Pharmacological Strategies to Block Rod Photoreceptor Apoptosis Caused by Calcium Overload: A Mechanistic Target-Site Approach to Neuroprotection

Abstract

Purpose

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca²⁺ overload in Pde6b^rd1 mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca²⁺ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca²⁺ overload/apoptosis.

Methods

Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca²⁺ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca²⁺ overload/apoptosis.

Results

Ca²⁺ overload preceded rod apoptosis in mice and rats, although the extent and kinetics in each differed significantly. The isolated rat model of rod Ca²⁺ overload/apoptosis showed that blockade of Ca²⁺ entry through rod cGMP-activated channels with L-cis diltiazem was partially neuroprotective, whereas blockade of Ca²⁺ entry into rods through L-type Ca²⁺ channels with D-cis diltiazem or verapamil provided no protection. Inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger with D-cis diltiazem provided no protection. CsA and NIM811, mitochondrial permeability transition pore (mPTP) inhibitors, blocked all Ca²⁺-induced apoptosis, whereas the caspase-3 inhibitor DEVD-fmk only blocked the downstream cytochrome c-induced apoptosis.

Conclusions

The successful pharmacological neuroprotective strategies for rod Ca²⁺ overload/apoptosis targeted the rod cGMP-activated channels or mPTP, but not the rod L-type Ca²⁺ channels.

Keywords

Retinal degeneration Rod photoreceptors Apoptosis Mitochondria Neuroprotection

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