Retinal Calcium Research Articles

Contribution of calpains to photoreceptor cell death in N-methyl- N-nitrosourea-treated rats

The purpose of the present study was to determine if proteolysis by the calcium-dependent enzyme calpains (EC 3.4.22.17) contributed to retinal cell death in a rat model of photoreceptor degeneration induced by intraperitoneal injection of N-methyl- N-nitrosourea (MNU). Retinal degeneration was evaluated by H&E staining, and cell death was determined by TUNEL assay. Total calcium in retina was measured by atomic absorption spectrophotometry. Activation of calpains was determined by casein zymography and immunoblotting. Proteolysis of α-spectrin and p35 (regulator of Cdk5) were evaluated by immunoblotting. Calpain inhibitor SNJ-1945 was orally administrated to MNU-treated rats to test drug efficacy. MNU decreased the thickness of photoreceptor cell layer, composed of the outer nuclear layer (ONL) and outer segment (OS). Numerous cells in the ONL showed positive TUNEL staining. Total calcium was increased in retina after MNU. Activation of calpains and calpain-specific proteolysis of α-spectrin were observed after MNU injection. Oral administration of SNJ-1945 to MNU-treated rats showed a significant protective effect against photoreceptor cell loss, confirming involvement of calpains in photoreceptor degeneration. Conversion of p35 to p25 was well correlated with calpain activation, suggesting prolonged activation of Cdk5/p25 as a possible downstream mechanism for MNU-induced photoreceptor cell death. SNJ-1945 reduced photoreceptor cells death, even though MNU is one of the most severe models of photoreceptor cell degeneration. Oral calpain inhibitor SNJ-1945 may be a candidate for testing as a medication against retinal degeneration in retinitis pigmentosa.

Presence of calpain‐induced proteolysis in retinal degeneration and dysfunction in a rat model of acute ocular hypertension

The purpose of this study was to determine if calpain-induced proteolysis was associated with retinal degeneration or dysfunction in the rat acute ocular hypertensive model. Acute glaucoma was produced by elevation of IOP to 120 mm Hg for 1 hr. Retinal degeneration was evaluated by H&E staining and apoptosis was determined by TUNEL staining in histologic sections of retina. Electroretinogram (ERG) was carried out to evaluate changes in functionality. Activation of calpains was determined by casein zymography and immunoblotting. Total calcium in retina was measured by atomic absorption spectrophotometry. Proteolysis of alpha-spectrin, tau, cdk5, and p35 (a regulator of cdk5) were evaluated by immunoblotting. The thickness of inner plexiform layer (IPL) and inner nuclear layer (INL), and the number of cells in the ganglion cell layer (GCL) decreased after ocular hypertension. Numerous cells in the INL stained positive for TUNEL and some cells in the outer nuclear layer (ONL) showed TUNEL staining. The a-wave in ERG was temporarily decreased after ocular hypertension and then recovered to normal. In contrast, the b-wave was completely lost. Calpains were activated after ocular hypertension. Activation of calpains was associated with increased calcium in retina. Calpain-dependent proteolysis of alpha-spectrin, tau, and p35 were observed in retina after ocular hypertension. The results suggested that increased calcium and subsequent proteolysis by activated calpains was associated with the death of inner retinal cells due to acute ocular hypertension in the rat model. Calpain inhibitors may be candidate drugs for treatment of retinal degeneration and dysfunction resulting from glaucoma.

Ultrastructural demonstration of calcium in retina, retinal pigment epithelium and choroid

Calcium has been demonstrated ultrastructurally in the retina, pigment epithelium and choroid of the frog using the potassium pyroantimonate (KPA) technique. Optimum tissue preservation was obtained with 2% potassium pyroantimonate in 1% osmium tetroxide at pH 9·2. With this fixative, electron-dense precipitate was found within the discs of the rod outer segments, as a dense band of granules along Bruch's membrane, in the extracellular space of the choroid, and to some extent within the nuclei and mitochondria of all cells. The precipitate has been identified as calcium antimonate by the use of EGTA pre-chelation and with the EMMA-4 electron microprobe. The possible significance of calcium in the various locations demonstrated by the KPA technique is discussed. This study establishes the specificity of the technique in demonstrating calcium in these tissues and suggests future applications in evaluating changes in the amount or location of this cation with both physiologic and pathologic processes.