Controlled elevation of intraocular pressure in anesthetized mice

Our purpose was to develop a protocol for prolonged anesthesia in mice and evaluate optic nerve axon injury in response to 4 h of controlled elevation of intraocular pressure (CEI). During CEI, C57BL/6 male mice (3-5 months old) were anesthetized with 1.5% isoflurane with 100% oxygen for 4 h and placed on a warm platform, with expired gas and anesthetic actively evacuated. Lactated ringers (0.5 ml) with 5% dextrose was administered subcutaneously at the start and end of CEI. Physiological parameters (oxygen saturation = O₂, heart rate = HR, systolic blood pressure = SBP, and temperature) were monitored throughout the 4-h CEI. One eye was cannulated with polyurethane tubing connected to a balanced salt solution reservoir and IOP elevated to 20 (N = 18), 30 (N = 13), 50 (N = 14), and 60 mmHg (N = 16). An additional group of 22 female mice was exposed to CEI of 60 mmHg. Fourteen days after CEI, optic nerves were assessed for axonal injury by masked observers that assigned a grade on a scale from 1 (normal) to 5 (>50% of axons degenerating). CEI optic nerve injury was compared to injury assessed in contralateral optic nerves (N = 84) and naïve optic nerves (N = 18) using a one-way ANOVA followed by Kruskal-Wallis test for multiple comparisons. The relationship between optic nerve injury, physiological parameters, and IOP were assessed by linear regression analyses. Physiologic parameters remained stable throughout CEI (O₂ = 95 ± 9%; HR = 450 ± 39; SBP = 102 ± 15 mmHg, and temperature = 38 ± 0.7 °C) and were not statistically different between groups (all comparisons had P > 0.5). Mean optic nerve injury grades (±SD) for naïve optic nerves (1.01 ± 0.02) were not significantly different from fellow/contralateral optic nerves (1.03 ± 0.07, P > 0.99), or from CEI of 20 mmHg (1.04 ± 0.08, P > 0.99) or 30 mmHg (1.05 ± 0.06, P = 0.6). However, animals exposed to CEI of 50 mmHg (2.09 ± 1.43, P = 0.0005) and 60 mmHg (male: 2.86 ± 1.30, P < 0.0001, female: 1.63 ± 1.00, P = 0.0006) developed significant optic nerve injury relative to their fellow/contralateral optic nerves. Axonal injury grades following a CEI of 60 mmHg were not significantly different between male and female mice (P = 0.19). Optic nerve injury positively correlated (P < 0.0001) with IOP and not with physiological parameters, indicating that the optic nerve injury is IOP-related. In conclusion, prolonged anesthesia in mice requires careful attention to animal physiology. With this, a 4-h exposure to elevated IOP can produce significant optic nerve injury with IOPs equal to or greater than 50 mmHg. We provide detailed descriptions of methods and materials for producing prolonged elevations of IOP in mice while maintaining and monitoring their physiology, as well as a unique, cost-effective transducer system for monitoring pressure delivery.