becker and Constant (1956) to be 18 mm Hg, with a standard deviation of ±3.7. They used a Mueller electronic tonometer and took their measure ments in vivo. This was repeated while doing tonographies in vitro and found to be 19 mm Hg, with a standard deviation of ±4.8, a remarkably close agreement. Kornblueth and Linnér (1955) and Becker and Constant (1956) showed that tonography could be applied to the rabbit eye with valid results. The latter did a series on 28 rabbit eyes in vivo and found an average facility of outflow C of 0.33 ( standard devia tion ±0.08). These eyes were then perfused with isotonic saline in vivo and an average C value of 0.34 was obtained (standard de viation ±0.10). When perfused in vitro, the average C value was 0.37 (standard devia tion ±0.11). Considering the experimental error inherent in tonography, the C values obtained by all methods are surprisingly similar. SCLEROSING MATERIALS
The rabbits were given subconjunctival injections of a sclerosing fluid, using either a 0.175% solution of silver nitrate, a 1% so lution of ethanolamide (Ethanolamine Oleate) or a 5% solution of phenol in al mond oil. The latter two preparations are standard sclerosing agents used in the treat ment of varicosities (for example, in the rectum). Of these only the phenol solution proved to be satisfactory, causing a rise of intraocular pressure but no apparent macro scopic or microscopic damage to the eye. The silver nitrate was prepared by the hospital dispensary (Groote Schuur Hos pital). The ethanolamide and phenol came as commercial preparations in 5-cc am pules; the former prepared by May and Baker (S.A.) Pty. Limited, Port Elizabeth; the latter by Glaxo-Allenbury (S.A.) Pty. Limited, Durban.
MAURICE H. LUNTZ
668 D E S C R I P T I O N OF E X P E R I M E N T A L
TECHNIQUE a. TONOMETRY
The animal was tied in a duster so that only its head and neck protruded. Both eyes were anesthetized using two drops topically of a 1% solution of tetracaine in aqueous. The technician then placed the animal on his knee and held its head with the right cornea facing vertically upward. The tonometer (a standardized Schip'tz tonometer) was cleaned with spirits and checked on a hard convex surface, supplied with the instru ment : the pointer should be deflected to the zero position on the calibrated scale. If cor rect, the instrument was placed vertically on the cornea, the operator carefully holding the eyelids apart with one hand, being care ful not to press on the globe, and the read ing on the calibrated scale noted. This was recorded in mm Hg by consulting Fri eden wald's nomograms (1957). Two read ings were made, one using a 5.5-gm weight Ui RABBIT NO. 3 RIGHT EYE
and another using a 7.5-gm weight. Occasionally, the nictitating membrane gave trouble by covering the cornea, over come by patiently waiting for it to retract and then proceeding. Having measured the intraocular pressure of the right eye, the same procedure was re peated on the left eye. The intraocular pressure was recorded in each eye of every rabbit on at least three oc casions before the first subconjunctival in jection was given and at weekly intervals thereafter until the experiment was termi nated. Where subsequent tonometries coin cided with a subconjunctival injection, the tonometry was done first. Intraocular pres sures in the first series are followed graphi cally in Figures 1 to 5 and the initial sub conjunctival injection is indicated. Figures 6 to 10 are graphs of the intraocular pressures in the second series. These also indicate all the subconjunctival injections and also the intraocular pressures before the initial in jection. $
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EXPERIMENTAL GLAUCOMA IN T H E RABBIT SUBCONJUNCTIVAL INJECTION PHENOL 5% IN ALMOND OIL 4 QUADRANTS
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RABBIT NO. 4 METHOD:- SUBCONJUNCTIVAL INJECTION PHENOL S% IN ALMOND OIL 17 MARCH 1961
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Fig. 3 (Luntz). The intraocular pressure in both eyes of Rabbit 4 is plotted against time in weeks. The initial subconjunctival injection given in the right eye is indicated. This eye did not become glaucomatous. The third of three pressure readings made before the initial subconjunctival injection is plotted as the first reading.
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Fig. S (Luntz). Intraocular pressure of Rabbit 6 plotted against time in weeks. The initial subcon junctival injection in the left eye is indicated. The right eye is the control. The first plotted reading is the third of three measurements of intraocular pressure made before giving the initial subconjunctival injection.
EXPERIMENTAL GLAUCOMA IN THE RABBIT b. T H E SUBCONJUNCTIVAL INJECTION
The method of injection was the ap proved technique for a subconjunctival in jection, the aim being to deliver the sclerosing fluid into the episcleral tissues. Two min ims of sclerosing fluid were drawn up into a standard 2-cc syringe fitted with a No. 1 needle. The needle was then removed and a No. 18 needle fitted to the syringe. The animal was immobilized and anesthe tized in the same way as described for tonometry and the initial intraocular pressure measurement was made. A conjunctival spe culum was now used to hold the lids apart, the operator fixated the conjunctiva in the desired quadrant with a pair of Lister's for ceps and the No. 18 needle was pushed through the conjunctiva into the episcleral tissue. One minim of the sclerosing fluid was injected, the needle was withdrawn and the speculum removed. If more than one quadrant was to be injected, the procedure was repeated in each quadrant at the same sitting. There was no after-treatment. With only one exception (Rabbit 3) one eye in each rabbit was given a subconjunctival in jection of sclerosing fluid (the "treated" eye) while the other eye was left as a con trol (the "control" eye). Hence treated and control eyes could be compared in the same animal. Work on the first series of rabbits was done in the Department of Surgery, Univer sity of Cape Town. These were numbered 1 to 6. The first two animals were given sub conjunctival injections of the 0.175% solu tion of silver nitrate. This resulted in a se vere local reaction, the method was aban doned and the rabbits withdrawn from the experiment. In the remaining rabbits in this series one eye was injected subconjunctivally with the 5% phenol in almond oil. At first one quad rant of the globe, then two and then three were injected; this failed to cause a rise in intraocular pressure. Finally, all four quad rants were injected, using four minims of
671
fluid. Positive results were obtained at this stage. In Rabbit 3, both eyes were injected sub conjunctivally with 5% phenol in almond oil (four quadrants) : a similar injection but of almond oil was given in the control eye of Rabbit 4. The control eyes of the remaining rabbits in this series were left untreated. These injections were repeated at regular intervals at two and again at four weeks after the initial injection. c. TREATMENT OF GLAUCOMATOUS EYES IN THE FIRST SERIES
This part of the experiment was termi nated after some weeks by killing the rab bits and removing their eyes for histologie study. Before doing this, an iris-inclusion operation was done in some eyes; in other rabbits pilocarpine nitrate drops in l%-4% solution was given together with acetazolamide (Diamox) 125 mg daily by mouth in tablet form. These are established methods of treating hypertension in man and the effect on the intraocular pressure in these glaucomatous rabbit eyes was recorded. Al though the purpose of this paper is to de scribe a method of reproducing chronic glaucoma in rabbits, the results of treating their glaucomatous eyes offers an interesting digression and has useful research possibili ties. The results of treatment and a discus sion of its possible research application are presented later. The iris inclusion was performed by an ab externo technique. The animal was an esthetized by the open-mask ether method and an attempt was made to control the depth of anesthesia at a level just deep enough for the operative procedure. The an esthetized animal was placed on its side with the eye to be operated on uppermost. The operator scrubbed and donned sterile gown, cap, mask and gloves. The rabbit was dressed in sterile towels so that only the eye to be operated on was exposed. A conjuncti val speculum was inserted and a few drops of 1% tetracaine were instilled topically.
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MAURICE H. LUNTZ
A limbal-based conjunctival flap was fashioned and retracted over the cornea. A scierai incision was made with a Bard-Park er knife and No. 11 blade into the anterior chamber from the limbus at the 12-o'clock position. The iris prolapsed through this in cision and was pulled out until the pupillary border became visible. A vertical incision was then made with de Wecker's scissors from the pupillary edge to the root of the iris. The iris was torn from its root for a short distance on either side of this incision, fashioning two iris pillars. These were left passing through the scierai incision at each edge and the conjunctiva was sutured in place over them with 6-0 catgut sutures. A few drops of 1% chloramphenicol were instilled into the conjunctival sac and an eyepad applied which was removed immediate ly the animal regained consciousness. Within a few days the conjunctiva had healed and a bleb of aqueous had formed under it. The second series of six rabbits was treated by a similar experimental technique at the Nuffield Laboratory of Ophthalmol
10
ogy, University of Oxford. In five of these the subconjunctival injection contained phe nol in almond oil as before. In one, four min ims of the ethanolamide solution was in jected. This resulted in a violent reaction, with sloughing of the sciera. The rabbit was discarded, leaving five. These five were numbered 2(a) to 6 ( a ) . The subconjuncti val injections were repeated but at varying intervals (figs. 6 to 10). The intraocular pressure was measured by tonometry. The rabbits were not given treatment as in the first series, but were subjected to inflow and outflow studies. Outflow studies were done by a four-minute tonography ; inflow studies by the fluorescein appearance-time tech nique. These studies were done some time after the subconjunctival injections when the intraocular pressure in the treated eyes was raised. d. TONOGRAPHY
The Mueller tonometer was allowed to warm up for at least half an hour. The to nometer head and plunger were scrupulous ly cleaned with spirit and the scale was cali-
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Fig. 6 (Luntz). Intraocular pressure in Rabbit 2a plotted against time in weeks. The timing of the two subconjunctival injections in the right eye is indicated. The left eye is the control.
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coincide with this, and the recorder was checked to insure that it traced properly. The animal was immobilized and anesthe tized in the same way as described for to-
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Fig. 8 (Luntz). Intraocular pressure in Rabbit 4a plotted against time in weeks. Four subconjunctival injections given in the right eye are indicated. The left eye is the control.
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RABBIT NO.i (A)
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Fig. 9 (Luntz). Intraocular pressure in Rabbit Sa plotted against time in weeks. Three subconjunctival injections which were given in the right eye are indicated. The left eye is a control. nometry. The tonometer head was placed on the cornea of the eye to be measured and the reading begun. A four-minute tonography was the rule in each eye. During this
period, the animal had to remain perfectly quiet and this was best achieved in a closed, isolated room free from outside interfer ences. Drugs were not used.
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Fig. 10 (Luntz). Intraocular pressure in Rabbit 6a plotted against time in weeks. Two subconjunctival injections given to the right eye are indicated. The left eye is a control.
EXPERIMENTAL GLAUCOMA IN THE RABBIT e. FLUORESCEIN-APPEARANCE TIME
A sterile 10% solution of fluorescein in normal saline was prepared by the Oxford Eye Hospital pharmacist. The rabbit to be investigated was given a few drops of a mixture of Cyclogyl 1% (cyclopentolate hydrochloride) and phenylephrine 10% into one conjunctival sac. Half an hour later when the pupil was maximally dilated, the animal was bound in a duster with the head and neck protruding and was placed in posi tion in front of a slitlamp. Its head was held with the dilated pupil facing the lamp by a technician who also operated a stopwatch. One ear had already been shaved to expose a vein. Five cc of the fluorescein solution were drawn up into a 5-cc Luer-lock syringe with a No. 1. needle. A No. 20 needle was fitted and the fluorescein injected intravenously into the prepared ear vein as rapidly as pos sible. As soon as the injection was started, the stopwatch was released and the eye ob served under slitlamp magnification, using the low power. The end-point occurred when the fluorescein was seen at the edge of the pupil, easily recognized by its green color; the time taken between the injection of fluorescein intravenously and the endpoint was recorded as the fluroescein-appearance time (table 2). The two eyes of each rabbit were investi gated on separate occasions, as it was im possible for one observer to visualize both eyes simultaneously through the slitlamp. An identical technique was used each time and the measurement was done twice in each eye. RESULTS A. APPEARANCE OF TREATED EYES
Immedately following the subconjunctival injection, the treated eyes were red and congested but no flare or cells were present when examined on the slitlamp. They settled within a few days, the external appearance being relatively normal. The conjunctiva re
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mained freely mobile. There was no cupping of the optic discs, the fundi remaining nor mal in appearance. B. INTRAOCULAR PRESSURE 1. FIRST SERIES
This is presented graphically (figs. 1-5), the tension recorded at weekly intervals and plotted on the ordinate, the time in weeks along the abscissa. In all the treated eyes except Rabbit 4, the intraocular pressure rose steadily, with some fluctuations, to 60 mm Hg where the graph flattens. The average time taken to reach this level was 7.5 weeks. This period represents an adjustment of ocular dynam ics to the steadily increasing outflow resis tance, a consequence presumably of slowly progressive episcleral fibrosis (or episcleral vascular sclerosis). As outflow resistance increases (demonstrated by the tonography records), so aqueous formation and inflow decreases (shown by the fluorescein appear ance-time test). Ultimately compensation fails and the intraocular pressure rises. The level of ocular hypertension attained and the rapidity with which it is reached would de pend on the degree of episcleral pathology and the availability of other routes for aque ous drainage from the eye (for example, the demonstration by Ruskell and Nemetz of an alternative route to the choroidal vessels and thence to the vorticose veins). This is prob ably why the treated eye in Rabbit 4 did not become glaucomatous. RABBIT 3 :
a. Right eye (fig 1). Baseline ocular tension before treatment with phenol 5% in almond oil was 21 mm Hg. The first four-quadrant subconjunctival injection was given on February 27, 1961, the second on March 3, 1961, and the third, March 17, 1961. Ocular tension rose gradually, reaching 60 mm Hg after five weeks and then fluctuated between 35 mm Hg and 60 mm Hg over the following seven weeks. At this stage, pilocarpine nitrate ( 1 % drops twice daily) was applied topically. This had no effect and was increased to 4% twice daily, with an immediate fall in tension to 22 mm Hg. Two weeks later Diamox (acetazolamide) 125 mg twice daily was given by mouth. Ocular tension
MAURICE H. LUNTZ
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fell further to IS mm Hg for two weeks and then rose gradually to 24 mm Hg. Medication was stopped after seven weeks and ocular tension returned to 60 mm Hg. It is noteworthy that there was a latent period of two weeks without drugs during which the in traocular pressure remained at normal levels be fore starting to rise. I am at a loss to explain this. A cumulative effect of Diamox, which could explain it, has never been demonstrated in the rabbit and does not occur in man. Little is known of the effects of pilocarpine nitrate drops on the rabbit eye. b. Left eye (fig 2). Baseline ocular tension be fore treatment with phenol 5% in almond oil was 21 mm Hg. The first four-quadrant injection was given on February 17, 1961, the second on March 3, 1961, and the third on March 17, 1961. Ocular ten sion rose gradually reaching 60 mm Hg after five weeks then fluctuated between 35 mm Hg and 60 mm Hg over the following weeks. Pilocarpine nitrate drops ( 1 % twice daily) were administered at this stage but, as this dosage had no effect, it was increased to 4% twice daily, still without any effect. Diamox (125 mg twice daily) by mouth was added and the tension dropped im mediately to 27 mm Hg, but a week later was back to 60 mm Hg and subsequently remained about 30 mm Hg until the drugs were discon tinued two weeks later. Presumably outflow resis tance was greater in the left eye than in the right eye. RABBIT 4 (fig
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a. Right eye. Phenol 5% in almond oil was in jected into all four quadrants on March 17, 1961, on March 31, 1961, and on April 14, 1961. Ocular tension fluctuated between 12 mm Hg and 27 mm Hg. This was the only treated eye in the series which failed to become hypertensive, possibly be cause of a well-developed choroidal aqueous outflow route (Nemetz and Ruskell). b. Left eye. This was a control eye and re ceived subconjunctival injections of almond oil only on March 17 and the next two at fortnightly intervals. Intraocular pressure fluctuated between 12 mm Hg and 23 mm Hg. RABBIT 5 (fig
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a. Right eye. This was used as a control eye. The ocular tension remained constant at 19 mm Hg, except on two occasions when it was re corded as 18 mm Hg. b. Left eye. The first injection of phenol in al mond oil in all four quadrants was given on March 17, 1961, and the next two at fortnightly intervals. The baseline ocular tension was 20.5 mm Hg and this rose over 10 weeks to 60 mm Hg, remaining at this level for four weeks. At this point an iris inclusion done on the control eye resulted in a fall of intraocular pressure in the operated eye, as well as in the left eye, the latter subsequently returning to 60 mm Hg. At
the 16th week an iris inclusion was done in this eye and the ocular tension dropped to normal val ues. RABBIT 6 (fig
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a. Right eye. This was the control eye. Intraoc ular pressure remained at 19 mm Hg with minor fluctuations except on two occasions, one of these at the 16th week, the result of an iris inclusion. b. Left eye. The first injection of phenol in al mond oil in all four quadrants was given on March 17, 1961, and repeated twice at fortnightly intervals. This eye reacted in an identical way to the left eye in Rabbit 5. There was a fall in the intraocular pressure in this eye at the 16th week in sympathy with a fall in pressure in the right eye following an iris inclusion. This is an interesting phenomenon, well illustrated in these two eyes. 2. RABBIT 2(a)
(fig
S E C O N D SERIES 6)
a. Right eye. This was the control eye. Intraoc ular pressure at the start of the experiment was 21 mm Hg but fell within a few weeks to 16 mm Hg and remained fairly constant for the 34 weeks of the test. b. Left eye. Phenol in almond oil was injected on September 22, 1961, and repeated at the fifth week Intraocular pressure was consistently higher than in the left eye, fluctuating between 22 mm Hg and 33 mm Hg, from a baseline intraoc ular pressure of 22 mm Hg. The rabbit developed ear canker and had to be killed at the 34th week. RABBIT 3(a)
(fig
7)
a. Right eye. The control eye, duration of the test 42 weeks. Ocular tension fluctuated around 15 mm Hg. , b. Left eye. Subconjunctival phenol 5% in al mond oil was injected on September 22, 1961, and repeated at the fifth and again at the eighth week. The intraocular pressure remained consis tently higher than the control eye, the baseline intraocular pressure before injection being 16 mm Hg. For the most part the intraocular pressure fluctuated between 25 and 30 mm Hg. RABBIT 4(a)
(fig
8)
The baseline intraocular pressure was 19 mm Hg in the left eye and 20 mm Hg in the right eye. a. Right eye. This was the injected eye. The first four-quadrant injection was given on September 22, 1961, and repeated at the 4th, 8th and 10th week. Again the intraocular pressure remained consis tently higher than in the left eye, fluctuating be tween 25 mm Hg and 30 mm Hg, rarely exceed ing this figure. At the 34th week the rabbit de veloped ear canker and had to be killed. b. Left eye. This was the control eye.
677
E X P E R I M E N T A L GLAUCOMA IN T H E RABBIT RABBIT 5 ( a )
(fig
9)
The baseline intraocular pressure was 19 mm H g in the left eye and 20 mm H g in the right eye. a. Right eye. Phenol was injected subconjunctivally in four quadrants on September 25, 1961, and repeated twice, at the 11th and 22nd week. Intraocular pressure varied between 20 mm H g and 35 mm H g , remaining consistently higher than that in the left eye. b. Left eye. The left eye was the control eye, the intraocular pressure remaining constant at 15 mm Hg. The rabbit was killed after 48 weeks. RABBIT 6 ( a )
(fig
10)
a. Right eye. Subconjunctival phenol in almond oil was injected on September 25, 1961, into four quadrants and repeated after eight weeks. Intra ocular pressure was 19 mm H g and rose gradual ly, with a few fluctuations, to 45 mm H g at the 46th week. The experiment was terminated at the 48th week. b. Left eye. This was the control eye and had a baseline ocular tension of 19 mm Hg. This fell gradually over the test period to 15 mm H g at the 36th week, where it remained until the test was terminated at the 48th week. C. TONOGRAPHY
The test was carried out on all the rabbits in the second series. The results, expressed as the C value, are summarized in Table 1. The C values in all the treated eyes were well below the average normal value, and there was a significant difference between these values and the C
values in the control eyes, which were with in normal limits. These results indicate an increased resis tance to aqueous outflow in the treated eyes. D. FLUORESCEIN APPEARANCE-TIME TEST
The results of this test are summarized in Table 2. Two results are recorded from each eye of the rabbits in the second series. Taking into account the inevitable crudeness of this test, these are remarkably constant. The treated eyes all showed prolonged TABLE 2 FLUORESCEIN APPEARANCE-TIME TEST
Appearance Time
Rabbit Eyes
First Result
Second Result
(min) 2 1
(sec) 20 45
(min) 3 2
(sec)
3 (a) Right (c) Left
1 3
30 30
1 3
50 10
4 (a) Right Left (c)
2 1
5 35
2 1
20 30
5 (a) Right Left (c)
3 2
10 5
3 2
10 20
6 (a) Right Left (c)
3 2
30
3 1
50
2 (a) Right Left (c)
5
(c) = control eye.
TABLE 1 TONOGRAPHY
Mean C
Rabbit
Eye
2(a)
Right Left (c)
0.06 0.19
(a) Controls
(a) Controls
3(a)
Right (c) Left
0.29 0.08
0.21
±0.03
4(a)
Right Left (c)
0.06 0.20
(b) Treated
5(a)
Right Left (c)
0.08 0.15
0.07
6(a)
Right Left (c)
0.08 0.18
C = coefficient of resistance to outflow ( c ) = control eye
Standard Deviation
(b) Treated ±0.02
MAURICE H. LUNTZ
678
fluorescein appearance time when compared to the controls (comparing treated and con trol eyes in the same animal), and this difference is obviously significant without having to resort to further statistical analy sis. This observation showed that there had been a reduction in aqueous inflow in the treated eyes, presumably as an attempt to compensate for the increased resistance to outflow. This had failed with the result that these eyes became glaucomatous. It was this compensatory process that dictated the slow rise of ocular tension, a situation analagous to the onset of chronic simple glaucoma in man. There is no absolute normal value for this test as it varies widely in different subjects so that one can only compare an abnormal eye with the normal, preferably in the same animal. DISCUSSION OF RESULTS
Results have been most encouraging. Glaucoma supervened in four out of five eyes treated with phenol in almond oil in the first series, and in all treated eyes in the sec ond series. A high level of intraocular pres sure was reached in the first series and was maintained for an average period, in the four eyes, of 9.5 weeks, when the experi ment was terminated. The reactions to pilocarpine and Diamox in two eyes in the first series and iris inclu sion in the other two were in accord with what one would expect. This raises the pos sibility of using animals eyes prepared by this method to investigate the effectiveness of new preparations for the treatment of glaucoma, before applying them to man. The level of ocular hypertension in the second series was not as striking as in the first. This may have been due to the different spacing in time of the injections, or to the use of a different breed of animal. Nevertheless, consistently raised pressures often reaching over 30 mm Hg were main tained in the treated eyes for an average pe riod of 40 weeks, when the experiment was
terminated. During the same time, aqueous outflow resistance and aqueous inflow time were increased in these eyes. I attempted to produce an outflow glauco ma by obliterating the aqueous outflow channels. That I succeeded in doing this is shown by the tonography results which re corded low C values in the treated eyes, and indirectly by the delayed fluorescein-appearance time, suggesting a reduced aqueous inflow and turnover in the glaucomatous eyes. No gross ocular damage could be detected by either macroscopic or microscopic exami nation of the treated eyes. The intraocular pressure in the control eyes remained constant. The average tension readings compared well with those from other series. This indicated that the methods used for measuring intraocular pressure and aqueous outflow are valid. It would be ap propriate at this stage to examine in more detail the validity of these methods. VALIDITY OF METHODS
The methods used for measuring intraoc ular pressure and aqueous outflow have al ready been described. I have earlier in this paper submitted that they can be used with a high degree of clinical accuracy. I now propose to show that the results obtained in this experiment are valid, by comparing the measurements in my control eyes with mea surements obtained from normal rabbit eyes by other investigators. There should be close agreement and, in fact, this is what one finds. Secondly, the measurements obtained from the control eyes show very little scat ter around the mean value when expressed by the standard deviation from the mean. a. Intraocular pressure. In Table 3, the mean ocular tension (Po) in the control eyes in my series is compared with the fig ures obtained by Becker and Constant ( 1956) when they measured the intraocular pres sure in normal rabbit eyes in vivo, using a standardized, weighted Schi^tz tonometer. There is good agreement.
EXPERIMENTAL GLAUCOMA IN THE RABBIT TABLE 3 COMPARISON OF MEAN OCULAR TENSION
Mean Ocular Standard Tension (Po) Deviation (mm Hg)
Series Present series Becker & Constant Series
17.8 18.0
±1.9 ±3.7
The standard deviations are not large, striking evidence for the accuracy of the Schi^tz tonometer, used under these condi tions, for measuring intraocular pressure in rabbits. b. Tonography. Table 4 compares the mean C value in my series of control rabbit eyes with the mean C values obtained by Becker and Constant (1956) and by Kornblueth and Linnér (1955) and Levene and Bloomgarden (1961) when measuring facility of outflow in normal rabbit eyes, using a similar technique as in the present study. The latter authors also showed that there was no significant difference in results be tween right and left eyes; the right eyes were measured first. The standard deviation about the mean is small, underlining the consistency of the measurements. The C value in the present series is lower than in the other three, but well within the range of normal, which is 0.14 to 0.60. This emphasizes very clearly that tonography is not a scientifically ac curate measurement of resistance to outflow but is nevertheless a useful clinical test as long as it is accepted that there is a large range for normal values. The value obtained depends to a great extent on environmental conditions pertaining at the time of the test. TABLE 4 COMPARISON OF C VALUES
Series Present series (1962) Kornbluth & Linner (1955) Becker & Constant (1956) Levene & Bloomgarden (1961)
Mean Standard C Value Deviation 0.21 0.30 0.34 0.26
±0.03 ±0.04 ±0.10 ±0.06
679
In the four series quoted in Table 4, al though the test was done by different inves tigators under vastly differing conditions, the mean values all lie within the normal range and the standard deviations for each series are small. In my opinion Table 3 provides sufficient evidence that the methods employed in this experiment have provided valid results. If one accepts that the results are valid, then the technique that I have described is a use ful method of producing chronic outflow glaucoma in animals, which closely resem bles primary glaucoma in man. The possibil ities of studying the effects of various drugs or surgical procedures on these eyes is im mediately obvious. One hopes that these ex periments will emphasize the importance of the episclera and in particular the episcleral vessels in the regulation of aqueous-humor flow and dynamics and its possible role in the etiology of primary glaucoma. SUMMARY
A method of producing primary glaucoma in rabbits by means of the subconjunctival injection of phenol is described. In nine out of 10 rabbits treated in this way, a chronic glaucoma (as described in the introduction) resulted. The effects of pilocarpine drops and Diamox were studied in two rabbits, and of iris inclusion operations on four eyes, two control and two treated. Aqueous outflow and inflow studies sug gest that the inflow of aqueous was reduced and the resistance to outflow increased in "treated" eyes with raised intraocular pres sure. The methods have been described in de tail, and a critical analysis of the validity of the results has been offered. Hospital Street. ACKNOWLEDGMENT
I wish to acknowledge technical assistance from the Nuffield Laboratory of Ophthalmology, Oxford, and the Surgical Laboratory of the Uni versity of Cape Town, as well as the Photo Unit, De partment of Medicine, University of the Witwatersrand.
MAURICE H. LUNTZ
680 REFERENCES
Ashton, N.: Brit. J. Ophth., 35: 291, 1951. : Brit. J. Ophth., 38: 129, 1952. : Glaucoma symposium (Council Inter nat. Organiz. M. Sei). (Edited by Duke-Elder, S.) Oxford, Blackwell, 1955. Becker, B., and Constant, M. : Arch. Ophth., 55 : 305, 1956. Davis, F. A.: Tr. Am. Ophth. Soc, 27: 401, 1929.
Kornblueth, W. and Linnér, E. : Arch. Ophth., 54: 717, 1955. Leber, T. : Arch. f. Ophth., 19: 87 (pt. 2) 1873. Levene, R. Z., and Bloomgarden, C. : Arch. Ophth., 66: 149, 1961. Nemetz, U. R. : Ber. Deutsch. Ophth. Ges., 60: 60, 1956. Pickering, G. W. : High Blood Pressure. Longon, Churchill, 1955, p. 85. Ruskell, G. L.: Arch. Ophth., 66: 861, 1961. Smith, P. : On the Pathology and Treatment of Glaucoma. Churchill, London, 1891.
TONOGRAPHIC SURVEY* LORAND V.
JOHNSON,
M.D.
Cleveland, Ohio
A tonographic survey was made of 7,577 eyes, consecutive and unselected except for the exclusion of all individuals who had a1 previous diagnosis of or treatment for ex isting glaucoma. These persons, aged over 41 years, consecu tively passed one point in this city, namely the front door to my office. It is by coinci dence that they are mainly Caucasians. Tonography was performed by skilled techni cians, using standard tonographic equip ment. Initial pressure was obtained by not ing the initial scale readings of the tonogram to the nearest 0.25 scale division and1 the intraocular pressure read to the nearestt mm Hg from a graph of the 1955 Schlitz: tonometer scale of the Committee on Stan dardization of Tonometers of the American1 Academy of Ophthalmology and Otolaryn-\ gology. Schi^tz pressure and facility of1 outflow were recorded in categories of male5 or female and tabulated into five-year age; delineations (table 1). The descending,> stair-step lines separate those eyes with P 0 / C greater than 100 and those with less than1 100. From these charts, the data were statis tically analyzed.
A.
FACILITY OF AQUEOUS OUTFLOW
The facility of aqueous outflow was statistically analyzed to answer the following questions : Question 1. At each age classification is there a real difference in the average outflow between male and female ? Answer. There is no difference in the av erage outflow between men and women in each age classification except for the age group of 66-70 years. This result is based on the use of Student t-test and Kolmogorov-Smirnov test. In the age group of 66-70 years, the female average facility of outflow is 0.2688 and the male average is 0.2514. Although this difference is statistically significant, the difference has no clinical interpretation. Thus, it is concluded that there is no real difference in the average outflow between male and female in any of the age classifications. Therefore, the facility of outflow data for men and women were combined into one group for each age clas sification. Question 2. Are the combined facility of outflow data for men and women normally distributed in each age classification? Answer. The data were plotted on normal * Presented at the 101st annual meeting of thee probability paper. Chart 1 shows the plot American Ophthalmological Society, Hot Springs,I for the age group 56-60 years. Virginia, May, 1965. The statistical analyses were done by Julio N. Berrettoni.
This chart shows three distinct straight
665
Michels, V. : L'hérédité dans le glaucome pri Strehlar, E. : Zwei Stammbäume von Glaucoma maire. Thèse, Lausanne, 1957. simplex. Thèse Zurich 1938. Miller, S. J. H., and Paterson, G. D. : Studies Sveinsson, K. : Glaucoma and heredity in Ice on glaucoma relatives. Brit J. Ophth., 46:513, land. Acta Ophth., 37 :191, 1959. 1962. Tiscornia, B. : Erblichkeit und Blutsverwandt Olivier, W. : Familiäres Glaukom über zehn schaft in der Augenheilkunde. Zentralbl. Ges. Generationen. Deutsche Ophth. Ges. Heidelberg, Ophth., 38:339, 1937. 65:547, 1963. Törnquist, R. : Genetics of senile glaucoma. Acta Penzani, B. : Conbributo allô studio dell'eredità nel Ophth., 32 :513. 1954. glaucoma giovanile tardivo. Giorn. Ital. OftaL, 8: Waardenburg, P. J. : Ueber das Familiäre Vor 53, 1955. kommen und den Erbgang des praesenilen und Pimentel, P. C. : Consanguinity and morbid he senilen Glaukoms. Genetica, The Hague, 25:79, redity. Ophthalmos, 2:329, 1941. 1950. Politzer, M. : The gonioscopic picture of he Waardenburg, P. J., Franceschetti, A., and reditary glaucoma. (In Czech.) Csl. oftal., 18:446, Klein, D. : Genetics and Ophthalmology (Glau 1962. coma). Assen, Von Gorcum. 1961, v. 1, pp. 601Posner, A., and Sohlossman, A. : Role of in 626. heritance in glaucoma. Arch. Ophth., 41:125, 1949. Weekers, R., Gougnard-Rion, C, and Gougnard, Probert, L. A. : A survey of hereditary glau L. : Considérations cliniques sur l'hérédité des coma. Canad. M. A. J.. 66:563, 1952 glaucomes. Bull. Soc. Belge Opht, 110:255, 1955. Rossi, V. : Glaucoma primario e constituzione. Westerlund, E. : Clinical and genetic studies on Arch. Ottal., 39:1, 1932. the primary glaucoma diseases. Vol. XII, opera ex Sedan, J., and Sédan-Bauby, S. : Dépistage d'une Domo biologiae Heredit. Hum. Univ. Hafmensis. "glaucomatose" latente chez deux jeunes gens Copenhague, Munksgaard, 1947. ayant une hérédité de trois et quatre générations Woerner, E. : Primäres Glaukom und Konstitu pour cette affection. Bull. Soc. Ophtal. France, tion. Thèse Tübingen, 1936. 1949, pp. 29-36. Wolf söhn- Jaffe, E.: Ueber 9 Fälle von hereditä Stankovic, L. : Ein Beitrag zur Kenntnis der rem Glaukom der Erwachsenen in 3 Generationen. Vererbung des Pigmentglaukoms. Klin. Mbl. Klin. Mbl. Augenh, 94:622, 1935. Augenh., 139:165, 1961. Zorab, A. : Glaucoma simplex familialis. Tr. Stokes, W. H. : Hereditary primary glaucoma : Ophth. Soc. U. Kingdom, 52:446, 1932. A pedigree with five generations. Arch. Ophth., 24:885, 1940.
E X P E R I M E N T A L GLAUCOMA IN T H E RABBIT* MAURICE H. LUNTZ, F.R.C.S. Johannesburg, South Africa Our knowledge and understanding of primary simple glaucoma in man has in re cent years been enriched by a vast amount of research undertaken on a world-wide basis. Nevertheless, there are still many un solved problems, especially as regards etiolo gy and natural history. Progress has been hampered because the disease cannot be sat isfactorily reproduced in animals for exper imental study. Sir George Pickering (1955) wrote : "A time-honoured method of investi gating disease is to try and reproduce it in animals. This method has proved of the ut* From the Department of Ophthalmology, University of the Witwatersrand Medical School.
most value and without it the advances that have taken place all over the front of medi cine would not have occurred." This state ment will no doubt prove as true of glauco ma as of other diseases. One essential in any attempt to reproduce disease experimentally is that it should re semble the malady as it occurs in man. Pre vious workers, attempting to reproduce glau coma and bearing this in mind, have adapt ed the current theories for the etiology of primary simple glaucoma in man to their techniques for reproducing it in the experi mental animal. Some managed to produce raised intraocular pressures over a period as long as six months, but always at the price
666
MAURICE H. LUNTZ
of gross structural and functional damage to the eye. However, affected eyes in man characteristically appear to be clinically and histologically normal apart from the effects of prolonged ocular hypertension, for exam ple, cupping of the optic disc. What is re quired therefore is a technique which pro duces ocular hypertension in an experimen tal eye that is maintained over some months or longer but which does not cause any ap parent structural or functional damage to the eye. Such damage may occur later secon dary to the ocular hypertension and would be permissible and indeed expected. It is the purpose of this paper to describe such a technique and to discuss the results ob tained.
where it was absorbed into the general cir culation. Aqueous drainage from the anterior chamber is considered to take place predom inantly at the angle of the anterior chamber of the eye via Schlemm's canal. The aque ous gains access to Schlemm's canal by per colating through the trabecular tissue and leaves Schlemm's canal mainly through col lector channels reaching the episcleral ve nous plexus and the venous circulation by way of the deep scierai and intrascleral plexuses. A limited proportion of this drain age from Schlemm's canal reaches the epis cleral venous plexus directly via aqueous veins which bypass the deep scierai and in trascleral plexuses.
The cause of primary simple glaucoma in man is now widely accepted as being an in creased resistance to the outflow of aqueous humor at the anterior chamber angle. This concept has assumed pride of place since Leber (1873) described the angle of the an terior chamber and postulated that aqueous drainage took place mainly at this site. This was confirmed by Priestley Smith (1891) in a brilliant series of perfusion experiments on enucleated eyes. He went further and stressed that obstruction at the angle of the anterior chamber was primarily responsible for the raised ocular tension. Gonioscopy has shown that acute closed-angle glaucoma oc curs when the iris obstructs the entry of aqueous into the angle.
Considering the anatomy of the anterior chamber angle in the light of present-day knowledge, it is evident that obstruction to the aqueous outflow in primary simple glau coma in man could occur at one of three sites :
Investigations have made it clear that the aqueous humor from Schlemm's canal drained into the episcleral venous plexus by a series of intercommunicating plexuses in the sciera and episclera, eventually reaching the superficial vessels of the episclera (Ashton 1951, 1952). Approximately 30 external collector channels, irregularly distributed and varying in size and shape, conveyed aqueous from Schlemm's canal to the deep scierai plexus which in turn anastomosed with the intrascleral plexus, the aqueous finally reaching the episcleral venous plexus
a. The trabecular tissue, immediately proximal to Schlemm's canal. b. At Schlemm's canal (rare). c. The outflow channels from Schlemm's canal of which the epsicleral venous plexus forms the ultimate stage. The purpose of this paper is to describe a technique for producing chronic simple glaucoma in rabbits by injecting phenol into the episcleral tissue. PRESENT EXPERIMENTS MATERIALS
Twelve gray female rabbits were used be cause they are more docile and easier to handle than males. Six were bred from the stock at the Cape Town University Medical School, which are not a pure breed and are closest to pure-bred chinchillas. A second series of six were chinchilla rabbits obtained at Oxford University. Rabbits are docile animals, have relatively large eyes and cor-
EXPERIMENTAL GLAUCOMA IN THE RABBIT neas and submit uncomplainingly to tonometry. Histologically the rabbit anterior cham ber angle has structural differences from those of man. These were well described by Davis (1929). Essentially these differences occur in the meshwork which is better developed in the rabbit. The collagenous fibers are much finer and not enclosed by elastic fibers, as in man. They run as isolated fibers. Schlemm's canal is not a single channel, as in man, but rather a series of channels forming a plex us. In spite of these anatomic differences Ruskell (1961) showed that the rabbit has a close functional similarity to man, which justifies its use as an experimental animal. Using the Neoprene cast technique, as de scribed by Ashton (1951), he showed that in rabbits the major outflow of aqueous is to the episcleral vessels, as in man. Ruskell's results also supported the idea of a secondary route for aqueous otflow as ad vocated by Nemetz (1956) from the filtra tion angle through the intrascleral plexus to the choroid. These connections had also been demonstrated in man by Ashton (1955). There is considerable disagreement about the extent to which this route could be used in rabbits under physiologic and patho logic conditions. The fact that prolonged ocular hypertension can be produced by blocking the episcleral outflow strongly im plies this route is of minor importance as an alternative outflow channel in the breed of rabbit used in these experiments. It is gen erally agreed that this is also the case in man. The rabbit, then, has an aqueous drainage system which is similar to that in man, apart from some minor anatomic dif ferences. Monkeys have an angle very similar to that in man but, unfortunately, it is impossible to do tonometries on them with local anesthetics. Another argument in favor of using rab bits was that normal rabbits produce fairly constant intraocular pressure readings with
667
repeated indentation tonometries, using local anesthetics. The average tension in 28 rabbit eyes was found by Becker and Constant (1956) to be 18 mm Hg, with a standard deviation of ±3.7. They used a Mueller electronic tonometer and took their measure ments in vivo. This was repeated while doing tonographies in vitro and found to be 19 mm Hg, with a standard deviation of ±4.8, a remarkably close agreement. Kornblueth and Linnér (1955) and Becker and Constant (1956) showed that tonography could be applied to the rabbit eye with valid results. The latter did a series on 28 rabbit eyes in vivo and found an average facility of outflow C of 0.33 ( standard devia tion ±0.08). These eyes were then perfused with isotonic saline in vivo and an average C value of 0.34 was obtained (standard de viation ±0.10). When perfused in vitro, the average C value was 0.37 (standard devia tion ±0.11). Considering the experimental error inherent in tonography, the C values obtained by all methods are surprisingly similar. SCLEROSING MATERIALS
The rabbits were given subconjunctival injections of a sclerosing fluid, using either a 0.175% solution of silver nitrate, a 1% so lution of ethanolamide (Ethanolamine Oleate) or a 5% solution of phenol in al mond oil. The latter two preparations are standard sclerosing agents used in the treat ment of varicosities (for example, in the rectum). Of these only the phenol solution proved to be satisfactory, causing a rise of intraocular pressure but no apparent macro scopic or microscopic damage to the eye. The silver nitrate was prepared by the hospital dispensary (Groote Schuur Hos pital). The ethanolamide and phenol came as commercial preparations in 5-cc am pules; the former prepared by May and Baker (S.A.) Pty. Limited, Port Elizabeth; the latter by Glaxo-Allenbury (S.A.) Pty. Limited, Durban.
MAURICE H. LUNTZ
668 D E S C R I P T I O N OF E X P E R I M E N T A L
TECHNIQUE a. TONOMETRY
The animal was tied in a duster so that only its head and neck protruded. Both eyes were anesthetized using two drops topically of a 1% solution of tetracaine in aqueous. The technician then placed the animal on his knee and held its head with the right cornea facing vertically upward. The tonometer (a standardized Schip'tz tonometer) was cleaned with spirits and checked on a hard convex surface, supplied with the instru ment : the pointer should be deflected to the zero position on the calibrated scale. If cor rect, the instrument was placed vertically on the cornea, the operator carefully holding the eyelids apart with one hand, being care ful not to press on the globe, and the read ing on the calibrated scale noted. This was recorded in mm Hg by consulting Fri eden wald's nomograms (1957). Two read ings were made, one using a 5.5-gm weight Ui RABBIT NO. 3 RIGHT EYE
and another using a 7.5-gm weight. Occasionally, the nictitating membrane gave trouble by covering the cornea, over come by patiently waiting for it to retract and then proceeding. Having measured the intraocular pressure of the right eye, the same procedure was re peated on the left eye. The intraocular pressure was recorded in each eye of every rabbit on at least three oc casions before the first subconjunctival in jection was given and at weekly intervals thereafter until the experiment was termi nated. Where subsequent tonometries coin cided with a subconjunctival injection, the tonometry was done first. Intraocular pres sures in the first series are followed graphi cally in Figures 1 to 5 and the initial sub conjunctival injection is indicated. Figures 6 to 10 are graphs of the intraocular pressures in the second series. These also indicate all the subconjunctival injections and also the intraocular pressures before the initial in jection. $
SUBCONJUNCTIVAL INJECTION PHENOL S% IN ALMOND OIL 4 QUADRANTS
N H
o X
13 I Z Σ Z O
NORMAL
Fig. 1 (Luntz). Intraocular pressure of right eye of Rabbit 3 plotted against time in weeks. The timing of initial subconjunctival injection is indicated. The stippled line is of no significance. Three measurements of intraocular pressure were made before the initial subconjunctival injection, but only the third is plotted.
EXPERIMENTAL GLAUCOMA IN T H E RABBIT SUBCONJUNCTIVAL INJECTION PHENOL 5% IN ALMOND OIL 4 QUADRANTS
669 RABBIT NO. LEFT EYE
60'
/ SO N I-
g z u X Σ Σ Z
40
111
°-z zo
z " /\
\/
/
V
< /
/ \
2«
!ÜZ 30
\
< .
w
O
NORMAL
yo 20 10
10 12 TIME - WEEK
16
14
18
20
Fig. 2 (Luntz). The intraocular pressure of the left eye of Rabbit 3 plotted against time in weeks. The initial subconjunctival injection is indicated. The stippled line is of no significance. Only the third of three measurements of intraocular pressure before the initial subconjunctival injection is plotted.
RABBIT NO. 4 METHOD:- SUBCONJUNCTIVAL INJECTION PHENOL S% IN ALMOND OIL 17 MARCH 1961
8 10 TIME - WEEK
12
Fig. 3 (Luntz). The intraocular pressure in both eyes of Rabbit 4 is plotted against time in weeks. The initial subconjunctival injection given in the right eye is indicated. This eye did not become glaucomatous. The third of three pressure readings made before the initial subconjunctival injection is plotted as the first reading.
MAURICE H. LUNTZ
670
RABBIT NO. S RIGHT AND LEFT EYES
60
8S
h U
50
û Z
il
—I
< >^ z ,5 -w _ _ ^, Z "> f»
X
2 30
o9z
U!" Σ Σ ■» ï Z Q 20
/
10
8
10 U TIME - WEEK
14
16
18
20
Fig. 4 (Luntz). The intraocular pressure in Rabbit S plotted against time in weeks. The initial subconjunctival injection into the left eye is indicated. The right eye is the control. The first reading plotted is the third of three measurements of the intraocular pressure made before giving the initial subconjunctival injection. METHOD;-INJECTION S% PHENOL IN ALMOND OIL SUBCONJUNCTIVAL 4 QUADRANTS L.E. 17 MARCH 1961
RABBIT NO. 6
8
10 12 TIME - WEEK
14
16
18
20
Fig. S (Luntz). Intraocular pressure of Rabbit 6 plotted against time in weeks. The initial subcon junctival injection in the left eye is indicated. The right eye is the control. The first plotted reading is the third of three measurements of intraocular pressure made before giving the initial subconjunctival injection.
EXPERIMENTAL GLAUCOMA IN THE RABBIT b. T H E SUBCONJUNCTIVAL INJECTION
The method of injection was the ap proved technique for a subconjunctival in jection, the aim being to deliver the sclerosing fluid into the episcleral tissues. Two min ims of sclerosing fluid were drawn up into a standard 2-cc syringe fitted with a No. 1 needle. The needle was then removed and a No. 18 needle fitted to the syringe. The animal was immobilized and anesthe tized in the same way as described for tonometry and the initial intraocular pressure measurement was made. A conjunctival spe culum was now used to hold the lids apart, the operator fixated the conjunctiva in the desired quadrant with a pair of Lister's for ceps and the No. 18 needle was pushed through the conjunctiva into the episcleral tissue. One minim of the sclerosing fluid was injected, the needle was withdrawn and the speculum removed. If more than one quadrant was to be injected, the procedure was repeated in each quadrant at the same sitting. There was no after-treatment. With only one exception (Rabbit 3) one eye in each rabbit was given a subconjunctival in jection of sclerosing fluid (the "treated" eye) while the other eye was left as a con trol (the "control" eye). Hence treated and control eyes could be compared in the same animal. Work on the first series of rabbits was done in the Department of Surgery, Univer sity of Cape Town. These were numbered 1 to 6. The first two animals were given sub conjunctival injections of the 0.175% solu tion of silver nitrate. This resulted in a se vere local reaction, the method was aban doned and the rabbits withdrawn from the experiment. In the remaining rabbits in this series one eye was injected subconjunctivally with the 5% phenol in almond oil. At first one quad rant of the globe, then two and then three were injected; this failed to cause a rise in intraocular pressure. Finally, all four quad rants were injected, using four minims of
671
fluid. Positive results were obtained at this stage. In Rabbit 3, both eyes were injected sub conjunctivally with 5% phenol in almond oil (four quadrants) : a similar injection but of almond oil was given in the control eye of Rabbit 4. The control eyes of the remaining rabbits in this series were left untreated. These injections were repeated at regular intervals at two and again at four weeks after the initial injection. c. TREATMENT OF GLAUCOMATOUS EYES IN THE FIRST SERIES
This part of the experiment was termi nated after some weeks by killing the rab bits and removing their eyes for histologie study. Before doing this, an iris-inclusion operation was done in some eyes; in other rabbits pilocarpine nitrate drops in l%-4% solution was given together with acetazolamide (Diamox) 125 mg daily by mouth in tablet form. These are established methods of treating hypertension in man and the effect on the intraocular pressure in these glaucomatous rabbit eyes was recorded. Al though the purpose of this paper is to de scribe a method of reproducing chronic glaucoma in rabbits, the results of treating their glaucomatous eyes offers an interesting digression and has useful research possibili ties. The results of treatment and a discus sion of its possible research application are presented later. The iris inclusion was performed by an ab externo technique. The animal was an esthetized by the open-mask ether method and an attempt was made to control the depth of anesthesia at a level just deep enough for the operative procedure. The an esthetized animal was placed on its side with the eye to be operated on uppermost. The operator scrubbed and donned sterile gown, cap, mask and gloves. The rabbit was dressed in sterile towels so that only the eye to be operated on was exposed. A conjuncti val speculum was inserted and a few drops of 1% tetracaine were instilled topically.
672
MAURICE H. LUNTZ
A limbal-based conjunctival flap was fashioned and retracted over the cornea. A scierai incision was made with a Bard-Park er knife and No. 11 blade into the anterior chamber from the limbus at the 12-o'clock position. The iris prolapsed through this in cision and was pulled out until the pupillary border became visible. A vertical incision was then made with de Wecker's scissors from the pupillary edge to the root of the iris. The iris was torn from its root for a short distance on either side of this incision, fashioning two iris pillars. These were left passing through the scierai incision at each edge and the conjunctiva was sutured in place over them with 6-0 catgut sutures. A few drops of 1% chloramphenicol were instilled into the conjunctival sac and an eyepad applied which was removed immediate ly the animal regained consciousness. Within a few days the conjunctiva had healed and a bleb of aqueous had formed under it. The second series of six rabbits was treated by a similar experimental technique at the Nuffield Laboratory of Ophthalmol
10
ogy, University of Oxford. In five of these the subconjunctival injection contained phe nol in almond oil as before. In one, four min ims of the ethanolamide solution was in jected. This resulted in a violent reaction, with sloughing of the sciera. The rabbit was discarded, leaving five. These five were numbered 2(a) to 6 ( a ) . The subconjuncti val injections were repeated but at varying intervals (figs. 6 to 10). The intraocular pressure was measured by tonometry. The rabbits were not given treatment as in the first series, but were subjected to inflow and outflow studies. Outflow studies were done by a four-minute tonography ; inflow studies by the fluorescein appearance-time tech nique. These studies were done some time after the subconjunctival injections when the intraocular pressure in the treated eyes was raised. d. TONOGRAPHY
The Mueller tonometer was allowed to warm up for at least half an hour. The to nometer head and plunger were scrupulous ly cleaned with spirit and the scale was cali-
12 u TIME -- WEEK
20
24
28
32 34
Fig. 6 (Luntz). Intraocular pressure in Rabbit 2a plotted against time in weeks. The timing of the two subconjunctival injections in the right eye is indicated. The left eye is the control.
EXPERIMENTAL GLAUCOMA IN T H E RABBIT
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,
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12 16 TIME - WEEK
20
24
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34
Fig. 7 (Luntz). Intraocular pressure in Rabbit 3a plotted against time in weeks. Three subconjunctival injections were given in the left eye and this is indicated in the figure. The right eye is the control. brated in the usual way to 0 and 7, using a 5.5-gm weight on the tonometer head. The reading on the recorder scale was checked against the tonometer scale and adjusted to
10
coincide with this, and the recorder was checked to insure that it traced properly. The animal was immobilized and anesthe tized in the same way as described for to-
12 16 TIME - WEEK
20
24
28
32
34
Fig. 8 (Luntz). Intraocular pressure in Rabbit 4a plotted against time in weeks. Four subconjunctival injections given in the right eye are indicated. The left eye is the control.
674
MAURICE H. LUNTZ
RABBIT NO.i (A)
10
12 16 TIME - WEEK
32
20
34
Fig. 9 (Luntz). Intraocular pressure in Rabbit Sa plotted against time in weeks. Three subconjunctival injections which were given in the right eye are indicated. The left eye is a control. nometry. The tonometer head was placed on the cornea of the eye to be measured and the reading begun. A four-minute tonography was the rule in each eye. During this
period, the animal had to remain perfectly quiet and this was best achieved in a closed, isolated room free from outside interfer ences. Drugs were not used.
?_, RABBIT NO. 6 (A)
10
12 16 TIME - WEEK
20
24
28
32 34
Fig. 10 (Luntz). Intraocular pressure in Rabbit 6a plotted against time in weeks. Two subconjunctival injections given to the right eye are indicated. The left eye is a control.
EXPERIMENTAL GLAUCOMA IN THE RABBIT e. FLUORESCEIN-APPEARANCE TIME
A sterile 10% solution of fluorescein in normal saline was prepared by the Oxford Eye Hospital pharmacist. The rabbit to be investigated was given a few drops of a mixture of Cyclogyl 1% (cyclopentolate hydrochloride) and phenylephrine 10% into one conjunctival sac. Half an hour later when the pupil was maximally dilated, the animal was bound in a duster with the head and neck protruding and was placed in posi tion in front of a slitlamp. Its head was held with the dilated pupil facing the lamp by a technician who also operated a stopwatch. One ear had already been shaved to expose a vein. Five cc of the fluorescein solution were drawn up into a 5-cc Luer-lock syringe with a No. 1. needle. A No. 20 needle was fitted and the fluorescein injected intravenously into the prepared ear vein as rapidly as pos sible. As soon as the injection was started, the stopwatch was released and the eye ob served under slitlamp magnification, using the low power. The end-point occurred when the fluorescein was seen at the edge of the pupil, easily recognized by its green color; the time taken between the injection of fluorescein intravenously and the endpoint was recorded as the fluroescein-appearance time (table 2). The two eyes of each rabbit were investi gated on separate occasions, as it was im possible for one observer to visualize both eyes simultaneously through the slitlamp. An identical technique was used each time and the measurement was done twice in each eye. RESULTS A. APPEARANCE OF TREATED EYES
Immedately following the subconjunctival injection, the treated eyes were red and congested but no flare or cells were present when examined on the slitlamp. They settled within a few days, the external appearance being relatively normal. The conjunctiva re
67S
mained freely mobile. There was no cupping of the optic discs, the fundi remaining nor mal in appearance. B. INTRAOCULAR PRESSURE 1. FIRST SERIES
This is presented graphically (figs. 1-5), the tension recorded at weekly intervals and plotted on the ordinate, the time in weeks along the abscissa. In all the treated eyes except Rabbit 4, the intraocular pressure rose steadily, with some fluctuations, to 60 mm Hg where the graph flattens. The average time taken to reach this level was 7.5 weeks. This period represents an adjustment of ocular dynam ics to the steadily increasing outflow resis tance, a consequence presumably of slowly progressive episcleral fibrosis (or episcleral vascular sclerosis). As outflow resistance increases (demonstrated by the tonography records), so aqueous formation and inflow decreases (shown by the fluorescein appear ance-time test). Ultimately compensation fails and the intraocular pressure rises. The level of ocular hypertension attained and the rapidity with which it is reached would de pend on the degree of episcleral pathology and the availability of other routes for aque ous drainage from the eye (for example, the demonstration by Ruskell and Nemetz of an alternative route to the choroidal vessels and thence to the vorticose veins). This is prob ably why the treated eye in Rabbit 4 did not become glaucomatous. RABBIT 3 :
a. Right eye (fig 1). Baseline ocular tension before treatment with phenol 5% in almond oil was 21 mm Hg. The first four-quadrant subconjunctival injection was given on February 27, 1961, the second on March 3, 1961, and the third, March 17, 1961. Ocular tension rose gradually, reaching 60 mm Hg after five weeks and then fluctuated between 35 mm Hg and 60 mm Hg over the following seven weeks. At this stage, pilocarpine nitrate ( 1 % drops twice daily) was applied topically. This had no effect and was increased to 4% twice daily, with an immediate fall in tension to 22 mm Hg. Two weeks later Diamox (acetazolamide) 125 mg twice daily was given by mouth. Ocular tension
MAURICE H. LUNTZ
676
fell further to IS mm Hg for two weeks and then rose gradually to 24 mm Hg. Medication was stopped after seven weeks and ocular tension returned to 60 mm Hg. It is noteworthy that there was a latent period of two weeks without drugs during which the in traocular pressure remained at normal levels be fore starting to rise. I am at a loss to explain this. A cumulative effect of Diamox, which could explain it, has never been demonstrated in the rabbit and does not occur in man. Little is known of the effects of pilocarpine nitrate drops on the rabbit eye. b. Left eye (fig 2). Baseline ocular tension be fore treatment with phenol 5% in almond oil was 21 mm Hg. The first four-quadrant injection was given on February 17, 1961, the second on March 3, 1961, and the third on March 17, 1961. Ocular ten sion rose gradually reaching 60 mm Hg after five weeks then fluctuated between 35 mm Hg and 60 mm Hg over the following weeks. Pilocarpine nitrate drops ( 1 % twice daily) were administered at this stage but, as this dosage had no effect, it was increased to 4% twice daily, still without any effect. Diamox (125 mg twice daily) by mouth was added and the tension dropped im mediately to 27 mm Hg, but a week later was back to 60 mm Hg and subsequently remained about 30 mm Hg until the drugs were discon tinued two weeks later. Presumably outflow resis tance was greater in the left eye than in the right eye. RABBIT 4 (fig
3)
a. Right eye. Phenol 5% in almond oil was in jected into all four quadrants on March 17, 1961, on March 31, 1961, and on April 14, 1961. Ocular tension fluctuated between 12 mm Hg and 27 mm Hg. This was the only treated eye in the series which failed to become hypertensive, possibly be cause of a well-developed choroidal aqueous outflow route (Nemetz and Ruskell). b. Left eye. This was a control eye and re ceived subconjunctival injections of almond oil only on March 17 and the next two at fortnightly intervals. Intraocular pressure fluctuated between 12 mm Hg and 23 mm Hg. RABBIT 5 (fig
4)
a. Right eye. This was used as a control eye. The ocular tension remained constant at 19 mm Hg, except on two occasions when it was re corded as 18 mm Hg. b. Left eye. The first injection of phenol in al mond oil in all four quadrants was given on March 17, 1961, and the next two at fortnightly intervals. The baseline ocular tension was 20.5 mm Hg and this rose over 10 weeks to 60 mm Hg, remaining at this level for four weeks. At this point an iris inclusion done on the control eye resulted in a fall of intraocular pressure in the operated eye, as well as in the left eye, the latter subsequently returning to 60 mm Hg. At
the 16th week an iris inclusion was done in this eye and the ocular tension dropped to normal val ues. RABBIT 6 (fig
5)
a. Right eye. This was the control eye. Intraoc ular pressure remained at 19 mm Hg with minor fluctuations except on two occasions, one of these at the 16th week, the result of an iris inclusion. b. Left eye. The first injection of phenol in al mond oil in all four quadrants was given on March 17, 1961, and repeated twice at fortnightly intervals. This eye reacted in an identical way to the left eye in Rabbit 5. There was a fall in the intraocular pressure in this eye at the 16th week in sympathy with a fall in pressure in the right eye following an iris inclusion. This is an interesting phenomenon, well illustrated in these two eyes. 2. RABBIT 2(a)
(fig
S E C O N D SERIES 6)
a. Right eye. This was the control eye. Intraoc ular pressure at the start of the experiment was 21 mm Hg but fell within a few weeks to 16 mm Hg and remained fairly constant for the 34 weeks of the test. b. Left eye. Phenol in almond oil was injected on September 22, 1961, and repeated at the fifth week Intraocular pressure was consistently higher than in the left eye, fluctuating between 22 mm Hg and 33 mm Hg, from a baseline intraoc ular pressure of 22 mm Hg. The rabbit developed ear canker and had to be killed at the 34th week. RABBIT 3(a)
(fig
7)
a. Right eye. The control eye, duration of the test 42 weeks. Ocular tension fluctuated around 15 mm Hg. , b. Left eye. Subconjunctival phenol 5% in al mond oil was injected on September 22, 1961, and repeated at the fifth and again at the eighth week. The intraocular pressure remained consis tently higher than the control eye, the baseline intraocular pressure before injection being 16 mm Hg. For the most part the intraocular pressure fluctuated between 25 and 30 mm Hg. RABBIT 4(a)
(fig
8)
The baseline intraocular pressure was 19 mm Hg in the left eye and 20 mm Hg in the right eye. a. Right eye. This was the injected eye. The first four-quadrant injection was given on September 22, 1961, and repeated at the 4th, 8th and 10th week. Again the intraocular pressure remained consis tently higher than in the left eye, fluctuating be tween 25 mm Hg and 30 mm Hg, rarely exceed ing this figure. At the 34th week the rabbit de veloped ear canker and had to be killed. b. Left eye. This was the control eye.
677
E X P E R I M E N T A L GLAUCOMA IN T H E RABBIT RABBIT 5 ( a )
(fig
9)
The baseline intraocular pressure was 19 mm H g in the left eye and 20 mm H g in the right eye. a. Right eye. Phenol was injected subconjunctivally in four quadrants on September 25, 1961, and repeated twice, at the 11th and 22nd week. Intraocular pressure varied between 20 mm H g and 35 mm H g , remaining consistently higher than that in the left eye. b. Left eye. The left eye was the control eye, the intraocular pressure remaining constant at 15 mm Hg. The rabbit was killed after 48 weeks. RABBIT 6 ( a )
(fig
10)
a. Right eye. Subconjunctival phenol in almond oil was injected on September 25, 1961, into four quadrants and repeated after eight weeks. Intra ocular pressure was 19 mm H g and rose gradual ly, with a few fluctuations, to 45 mm H g at the 46th week. The experiment was terminated at the 48th week. b. Left eye. This was the control eye and had a baseline ocular tension of 19 mm Hg. This fell gradually over the test period to 15 mm H g at the 36th week, where it remained until the test was terminated at the 48th week. C. TONOGRAPHY
The test was carried out on all the rabbits in the second series. The results, expressed as the C value, are summarized in Table 1. The C values in all the treated eyes were well below the average normal value, and there was a significant difference between these values and the C
values in the control eyes, which were with in normal limits. These results indicate an increased resis tance to aqueous outflow in the treated eyes. D. FLUORESCEIN APPEARANCE-TIME TEST
The results of this test are summarized in Table 2. Two results are recorded from each eye of the rabbits in the second series. Taking into account the inevitable crudeness of this test, these are remarkably constant. The treated eyes all showed prolonged TABLE 2 FLUORESCEIN APPEARANCE-TIME TEST
Appearance Time
Rabbit Eyes
First Result
Second Result
(min) 2 1
(sec) 20 45
(min) 3 2
(sec)
3 (a) Right (c) Left
1 3
30 30
1 3
50 10
4 (a) Right Left (c)
2 1
5 35
2 1
20 30
5 (a) Right Left (c)
3 2
10 5
3 2
10 20
6 (a) Right Left (c)
3 2
30
3 1
50
2 (a) Right Left (c)
5
(c) = control eye.
TABLE 1 TONOGRAPHY
Mean C
Rabbit
Eye
2(a)
Right Left (c)
0.06 0.19
(a) Controls
(a) Controls
3(a)
Right (c) Left
0.29 0.08
0.21
±0.03
4(a)
Right Left (c)
0.06 0.20
(b) Treated
5(a)
Right Left (c)
0.08 0.15
0.07
6(a)
Right Left (c)
0.08 0.18
C = coefficient of resistance to outflow ( c ) = control eye
Standard Deviation
(b) Treated ±0.02
MAURICE H. LUNTZ
678
fluorescein appearance time when compared to the controls (comparing treated and con trol eyes in the same animal), and this difference is obviously significant without having to resort to further statistical analy sis. This observation showed that there had been a reduction in aqueous inflow in the treated eyes, presumably as an attempt to compensate for the increased resistance to outflow. This had failed with the result that these eyes became glaucomatous. It was this compensatory process that dictated the slow rise of ocular tension, a situation analagous to the onset of chronic simple glaucoma in man. There is no absolute normal value for this test as it varies widely in different subjects so that one can only compare an abnormal eye with the normal, preferably in the same animal. DISCUSSION OF RESULTS
Results have been most encouraging. Glaucoma supervened in four out of five eyes treated with phenol in almond oil in the first series, and in all treated eyes in the sec ond series. A high level of intraocular pres sure was reached in the first series and was maintained for an average period, in the four eyes, of 9.5 weeks, when the experi ment was terminated. The reactions to pilocarpine and Diamox in two eyes in the first series and iris inclu sion in the other two were in accord with what one would expect. This raises the pos sibility of using animals eyes prepared by this method to investigate the effectiveness of new preparations for the treatment of glaucoma, before applying them to man. The level of ocular hypertension in the second series was not as striking as in the first. This may have been due to the different spacing in time of the injections, or to the use of a different breed of animal. Nevertheless, consistently raised pressures often reaching over 30 mm Hg were main tained in the treated eyes for an average pe riod of 40 weeks, when the experiment was
terminated. During the same time, aqueous outflow resistance and aqueous inflow time were increased in these eyes. I attempted to produce an outflow glauco ma by obliterating the aqueous outflow channels. That I succeeded in doing this is shown by the tonography results which re corded low C values in the treated eyes, and indirectly by the delayed fluorescein-appearance time, suggesting a reduced aqueous inflow and turnover in the glaucomatous eyes. No gross ocular damage could be detected by either macroscopic or microscopic exami nation of the treated eyes. The intraocular pressure in the control eyes remained constant. The average tension readings compared well with those from other series. This indicated that the methods used for measuring intraocular pressure and aqueous outflow are valid. It would be ap propriate at this stage to examine in more detail the validity of these methods. VALIDITY OF METHODS
The methods used for measuring intraoc ular pressure and aqueous outflow have al ready been described. I have earlier in this paper submitted that they can be used with a high degree of clinical accuracy. I now propose to show that the results obtained in this experiment are valid, by comparing the measurements in my control eyes with mea surements obtained from normal rabbit eyes by other investigators. There should be close agreement and, in fact, this is what one finds. Secondly, the measurements obtained from the control eyes show very little scat ter around the mean value when expressed by the standard deviation from the mean. a. Intraocular pressure. In Table 3, the mean ocular tension (Po) in the control eyes in my series is compared with the fig ures obtained by Becker and Constant ( 1956) when they measured the intraocular pres sure in normal rabbit eyes in vivo, using a standardized, weighted Schi^tz tonometer. There is good agreement.
EXPERIMENTAL GLAUCOMA IN THE RABBIT TABLE 3 COMPARISON OF MEAN OCULAR TENSION
Mean Ocular Standard Tension (Po) Deviation (mm Hg)
Series Present series Becker & Constant Series
17.8 18.0
±1.9 ±3.7
The standard deviations are not large, striking evidence for the accuracy of the Schi^tz tonometer, used under these condi tions, for measuring intraocular pressure in rabbits. b. Tonography. Table 4 compares the mean C value in my series of control rabbit eyes with the mean C values obtained by Becker and Constant (1956) and by Kornblueth and Linnér (1955) and Levene and Bloomgarden (1961) when measuring facility of outflow in normal rabbit eyes, using a similar technique as in the present study. The latter authors also showed that there was no significant difference in results be tween right and left eyes; the right eyes were measured first. The standard deviation about the mean is small, underlining the consistency of the measurements. The C value in the present series is lower than in the other three, but well within the range of normal, which is 0.14 to 0.60. This emphasizes very clearly that tonography is not a scientifically ac curate measurement of resistance to outflow but is nevertheless a useful clinical test as long as it is accepted that there is a large range for normal values. The value obtained depends to a great extent on environmental conditions pertaining at the time of the test. TABLE 4 COMPARISON OF C VALUES
Series Present series (1962) Kornbluth & Linner (1955) Becker & Constant (1956) Levene & Bloomgarden (1961)
Mean Standard C Value Deviation 0.21 0.30 0.34 0.26
±0.03 ±0.04 ±0.10 ±0.06
679
In the four series quoted in Table 4, al though the test was done by different inves tigators under vastly differing conditions, the mean values all lie within the normal range and the standard deviations for each series are small. In my opinion Table 3 provides sufficient evidence that the methods employed in this experiment have provided valid results. If one accepts that the results are valid, then the technique that I have described is a use ful method of producing chronic outflow glaucoma in animals, which closely resem bles primary glaucoma in man. The possibil ities of studying the effects of various drugs or surgical procedures on these eyes is im mediately obvious. One hopes that these ex periments will emphasize the importance of the episclera and in particular the episcleral vessels in the regulation of aqueous-humor flow and dynamics and its possible role in the etiology of primary glaucoma. SUMMARY
A method of producing primary glaucoma in rabbits by means of the subconjunctival injection of phenol is described. In nine out of 10 rabbits treated in this way, a chronic glaucoma (as described in the introduction) resulted. The effects of pilocarpine drops and Diamox were studied in two rabbits, and of iris inclusion operations on four eyes, two control and two treated. Aqueous outflow and inflow studies sug gest that the inflow of aqueous was reduced and the resistance to outflow increased in "treated" eyes with raised intraocular pres sure. The methods have been described in de tail, and a critical analysis of the validity of the results has been offered. Hospital Street. ACKNOWLEDGMENT
I wish to acknowledge technical assistance from the Nuffield Laboratory of Ophthalmology, Oxford, and the Surgical Laboratory of the Uni versity of Cape Town, as well as the Photo Unit, De partment of Medicine, University of the Witwatersrand.
MAURICE H. LUNTZ
680 REFERENCES
Ashton, N.: Brit. J. Ophth., 35: 291, 1951. : Brit. J. Ophth., 38: 129, 1952. : Glaucoma symposium (Council Inter nat. Organiz. M. Sei). (Edited by Duke-Elder, S.) Oxford, Blackwell, 1955. Becker, B., and Constant, M. : Arch. Ophth., 55 : 305, 1956. Davis, F. A.: Tr. Am. Ophth. Soc, 27: 401, 1929.
Kornblueth, W. and Linnér, E. : Arch. Ophth., 54: 717, 1955. Leber, T. : Arch. f. Ophth., 19: 87 (pt. 2) 1873. Levene, R. Z., and Bloomgarden, C. : Arch. Ophth., 66: 149, 1961. Nemetz, U. R. : Ber. Deutsch. Ophth. Ges., 60: 60, 1956. Pickering, G. W. : High Blood Pressure. Longon, Churchill, 1955, p. 85. Ruskell, G. L.: Arch. Ophth., 66: 861, 1961. Smith, P. : On the Pathology and Treatment of Glaucoma. Churchill, London, 1891.
TONOGRAPHIC SURVEY* LORAND V.
JOHNSON,
M.D.
Cleveland, Ohio
A tonographic survey was made of 7,577 eyes, consecutive and unselected except for the exclusion of all individuals who had a1 previous diagnosis of or treatment for ex isting glaucoma. These persons, aged over 41 years, consecu tively passed one point in this city, namely the front door to my office. It is by coinci dence that they are mainly Caucasians. Tonography was performed by skilled techni cians, using standard tonographic equip ment. Initial pressure was obtained by not ing the initial scale readings of the tonogram to the nearest 0.25 scale division and1 the intraocular pressure read to the nearestt mm Hg from a graph of the 1955 Schlitz: tonometer scale of the Committee on Stan dardization of Tonometers of the American1 Academy of Ophthalmology and Otolaryn-\ gology. Schi^tz pressure and facility of1 outflow were recorded in categories of male5 or female and tabulated into five-year age; delineations (table 1). The descending,> stair-step lines separate those eyes with P 0 / C greater than 100 and those with less than1 100. From these charts, the data were statis tically analyzed.
A.
FACILITY OF AQUEOUS OUTFLOW
The facility of aqueous outflow was statistically analyzed to answer the following questions : Question 1. At each age classification is there a real difference in the average outflow between male and female ? Answer. There is no difference in the av erage outflow between men and women in each age classification except for the age group of 66-70 years. This result is based on the use of Student t-test and Kolmogorov-Smirnov test. In the age group of 66-70 years, the female average facility of outflow is 0.2688 and the male average is 0.2514. Although this difference is statistically significant, the difference has no clinical interpretation. Thus, it is concluded that there is no real difference in the average outflow between male and female in any of the age classifications. Therefore, the facility of outflow data for men and women were combined into one group for each age clas sification. Question 2. Are the combined facility of outflow data for men and women normally distributed in each age classification? Answer. The data were plotted on normal * Presented at the 101st annual meeting of thee probability paper. Chart 1 shows the plot American Ophthalmological Society, Hot Springs,I for the age group 56-60 years. Virginia, May, 1965. The statistical analyses were done by Julio N. Berrettoni.
This chart shows three distinct straight