Trypsin dose optimization for glaucoma induction in rabbit model

Article highlights

1. Optimize appropriate dose of trypsin that cause constant and progressive elevation of intraocular pressure.
2. Two weeks’ elevation of intraocular pressure resulted in loss of retinal ganglionic cells and degeneration of optic nerve.

Trypsin dose optimization for glaucoma induction in rabbit model

Nour. M. Muharib and Hameed. A. Al- Timmemi

Corresponding author: Nour. M. Muharib

Department of Surgery and Obstetrics, College of veterinary medicine, University of Baghdad, Baghdad, Iraq

nour.muharib93@gmail.com, https://orcid.org/0000-0002-8022-8421

hameed_altmeme@yahoo.com, https://orcid.org/ 0000-0002-2263-3410

Abstract

Glaucoma is a neurodegenerative disease identified by optic nerve damage and loss of retinal ganglionic cells. The aim of the study was to test the precise dose of trypsin to induce glaucoma in a rabbit model. Nine White New Zealand rabbits were randomly divided into three groups: Group one (G1) normal animals as control, group two (G2) the animals were injected with 0.2 ml of 0.04% trypsin and group three (G3) injected with 0.2 ml of 0.05% trypsin into posterior chamber of eye. A schiotz tonometer was used to measure intraocular pressure (IOP) before the injection, two days after injection and once a week until the end of the second week. At the end of the second week, the histology of the retina and optic nerve was studied. G3's intraocular pressure increased by 43.7%, while G2's intraocular pressure increased by 0.7% compared to G1. Retinal ganglionic cells (RGCs) decreased in G3 by 91.5% and G2 by 43.8% in comparison with G1. Optic nerve histology in G3 displayed decreased Schwann cell quantity with vacuolation and low orientation of nerve fibers compared with G1 and G2. Group three had the highest increase in IOP due to obstruction of drainage canal and preventing outflow of aqueous humor. This resulted in mechanical compression on the retina, ending with loss of RGC and degeneration of the optic nerve. The conclusion is that 0.2ml of 0.05% trypsin is the proper dose for the induction of glaucoma.

Keywords: Glaucoma, Rabbit, Trypsin, Retinal ganglionic cells, Optic nerve

تحدید الجرعة المناسبة من التربسین لاستحداث الجلوکوما فی نموذج الأرنب

نور محمود محارب و حمید علی  التمیمی

فرع الجراحة والتولید، کلیة الطب البیطری، جامعة بغداد، بغداد، العراق

الخلاصة

الجلوکوما هو مرض تنکسی عصبی یتمیز بتلف العصب البصری وفقدان الخلایا العقدیة لشبکیة العین. کان الهدف من الدراسة هو اختبار الجرعة الدقیقة من التربسین لاستحداث الجلوکوما فی نموذج الأرنب. تسعة أرانب بیضاء نیوزیلیندیة تم تقسیمها عشوائیا إلى ثلاث مجامیع: المجموعة الأولى حیوانات طبیعیة کمجموعة سیطرة، المجموعة الثانیة تم حقن الحیوانات ب 0.2 مل من 0.04% من التربسین والمجموعة الثالثة تم حقنها ب 0.2 مل 0.05% من التربسین فی الغرفة الخلفیة للعین. تم قیاس ضغط العین قبل الحقن، بعد یومین من الحقن ومرة أسبوعیا حتى نهایة الأسبوع الثانی باستخدام مقیاس توتر العین، تم دراسة أنسجة الشبکیة والعصب البصری فی نهایة الأسبوع الثانی. ارتفع ضغط العین فی المجموعة الثالثة بنسبة 43.7% بینما ارتفع ضغط العین فی المجموعة الثانیة بنسبة 0.7% مقارنة مع المجموعة الأولى. انخفضت الخلایا العقدیة الشبکیة فی المجموعة الثالثة بنسبة 91.5% وانخفضت فی المجموعة الثانیة بنسبة 43.8% مقارنة مع المجموعة الأولى. أظهرت انسجه العصب البصری فی المجموعة الثالثة انخفاضا فی کمیة خلایا شوان مع تکوین فجوات وانخفاض توجه الألیاف العصبیة مقارنة مع المجموعة الأولى والمجموعة الثانیة. سجلت المجموعة الثالثة اعلى نسبة زیادة فی ضغط العین بسبب انسداد قناة الصرف ومنع التدفق الخارجی للخلط المائی مما سبب ضغط میکانیکی على شبکیة العین الذی أنتج فقدان فی الخلایا العقدیة الشبکیة وتنکس العصب البصری. الاستنتاج هو إن 0.02 مل من 0.05% من التربسین هی الجرعة المناسبة لاستحداث الجلوکوما.

Introduction

Glaucoma is a neurodegenerative disease identified by optic nerve damage and loss of retinal ganglionic cells (1). The most common risk factor of glaucoma is increased intraocular pressure (2). Rabbit is the proper model for study pharmacokinetic therapies of eye diseases furthermore the adequate size of eye ball that create easy handling (3). Glaucoma was induced by different methods and different models. (4) demonstrated that cauterization of episcleral vein in mouse model the intraocular pressure was increased until fourth week but this method may associate with burns of the sclera. Hyaluronic acid was injected into the anterior chamber in rat model the intraocular pressure increased up to 8 days with repeated injections the elevation of intraocular pressure extended to 10 weeks this technique may produce corneal problems due to the injection side effect (5). Laser photocoagulation was used for induction of glaucoma in monkey and this produced elevation of intra ocular pressure and changes in the optic nerve and retina but this method is expensive and need special expertise (6). Injection of 150 units of alpha-chemotrypsine into the posterior chamber resulted in increase of intraocular pressure some animals remained with high intraocular pressure till one year but there is no histological study that confirm loss of retinal ganglionic cells (7). In the present study demonstration, the proper dose of trypsin for inducing glaucoma in rabbit model.

Materials and methods    

Experimental animals

A total of 9 male White New Zealand rabbits, age 4-6 months, weight 1.2-1.8 all rabbits allowed for free access of food and water. The animals were randomly divided into three groups (n=3). Group one(G1) normal animals as control. Group two (G2) the animals were injected with 0.2 ml of 0.04% trypsin. Group three (G3) animals were injected 0.2 ml of 0.05% trypsin in to the posterior chamber.

Anesthetic protocol

Animals were anesthetized after intramuscular injection of acepromazine 1mg/kg followed after five minutes with xylazine 5mg/kg and ketamine 35mg/kg (8).

Induction of glaucoma

Glaucoma was induced by injection of trypsin into posterior chamber with using of 31gauge syringe (Figure 1). G2 injected with 0.2 ml of 0.04% trypsin while G3injected with 0.2ml of 0.05% trypsin.

Figure 1: injection of 0.2 ml of trypsin in to posterior chamber for induction of glaucoma

Tonometry

Intraocular pressure measured before induction, after two days from induction and once weekly until the end of second week with using of schiotz tonometer with 7.5g weight after application of local anesthetic eye drop 0.05% tetracaine.

Histopathology

Histopathological examination of retina and optic nerve. The eyes were collected after two weeks from induction and fixed in Davidson’s solution for 24h then transferred in to different concentrations of alcohol after that processed in paraffin (9). The tissue sectioned in sagittal line at 5 µm with microtome. Tissue were stained with hematoxylin and eosin.

Statistical analysis

Statistical analysis system- SAS program for data analysis to extract the means and SE, One-way ANOVA and Tukey’s-b of post hoc test were used for estimate the significant difference of means between groups when P < 0.05.

Results

Clinical Signs

Clinical signs of the G2 there was hyperemia of episcleral veins there was no luxation of lens, no edema of the cornea and no enlargement of the anterior chamber depth(ACD) (Figure 5-6). Conversely in G3 there was hyperemia of episcleral veins (Figure.3-4) enlargements of ACD in animals with extreme elevation of intraocular pressure (Figure.5-6), luxation of lens (figure 7-8), edema of cornea in addition to that friable cornea was established in two of three animals in the first seven days after induction. While animals with moderate elevation of intraocular pressure there is no luxation of lens. One animal in the G3 showed cloudy lens this animal was excluded from the experiment.

Figure 2: Photographs show hyperemia of episleral veins.

Figure 3: Enlargement of anterior chamber in G3 in the first week after injection.

Figure 4: Enlargement of anterior chamber in G3 in the second week after injection.

Figure 5: No enlargement of anterior chamber in G2 first week.

Figure 6: No enlargement of anterior chamber in G2 second week.

Figure 7: Photographs lateral view show luxation of lens due to extreme elevation of intraocular pressure in animal of G3.

Figure 8: Photographs Anterior view show luxation of lens due to extreme elevation of intraocular pressure in animal of G3.

Tonometry

Intraocular pressure results were increased 0.7% in G2 and 43.7% in G3 in comparison with G1. one of three animals in G2 showed increased IOP it reached to thirties and the other two animals showed the IOP increased but within normal range. In G3 intraocular pressure increased after two days from induction and continued in progressive elevation, intraocular pressure reached to forties. edematous and fragile cornea in the first seven days resulted in false negative recording of intraocular pressure. The statistical analysis revealed no significant difference between means of IOP in G2 and G1 P > 0.05 in contrast G3 showed significant difference of IOP in compare with G2 and G1 P< 0.01 (Table 1, Figure 9).

Table 1: The means, SE of intraocular pressure

* P<0.05, **P<0.01.

Figure 9: Diagram of means of IOP, G3 had the highest IOP among all groups.

Histopathology

Retinal Ganglion Cells

Histological sections of retina revealed decreased number of retinal ganglion cells 43.8% in G2and 91.5% in G3 in comparison with normal animals in G1 (Figures 10-12). Group three is significantly different from G2 and G1in number of RGCs P

Figure 10: Micrograph of normal retina of G1, appeared normal retinal ganglionic cells (black arrow) (40X).H&E stain.

Figure 11: Micrograph of retina in G2 after two weeks from induction of glaucoma. (40X). H&E stain.

Figure 12: Micrograph of retina in G3 after two weeks from induction of glaucoma, (40X). H&E stain.

Table 2: The means, SE and SD of number of retinal ganglionic cells

*P < 0.05, ** P < 0.01.

Figure 13: Diagram of means of RGC number, G3 display the lowest number of RGC among all groups, G2 also show mild decrease in number of RGC while G1 exhibit the highest number of RGC.

Optic nerve

There was increased of vacuolation, congested blood vessels, decreased quantity of schwann cells, segmental demyelination and low orientation of nerve fibers in G3(Figure 16). Optic nerve histology in G2 shown cells vaculation and constriction of blood vessels (Figure 15) instead of that in normal optic nerve in G1 there was no vaculation and obvious orientation of nerve fibers (Figure 14).

Figure.14: Micrograph of the longitudinal section of normal optic nerve. Normal myelination of nerve fibers, there is no Wallerian degeneration and good orientation of nerve fibers, (40X). H&E stain.

Figure 15: Micrograph of longitudinal section of optic nerve histology of G2 after two weeks from induction of glaucoma, there is vacuolation (black arrow) and disarrangement of optic nerve fibers (40X). H&E stain.

Figure 16: Micrograph of the longitudinal section of optic nerve histology of G3 after 2 weeks fom induction of glaucoma established low concentration of Schwann cells, congested blood vessel (white arrow), vacuolated degenerative nerve fibers (black arrow) and segmental demyelination (40X). H&E stain.

Discussion

Trypsin is a proteolytic enzyme (10) the main role of this substance was harnessed for digestion and get debris to stop or decrease aqueous out flow. Alpha chymotrypsin which is a sub type of trypsin was used by )7) for induction of glaucoma in rabbit model. Tonometry results of G2 displayed elevation of intraocular pressure in one animal while in G3 all animals had increase of IOP and this occur due to lysis of trabeculi that obstruct the drainage path way of the aqueous humor. The concentration of trypsin used in G2 was inadequate to get the proper lysis to block the out flow of aqueous fluid.

The sudden and progress increased of intraocular pressure accompanied with different changes in the eye structures that was noticed clinically include displacement of lens increase of anterior chamber depth(ACD). Increased IOP cause expansion of eye ball and scleral stretching that result in posterior luxation of lens and this sequel was confirmed by (11). Edema of cornea resulted from increased intraocular pressure that caused damaging of endothelium layer which is responsible of absorbance of water to the cornea. (12). ended with formation of keratoglobus which is limbus to limbus thinning of cornea. (13) this result agreed with (7) who documented displacement of lens due to extreme elevation of intraocular pressure so that increase of ACD. (14) reported enlargement of ACD and thin walled cornea in animals with chronic elevation of intraocular pressure after injection of autologous fixed red blood cells in to the anterior chamber. While (15) did not report displacement of lens or increase in ACD when 0.1ml of 0.3% carbomer injected into the anterior chamber resulted in increase of intraocular pressure in eleven of twelve eyes with intraocular pressure mean 29-35 mmHg extended from (20-50) days.

Trypsin enzyme that digest protein resulted in flexible cornea which is the main factor that effect on tonometry reading according to study by (16) (17) and this responsible of false negative readings of IOP in the first week in most rabbits in G3.

Histological findings presented loss and decreased number of retinal ganglionic cells (RGCs) in G 2 with 43.8% while in G3 the decrease of RGCs was 91.5% in compare with G1 due to increased intraocular pressure that cause mechanical compression which inhibit axonal transport resulted in enhancement of calcium influx in to the cells terminated with death of ganglion cells and this assured by (18).This disagree with study by (4)they demonstrated 20% loss of RGCs after two weeks from induction of glaucoma by occlusion episcleral veins in mouse model. (19) whom reported 20% loss of axons when polystyrene microbeads injected into the anterior chamber in mice and rat model resulted in elevation of intraocular pressure up to 2 weeks and the prolonged elevation of intra ocular pressure up to (4-5 weeks) made this loss of axons. While (20) revealed the weekly injection of microspheres with hudroxypylmethylcellulose in to anterior chamber until 30-week period resulted in 30% loss of RGC.

Conclusion

Using of 0.2ml of 0.05% trypsin is the proper dose for induction of glaucoma which resulted in constant increased of intraocular pressure with degenerative changes of retina and optic nerve with in two weeks.

Acknowledgments

We are grateful to the College of Veterinary Medicine/University of Baghdad for providing the required materials for the research, as well as a suitable location and circumstances for the animals to be housed.

Conflicts of interest

We declare that there are no conflicts of interest regarding the publication of this manuscript.