A key role for arginase in the prevention of cell death following spinal cord complete transected injury in the rat

I In nt tr ro od du uc ct ti io on n: : Oxidative and nitrosative stresses are important for mediating secondary damage following spinal cord injury (SCI). A key player in this process is likely to be nitric oxide (NO) because of its various physiological and pathological roles in the CNS. Arginase, a major metabolic enzyme of the liver, is involved in the regulation of NO production. It does this by competing with nitric oxide synthase (NOS) for the same substrate, L-arginine. However, the exact pathway by which arginase is linked to oxidative-nitrosative stress during SCI is unclear. For the successful reconstruction of spinal cord defects using biomaterials, it is important to first elucidate the mechanism of cellular damage following spinal cord transection. The aim of the present study was therefore to investigate NO, nNOS and endogenous arginase for possible roles in the mechanism of tissue damage following spinal cord transected injury (SCTI) in a rat model. M Ma at te er ri ia al ls s a an nd d M Me et th ho od ds s: : Experimental groups and surgical procedures Adult female rats were randomly assigned to 3 groups: the sham operated control group (Th9 laminectomy only), the vehicle group (SCTI + saline controls) and the treatment group (SCTI + arginase inhibitor Nw-Hydroxy-nor-L-arginine at 2.5 mg/kg/day for 3 days). Three days after the operation, a 1 cm length spinal cord segment containing the injury epicenter was removed. NOx measurement Generation of NO was expressed as NOx, nitrite (NO2-) and nitrate (NO3-). Nitrate reductase and NADPH were added to convert NO3-to NO2-. An NO analyzer (280i, Sievers, Boulder, CO) was then used to quantify NO2-. Western blot analysis Each sample was separated by SDS/PAGE and then transferred onto PVDF membranes. The membranes were incubated with the following primary antibodies: polyclonal rabbit anti-nNOS, monoclonal mouse anti-nitrotyrosine and polyclonal rabbit anti-arginase. Arginase activity assay 10mM MnCl2 was incubated with tissue homogenate for 10min. The mixture was then incubated in the presence of 0.5M arginine, pH9.7 for 1 hour. 9% isonitrosopropiopheanone was added and heating for 45min. The amount of urea formed was determined spectrophotometrically at 540 nm. TUNEL staining Frozen sections taken from a position 1 mm rostral to the injury site were incubated with an In Situ Apoptosis Detection Kit as per the man-ufacturer's instructions (TaKaRa, Japan) before counterstaining with hematoxylin. R Re es su …