Differential effects of two ROCK inhibitors, Fasudil and Y-27632, on optic nerve regeneration in adult cats.

A ROCK inhibitor Fasudil is widely administered to relieve vasospasm in patients after subarachnoid hemorrhage in Japan. We investigated the difference of Fasudil and Y-27632, a common ROCK inhibitor, on neurite regeneration in culture and axonal regeneration after injuring the optic nerve (OpN) in cats. The optimal dose of Y-27632, determined by counting the number and length of neurites in retinal explants, was found to be 100 microM: the only effect of Fasudil was to promote extension of glial processes. We next examined the effects of Fasudil (10 microM-100 microM) and Y-27632 (10 microM-300 microM) on axonal regeneration in the crushed OpN model in vivo. Immediately after crushing the left OpN, Fasudil or Y-27632 was injected into the vitreous and the crushed site. Injection of 10 microM and 100 microM Y-27632 induced extension of the optic axons beyond the crush site, with the latter dosage giving stronger regeneration. Very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection, and no axons elongated in the OpN with Fasudil injection.

Associations between serum C-reactive protein (CRP) levels and polymorphisms of CRP, interleukin 1B, and tumor necrosis factor genes among Japanese health checkup examinees.

C-reactive protein (CRP) is a sensitive marker of acute inflammation, which is associated with risk of cardiovascular and other chronic diseases. Some CRP polymorphisms are reported to affect the basal and stimulated CRP levels. Thus we conducted a population-based cross-sectional study to examine the associations of CRP levels with CRP C1444T polymorphism and two cytokine polymorphisms (IL-1B C-31T and TNF-A T-1031C), according to sex, age, smoking, alcohol, and BMI, in a total of 489 Japanese health checkup examinees (156 males and 333 females). Serum CRP levels were measured by high sensitivity latex-enhanced nephelometry. CRP C1444T, IL-1B C-31T and TNF-A T-1031C genotypes were genotyped by PCR-CTPP (polymerase chain reaction with confronting two-pair primers). Males, aged, smokers, and those with high BMI had a higher CRP on average. All genotype frequencies among the 489 subjects were in Hardy-Weinberg equilibrium. No significant associations of serum CRP levels with the genotypes of CRP C1444T and IL-1B C-31T were observed. TNF-A -1031CC polymorphism was significantly associated with high CRP values. For the females, those aged 61-69 years, never smokers, non-drinkers, or those with body mass index 24 or less, the association was remarkable. Since the biological mechanism is not clear, further investigations are required to confirm the association.

Regeneration of optic nerve fibers with unoprostone, a prostaglandin-related antiglaucoma drug, in adult cats.

PURPOSE: We investigated the effects of unoprostone on neurite extension of cultured retinal pieces and axonal regeneration of retinal ganglion cells in the crushed optic nerve of adult cats. METHODS: The retinal pieces were cultured with unoprostone or its primary metabolite, M1, dissolved in DMSO or polysorbate for 14 days, and the number and length of Tau-1-positive neurites and glial processes labeled with anti-glial fibrillary acidic protein antibodies were examined. After the optic nerve was crushed, unoprostone was injected into the vitreous body and the crushed site. On day 12, wheat germ agglutinin-conjugated horseradish peroxidase was injected into the vitreous body to anterogradely label the regenerated axons. On day 14, the optic nerve was excised and longitudinally sectioned. After peroxidase reaction, the number of axons regenerating beyond the crush site was examined. RESULTS: The greatest number of neurites protruded from the cultured retinal pieces in 3 µM unoprostone and 3 µM M1. The neurite length was also the longest at 3 µM unoprostone and 3 µM M1, in which no glial processes were detected. After injections of 3 µM unoprostone, the final concentration in the vitreous humor, into the vitreous body and the crush site, the optic nerve fibers regenerated and extended beyond the crush site. In contrast, almost no fibers extended beyond the crush site after injection of phosphate-buffered saline. CONCLUSIONS: The results indicate that intravitreal injection of unoprostone promotes regeneration of crushed optic nerve fibers in adult cats.

A novel ROCK inhibitor, Y-39983, promotes regeneration of crushed axons of retinal ganglion cells into the optic nerve of adult cats.

We investigated the effect of a novel ROCK inhibitor, Y-39983, on neurite regeneration in vitro and axonal regeneration in the crushed cat optic nerve in vivo. To determine the effective dose for neurite regeneration, retinal pieces were cultured with ROCK inhibitors, Y-39983 or Y-27632, a well-characterized ROCK inhibitor, and the number and length of TUJ-1-positive neurites were evaluated. The greatest number of neurites protruded at a dose of 3-10 microM Y-39983 and at a dose of 10-100 microM Y-27632, respectively. The neurite number at maximum effect of Y-39983 was greater than that of Y-27632. No significant difference was observed between values of neurite length with the inhibitors. Based on this finding, we examined the effect of Y-39983 on axonal regeneration in the crushed optic nerve in vivo. Immediately after crushing the left optic nerve, Y-39983 was injected into the vitreous and the crushed site. An injection of 10 microM Y-39983 induced the crushed axons to regenerate and pass over the crush site. In contrast, very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection. The second injection of 10 microM Y-39983 on day 7 doubled the number of regenerated axons, suggesting that new axons may have entered into the optic nerve after day 7 and that a continuous supply of the drug may make more axons to regenerate.