Method for constructing an animal model of open-angle glaucoma

A method for constructing an animal model of open-angle glaucoma disease, comprising:

• S1:
• Selecting a SPF C57BL/6J mouse, a SD rat, or a Japanese big-eared white rabbit as the model organism, wherein the C57BL/6J mouse is 6 to 8 weeks old, the SD rat is 4 to 8 weeks old, and the Japanese big-eared white rabbit is 12 to 14 months old;
• S2:
• Establishing an elevated intraocular pressure model:
• Anesthesia method:
• Weighing the model animals, inducing general anesthesia in the C57BL/6J mouse and SD rat via intraperitoneal injection of 1% pentobarbital sodium at a dose of 40 mg/kg to 50 mg/kg, and inducing general anesthesia in the rabbit via intramuscular injection of ketamine hydrochloride at a dose of 22 mg/kg to 44 mg/kg;
• Once deep anesthesia is confirmed, proparacaine hydrochloride eye drops are administered for ocular surface anesthesia;
• IFN-γ injection preparation:
• Diluting IFN-γ stock solutions specific to each species with PBS to final concentrations of 9000 Units/ml, 20000 Units/ml, and 30000 Units/ml, respectively;
• Anterior chamber injection:
• Mouse/rat:
• (1) Placing the anesthetized mouse/rat on the operating table of a surgical microscope, with a temperature-controlled mat beneath the table to maintain body temperature during the procedure;
• Positioning the eye to be treated upwards, disinfecting the eyelid margin using an iodophor-soaked cotton swab, arranging the eyelashes, and exposing the eyeball;
• The ocular surface is rinsed with sterile PBS or an ofloxacin antibiotic eye drop, followed by the removal of residual liquid and debris with a sterile dry cotton swab;
• (2) The eyeball is stabilized using microscopic smooth forceps, and a small puncture is made approximately 2 mm outside the pupil margin using a 34G needle to release a portion of the aqueous humor;
• A microsyringe (measuring range:
• 10 μl) connected to a 33G needle is inclined at an angle of 25° to inject IFN-γ (mouse:
• 1 μl, rat:
• 3 μl) through the corneal puncture;
• Subsequently, 2 μl of air is injected slowly to form a complete bubble in the anterior chamber;
• The needle of the microsyringe is retained in the anterior chamber for 30 seconds after the injection and then slowly withdrawn, allowing the bubble to seal the corneal puncture and prevent backflow of the injected reagent;
• The bubble is absorbed within a few hours;
• Throughout the puncture and injection procedure, care is taken to avoid puncturing the iris or the anterior lens capsule;
• The experimental group receives IFN-γ as the injection agent, while the control group is administered an equivalent volume of PBS;
• (3) Following the procedure, ofloxacin antibiotic eye ointment is applied to prevent infection;
• The mouse is placed on an animal temperature maintenance device to sustain body temperature during recovery;
• Once the mouse regains consciousness, it is returned to the animal housing facility;
• (4) Anterior chamber injections are repeated once per week to maintain the concentration of IFN-γ in the anterior chamber;
• Rabbit:
• (1) The anesthetized rabbit is placed directly on a surgical operating table;
• The disinfection procedure is identical to that of the mouse/rat;
• (2) The eyeball is stabilized using microscopic smooth forceps or a sterile cotton swab;
• A puncture is made at the corneoscleral limbus using a 30G needle to release a portion of the aqueous humor;
• A microsyringe (measuring range:
• 100 μl) connected to a 30G needle is inclined at an angle of 25° to inject IFN-γ (rabbit:
• 55 μl) through the corneal puncture;
• The needle remains in the anterior chamber for 30 seconds after the injection and is then slowly withdrawn;
• The puncture site is pressed and sealed with a sterile cotton swab to prevent backflow of the injected reagent;
• All other procedures are consistent with those for the mouse/rat;
• Intraocular Pressure Measurement:
• Mouse/Rat:
• (1) Gas Inhalation Anesthesia:
• Prior to each intraocular pressure measurement, the mouse/rat is anesthetized using 2%-4% isoflurane mixed with 95% oxygen;
• The animal is placed in a gas anesthesia chamber for 3 to 5 minutes;
• Anesthesia depth is assessed by toe pinch response or the presence of a blinking reflex;
• Upon confirming anesthesia, a proparacaine hydrochloride eye drop is applied for ocular surface anesthesia;
• (2) Use of TonoLab Rebound Tonometer:
• The TonoLab rebound tonometer, calibrated for both mice and rats (Π=mouse, r=rat), is used to measure intraocular pressure;
• During measurement, the rebound probe is aligned with the center of the cornea, ensuring the tonometer remains horizontal and perpendicular to the corneal surface;
• Care is taken to avoid applying pressure to the neck or orbit, as this may artificially elevate intraocular pressure;
• Measurements are conducted by the same operator within the same time frame;
• The tonometer automatically calculates a measurement by discarding the highest and lowest values from six consecutive readings;
• Five complete measurements are performed within two minutes after anesthesia induction, and the mean of these values is recorded as the final intraocular pressure;
• (3) Measurement Time Points:
• Baseline intraocular pressure is measured before the initial anterior chamber injection;
• Subsequent measurements are taken every other day after injection;
• Anterior chamber injections are repeated biweekly, and intraocular pressure is monitored for 4 weeks;
• If intraocular pressure remains elevated, measurements are performed biweekly for up to 12 weeks;
• Rabbit:
• (1) Intraocular pressure in rabbits is measured under topical anesthesia;
• The rabbit is restrained using a rabbit box for ease of handling;
• (2) Use of Tono Vet Rebound Tonometer:
• Intraocular pressure is measured using the Tono Vet rebound tonometer, which is pre-calibrated for rabbits, cats, and dogs and does not require manual adjustment;
• The probe is aligned with the center of the cornea, and the tonometer is held horizontally and perpendicularly to the corneal surface;
• Care is taken to avoid compressing the neck when using the rabbit box for restraint, as this can artificially elevate intraocular pressure;
• Other measurement procedures are identical to those used for mice and rats;
• S3:
• Removal of the Eye for Observation of Chamber Angle Opening, Trabecular Meshwork Injury, and Retinal Injury
• A group of experimental animals was euthanized at the end of the 4th, 8th, and 12th weeks following the initial anterior chamber injection, after successful establishment of the elevated intraocular pressure model;
• The eyeballs were then excised for resin sectioning and HE staining to evaluate the chamber angle opening, trabecular meshwork injury, and retinal injury;
• The excised eyeballs underwent fixation, embedding, sectioning (primarily including chamber angle trabecular meshwork and retinal sectioning), and HE staining;
• The prepared sections were photographed and examined under an optical microscope to assess the chamber angle opening, trabecular meshwork injury, and retinal injury;
• S4:
• Evaluation of Glaucoma-Induced Optic Nerve Injury Following the Establishment of the Elevated Intraocular Pressure Model
• A group of experimental animals was selected at the end of the 4th, 8th, and 12th weeks following the initial anterior chamber injection for perfusion;
• The retinas were isolated and prepared for flat-mounting to quantify RGCs;
• The loss of RGCs was assessed to evaluate optic nerve injury in the elevated intraocular pressure model and to determine whether the observed optic nerve damage corresponded to characteristic glaucomatous retinal degeneration;
• S5:
• Establishment of an Intraocular Pressure-Lowering Control Group to Evaluate the Effect of IFN-γ on Intraocular Pressure
• To exclude the influence of elevated intraocular pressure and to verify the direct effect of IFN-γ on retinal injury, an intraocular pressure-lowering control group was established;
• IFN-γ was injected into the anterior chambers of the experimental animals, followed by intervention using intraocular pressure-lowering eye drops;
• Specifically, 0.5% timolol maleate eye drops were administered twice daily, with one drop instilled into the conjunctival sac at 9:00 AM and 6:00 PM for 12 weeks;
• Intraocular pressure was monitored according to the previously described method and time points [3.4];
• The intraocular pressure was maintained at baseline levels, or within a fluctuation range comparable to that of the PBS control group receiving anterior chamber injections, to exclude the direct impact of elevated intraocular pressure on RGCs;
• A group of experimental animals was euthanized at the end of the 4th, 8th, and 12th weeks, and the eyeballs were removed for resin sectioning, HE staining, retinal flat-mount preparation, and RGC quantification (following the aforementioned methods);
• The extent of whole retinal layer injury and the RGC loss rate were evaluated and compared with the IFN-γ anterior chamber injection group without intraocular pressure-lowering intervention;
• The results indicated a significant reduction in the RGC loss rate and an absence of whole retinal layer injury after eliminating the intraocular pressure elevation factor caused by IFN-γ, thereby confirming the specific effect of IFN-γ on intraocular pressure.