Tonopen measurement of intraocular pressure in mice.

PURPOSE: To standardize a method of non-invasive measurement of intraocular pressure (IOP) in mice. METHODS: Cannulated-eye study: IOP was measured simultaneously with a Tonopen and by direct cannulation of the vitreous compartment while pressure was manipulated in steps between 10 and 45 mmHg by a saline reservoir via a second vitreal cannula (five mice, one rat). Non-cannulated-eye study: Tonopen and servo-null measurements were performed in independent groups (48 mice) to verify Tonopen measurements in non-cannulated-eyes. Topical brimonidine (0.15%) was used to decrease IOP. RESULTS: In the rat, there was a similar relationship between Tonopen readings and direct measurements via cannulation of the eye as previously reported. Although readings from mice eyes were higher in variability than those obtained from the rat, the measurements were reproducible and the correlation between the invasive and the non-invasive methods was good (r = 0.97). The IOP lowering effect of brimonidine was detected with Tonopen as well as servo-null measurements (p < 0.001) and the results with both techniques were similar. CONCLUSION: The Tonopen can be used for rapid and reproducible measurements of IOP in mice. The method is easy to apply and can provide a useful means for IOP measurement in mouse models of induced ocular hypertension, in knock-out and transgenic mice, or in pharmacological studies.

Baculoviral IAP repeat-containing-4 protects optic nerve axons in a rat glaucoma model.

Gene therapy represents an attractive approach for the treatment of eye diseases such as glaucoma. Ocular administration of viral vectors produces localized retinal gene expression with reduced risks of side effects reported with systemic administration of viral vectors. Recombinant adeno-associated viral (AAV) vectors have proven effective in producing long-term retinal gene expression, due to stable integration of DNA into the genome and lack of host immune response to the virus. Recently developed AAV constructs using the chicken beta-actin (CBA) promoter drive highly efficient transgene expression in retinal ganglion cells (RGCs), photoreceptors, and pigment epithelium. Rats were given unilateral intravitreal injections of AAV-CBA vector coding for human baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor. Ocular hypertension was induced in the same eye by sclerosis of aqueous humor outflow channels. After chronic exposure to elevated intraocular pressure, we performed optic nerve axon counts to determine the neuroprotective effects of retinal BIRC4 expression, and compared axon survival with vector and balanced salt solution control groups. Gene therapy delivering BIRC4 significantly promoted optic nerve axon survival in a chronic ocular hypertensive model of rat glaucoma. Blocking RGC apoptosis with caspase inhibitors represents a promising approach for treatment of human glaucoma.

Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension.

PURPOSE: Retinal ganglion cell (RGC) death in glaucoma involves apoptosis. Activation of caspases and abnormal processing of amyloid precursor protein (APP) are important events in other chronic neurodegenerations, such as Alzheimer's disease (AD). The retinal expression and activation of caspases and the patterns of caspase-3-mediated APP processing in ocular hypertensive models of rat glaucoma were investigated. METHODS: RGC death was produced in one eye by chronic exposure to increased intraocular pressure (IOP) or by optic nerve transection. Elevated IOP was produced by obstruction of aqueous humor outflow with laser coagulation or limbal hypertonic saline injection. Caspase activity and APP processing in the retina were examined by RNase protection assay (RPA), immunocytochemistry, immunoblot assay, and colorimetric assay. RESULTS: RPA revealed elevations of caspase-3 mRNA, as well as other apoptosis-related mRNAs. Immunocytochemistry showed caspase-3 activation in RGCs damaged by ocular hypertension. The generation of the caspase-3-mediated APP cleavage product (DeltaC-APP) was also increased in ocular hypertensive RGCs. Western immunoblot assay and colorimetry revealed significantly more activated caspase-3 in ocular hypertensive retinas than in control retinas. The activated form of caspase-8, an initiator caspase, and amyloid-beta, a product of APP proteolysis and a component of senile plaques in AD, were detected in RGCs by immunohistochemistry significantly more often in ocular hypertensive than in control retinas. The amounts of full-length APP were reduced and amyloid-beta-containing fragments were increased in ocular hypertensive retinas by Western immunoblot assay. CONCLUSIONS: Rat RGCs subjected to chronic ocular hypertension demonstrate caspase activation and abnormal processing of APP, which may contribute to the pathophysiology of glaucoma.