No abstract

Müller cell gliosis, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), is a universal response in many retinal pathological conditions. Whether down-regulation of inward rectifying K ϩ (Kir) channels, which commonly accompanies the enhanced GFAP expression, could contribute to Müller cell gliosis is poorly understood. We investigated changes of Kir currents, GFAP and Kir4.1 protein expression in Müller cells in a rat chronic ocular hypertension (COH) model, and explored the mechanisms underlying Müller cell gliosis. We show that Kir currents and Kir4.1 protein expression in Müller cells were reduced significantly, while GFAP expression was increased in COH rats, and these changes were eliminated by MPEP, a group I metabotropic glutamate receptors (mGluR I) subtype mGluR5 antagonist. In normal isolated Müller cells, the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppressed the Kir currents and the suppression was blocked by MPEP. The DHPG effect was mediated by the intracellular Ca 2ϩ -dependent PLC/IP 3 -ryanodine/PKC signaling pathway, but the cAMP-PKA pathway was not involved. Moreover, intravitreal injection of DHPG in normal rats induced changes in Müller cells, similar to those observed in COH rats. The DHPG-induced increase of GFAP expression in Müller cells was obstructed by Ba 2ϩ , suggesting the involvement of Kir channels. We conclude that overactivation of mGluR5 by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir channels.

EphB1, expressed in Müller cells, and ephrinB2, expressed in both Müller cells and retinal ganglion cells (RGCs), constitute an EphB/ ephrinB reverse signaling in RGCs. Whether and how this reverse signaling is involved in RGC apoptosis in a rat chronic ocular hypertension (COH) model was investigated. In the COH model, both EphB1 and ephrinB2 were significantly increased and the reverse signaling was activated, which was accompanied by increased protein levels of phosphorylated ( -permeable GluA2-lacking AMPA receptor inhibitor, remarkably inhibited RGC apoptosis in either EphB2-Fc-injected or COH rats. Together, elevated GluA2 trafficking induced by activated EphB2/ephrinB2 reverse signaling likely contributes to RGC apoptosis in COH rats.

We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5) signaling pathway in modifying NMDA receptors (NMDARs) in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5T15 protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs), which are both Ca2+-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5T15, calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2AS1232) and NR2A (p-NR2AS1232/NR2A), and caused a translocation of p-NR2AS1232 from the cytosol to the plasma membrane. The enhanced p-NR2AS1232 was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca2+ concentrations ([Ca2+]i) of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2AS1232 expression.

Microglial activation and the release of pro-inflammatory cytokines occur during early glaucoma. However, the exact mechanism underlying the initiation of the microglial activation process remains unclear. Thus, the present study investigated the potential role of a purine receptor subtype, the P2X purinoceptor 7 (P2X7) receptor, during microglial activation in the retinal tissues of a rat chronic ocular hypertension (COH) model. This was achieved by cauterizing 3 of the 4 episcleral veins. Microglial activation and caspase-1 upregulation were observed in COH rat retinas by immunohistochemical and western blotting techniques. Intravitreal injection of 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP), a P2X7 receptor agonist, induced microglial activation in normal rat retinal tissues, which was alleviated by pretreatment with the P2X7 receptor antagonist, Brilliant Blue G (BBG). BBG further attenuated caspase-1 increment in COH rat retinal tissues. The data demonstrated that BBG reduced TUNEL-positive retinal ganglion cells in whole-mount retinal tissues with COH and normal retinal tissues following intravitreal injection with BzATP. One may conclude that the P2X7 receptor may be involved in microglial activation in the COH retina and could be considered a target for neuronal protection in glaucoma.