Brimonidine Enhances the Electrophysiological Response of Retinal Ganglion Cells through the Trk-MAPK/ERK and PI3K Pathways in Axotomized Eyes.

PURPOSE: To investigate changes in retinal ganglion cell (RGC) activity by measuring the positive scotopic threshold response (pSTR) of the electroretinogram (ERG) in axotomized eyes after brimonidine injection. METHODS: In 50 adult Sprague-Dawley rats, the left eye was axotomized and injected with phosphate buffered saline (PBS) or brimonidine and the contralateral right eye was left untreated. Scotopic ERGs were recorded simultaneously from both eyes on days 1, 2, 3, 7, and 10 after the intravitreal injection, and the amplitude of the a- and b-waves and the pSTR were measured. Surviving RGCs in the flat-mounted retinas were counted 10 days after axotomy. In addition to brimonidine, K252a (an inhibitor of tyrosine kinase phosphorylation of the Trk receptors), U0126 (a MAPK/ERK kinase inhibitor), and LY294002 (phosphoinositide 3-kinases [PI3Ks]) were also injected intravitreally into the left eye, and ERGs were recorded using the same protocol. RESULTS: The pSTR amplitude increased significantly in the axotomized eyes with brimonidine, to 122.9 ± 5.0%, 161.8 ± 8.3%, and 133.6 ± 8.1% on days 1, 2, and 3 (P < 0.01), respectively, compared to the axotomized eyes treated with PBS (control). The increased pSTR amplitude returned to normal (103.6 ± 6.7%) on day 7, although there were a greater number of surviving RGCs in the treatment groups than in the controls. The intravitreal injection of K252a, U0126, or LY294002 significantly attenuated the increase in pSTR induced by intravitreal brimonidine (P < 0.01). CONCLUSION: Intravitreal brimonidine enhanced the survival and electrophysiological activity of the RGCs in rats. The mechanism of this electrophysiological change may involve activation of the Trk-MAPK/ERK and Trk-PI3K signals.

The neuroprotective effect of latanoprost acts via klotho-mediated suppression of calpain activation after optic nerve transection.

Latanoprost was first developed for use in glaucoma therapy as an ocular hypotensive agent targeting the prostaglandin F2α (FP) receptor. Subsequently, latanoprost showed a neuroprotective effect, an additional pharmacological action. However, although it is well-known that latanoprost exerts an ocular hypotensive effect via the FP receptor, it is not known whether this is also true of its neuroprotective effect. Klotho was firstly identified as the gene linked to the suppression of aging phenotype: the defect of klotho gene in mice results aging phenotype such as hypokinesis, arteriosclerosis, and short lifespan. After that, the function of klotho was also reported to maintain calcium homeostasis and to exert a neuroprotective effect in various models of neurodegenerative disease. However, the function of klotho in eyes including retina is still poorly understood. Here, we show that klotho is a key factor underlying the neuroprotective effect of latanoprost during post-axotomy retinal ganglion cell (RGC) degeneration. Importantly, a quantitative RT-PCR gene expression analysis of klotho in sorted rat retinal cells revealed that the highest expression level of klotho in the retina was in the RGCs. Latanoprost acid, the biologically active form of latanoprost, inhibits post-traumatic calpain activation and concomitantly facilitates the expression and shedding of klotho in axotomized RGCs. This expression profile is a good match with the localization, not of the FP receptor, but of organic anion transporting polypeptide 2B1, known as a prostaglandin transporter, in the ocular tissue. Furthermore, an organic anion transporting polypeptide 2B1 inhibitor suppressed latanoprost acid-mediated klotho shedding ex vivo, whereas an FP receptor antagonist did not. The klotho fragments shed from the RGCs reduced the intracellular level of reactive oxygen species, and a specific klotho inhibitor accelerated and increased RGC death after axotomy. We conclude that the shed klotho fragments might contribute to the attenuation of axonal injury-induced calpain activation and oxidative stress, thereby protecting RGCs from post-traumatic neuronal degeneration.

Molecular, anatomical and functional changes in the retinal ganglion cells after optic nerve crush in mice.

PURPOSE: Optic nerve crush (ONC) and subsequent axonal damage can be used in rodents to study the mechanism of retinal ganglion cell (RGC) degeneration. Here, we examined electroretinograms (ERGs) in post-ONC mice to investigate changes in the positive scotopic threshold response (pSTR). We then compared these changes with molecular and morphological changes to identify early objective biomarkers of RGC dysfunction. METHODS: Fifty 12-week-old C57BL/6 mice were included. ONC was used to induce axonal injury in the right eye of each animal, with the left eye used as a control. The expression of the RGC markers Brn3a and Brn3b was measured on days 1, 2, 3, 5 and 7 after ONC with quantitative real-time PCR. ERGs were recorded under dark adaptation with the stimulus intensity increasing from -6.2 to 0.43 log cd-s/m(2) on days 1, 2, 3, 5, 7 and 10 after ONC. The pSTR, a- and b-wave amplitudes were measured. Inner retinal thickness around the optic nerve head was measured with spectral-domain optical coherence tomography on days 0, 2, 5, 7 and 10 after ONC. RESULTS: The expression of Brn3a and Brn3b began to significantly decrease on day 1 and day 2, respectively (P < 0.01). The amplitude of the pSTR underwent rapid, significant deterioration on day 3, after which it fell gradually (P < 0.01), while the a- and b-wave amplitudes remained unchanged throughout the experiment. Inner retinal thickness gradually decreased, with the most significant reduction on day 10 (P < 0.01). CONCLUSIONS: Decrease in pSTR likely reflected the early loss of RGC function after ONC and that declining expression of RGC-specific genes preceded anatomical and functional changes in the RGCs.