Effect of Nafamostat on <i>N</i>-Methyl-<small>D</small>-aspartate-Induced Retinal Neuronal and Capillary Degeneration in Rats

Abstract

We examined the effects of the serine protease inhibitor nafamostat mesilate on neuronal and vascular injury in rat retinas treated with N-methyl-D-aspartate (NMDA). The degree of neuronal degeneration was assessed by measuring the number of cells in the ganglion cell layer and the thickness of the inner plexiform layer. The degree of capillary degeneration was assessed by measuring the number of empty basement membrane sleeves that were left as remnants of the vessels. Significant neuronal and capillary degeneration was observed 7 d after a single intravitreal injection of NMDA into the eye. Both forms of degeneration were significantly prevented by simultaneous injection of nafamostat mesilate with NMDA. These results indicate that nafamostat mesilate affords protection against the neuro/vascular injury seen in NMDA-treated retinas. Nafamostat mesilate may be considered as a candidate for neuro/vascular protective interventions in retinal diseases associated with glutamate-induced excitotoxicity, such as glaucoma and diabetic retinopathy.Key words endothelial cell; excitotoxicity; retinal blood vessel; retinal degeneration Neuronal injury and death play a critical role in the pathogenesis of retinal diseases such as diabetic retinopathy and glaucoma. Glutamate-induced excitotoxicity appears to play an important role in these retinal diseases. -activated proteolytic enzymes, including calpains.6,7) In addition, upregulation of pro-inflammatory cytokines and inflammatory adhesion molecules, 8,9) increase in matrix metalloproteinase (MMP) activity, [10][11][12] and recruitment of leukocytes into the retina 8) are also involved. These events precede the occurrence of retinal ganglion cell apoptosis, and preventive interventions have been shown to ameliorate NMDA-induced retinal ganglion cell death. Thus, excessive activation of NMDA receptors appears to affect retinal neuronal cell survival by indirect as well as direct mechanisms.A single intravitreal injection of NMDA into the eye is commonly used in vivo model to induce different forms of retinal damage, such as retinal ganglion cell apoptosis, thinning of the inner retina, and visual dysfunction. 13,14) More recently, injury to the retinal microvasculature, including capillary degeneration, has also been demonstrated in the same model. 15,16) Pathological changes in retinal circulation could contribute to the progression of retinal diseases, including diabetic retinopathy and glaucoma.17-21) Therefore, the NMDA-induced retinal injury model would be useful for screening novel interventions and approaches for treating such retinal diseases by protecting from neuro/vascular injury.Nafamostat mesilate (FUT-175, 6-amidino-2-napthyl-4-guanidinobenzoate) was originally developed as a complement inhibitor 22) and is used for the treatment of pancreatitis and disseminated intravascular coagulation.23,24) Nafamostat mesilate has also been shown to exert broad inhibitory effects against serine proteases, especially tryptase, 25) and decrease activities of MMP-9. 26,27) T...

New strategies for neuroprotection in glaucoma, a disease that affects the central nervous system

“…Moreover, nafamostat mesilate, a serine protease inhibitor showed protection against NMDA-induced neuronal and vascular damage in rat retinas (Tsuda et al, 2012).…”

Preventive Effects of Rapamycin on Inflammation and Capillary Degeneration in a Rat Model of NMDA-Induced Retinal Injury

“…15,16) Briefly, the rats were deeply anesthetized with pentobarbital sodium. The vasculature of each rat was perfused with 1% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 4 min at a pressure of 120 mmHg.…”

“…15,16) After the systemic perfusion was performed as described above, the eyes were removed and stored in fixative for 24 h at 4°C. Each retina was separated from the lens, vitreous, and pigment epithelium using 4 radial cuts and then incubated in blocking solution (5% normal hamster serum) in PBS-T for 0.5 to 1 h at room temperature.…”

Protective Effects of Everolimus against <i>N</i>-Methyl-D-aspartic Acid-Induced Retinal Damage in Rats

“…11,24) Seven days after the injection, rats were anesthetized with pentobarbital sodium, and their eyes were enucleated. Eyes were immersed in a fixative mixture (37.5% ethanol, 9.3% formaldehyde, 12.5% acetic acid, and 3% glutaraldehyde) for 12 h at room temperature.…”

“…The sections were stained with hematoxylin and eosin. We examined whether thicknesses of outer nuclear layer (ONL), which is unaffected by intravitreal NMDA injection, 11,12,24) are in the same range to check the consistency of the plane and the position of each cross-section. The number of cells in the ganglion cell layer (GCL) was counted between 1000 and 1250 µm from the center of the optic nerve head on both sides.…”