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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...

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We have previously demonstrated that both trypsin and the PAR-2 activating peptide (SLIGRL-NH 2 ) contract isolated rat urinary bladders by the activation of urothelium PARs and the subsequent release of prostaglandins.3) Furthermore, we found that contractions induced by PAR-2 agonists could be partly mediated through stimulation of capsaicin-sensitive sensory nerves.4) The PAR-2-mediated responses were enhanced after the induction of cystitis with cyclophosphamide (CYP).5) These findings suggested that PAR-2 plays a role in regulating bladder contractility under physiological and pathological conditions. Bradykinin, a metabolite of the kallikrein-kinin system, has multiple actions in urinary bladders, including the ability to contract the detrusor smooth muscle, 6) stimulate sensory nerves 6,7) and evoke the release of cyclooxygenase (COX) products.8) Also, bradykinin might play a key role in the pathogenesis of experimental cystitis 9) and activation of the kallikrein-kinin system in urinary bladders of patients with interstitial cystitis has been reported. 10,11) Thus, the actions of bradykinin in urinary bladders resemble those of PAR-2 agonists.In the present study, we examined the role of bradykinin in contractions induced by trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH 2 12) in the urinary bladders from normal and CYP-induced cystitis rats. MATERIALS AND METHODS AnimalsThe protocol for the use of animals was approved by the Committee on the Care and Use of Laboratory Animals of Kitasato University.Male Wistar rats weighing 250-280 g were maintained on standard rat chow and tap water ad libitum with a 12 h light/ dark cycle in a quiet environment. Rats were treated with vehicle (saline) or CYP (150 mg/kg, intraperitoneally (i.p.)). Experiments were performed 48 h after the treatment. Preparation of Rat Urinary Bladder StripsThe rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and sacrificed. The urinary bladder was removed and placed in ice-cold Krebs-Ringer bicarbonate buffer (KRB, composition in mmol/l: NaCl, 119; KCl, 4.8; MgSO 4 , 1.2; KH 2 PO 4 , 1.2; CaCl 2 , 2.5; NaHCO 3 , 25 and glucose, 10.0) (pHϭ7.4). Four longitudinal strips (approximately 1 mmϫ2 mmϫ10 mm) were isolated from the bladder body.Measurement of Mechanical Activity One end of each strip was attached to an isometric force displacement transducer (model TB-611T, Nihon Kohden, Tokyo) by a cotton thread, and the other end was tied to a stainless-steel holder. Tension was digitized at a sampling rate of 2 Hz (model 15BXW-H4, Dacs Giken, Okayama) and stored on the hard disk of a personal computer. Strips were mounted in 10-ml jacketed organ baths filled with KRB gassed with 95% O 2 -5% CO 2 at 37°C. The preparations were placed under initial load (9.8 mN) and the resting tension was adjusted every 15 min. The tissues were allowed to equilibrate for 60 min and the bath fluid was changed every 15 min with fresh KRB. After the equilibration period, all preparations were contracted with acetylcholine (ACh) (100 mmol/l) to c...