mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration.

Sciatic axotomy in 2-day-old rats (P2) causes lumbar motoneuron loss, which could be associated with nitric oxide (NO) production. NO may be produced by three isoforms of synthase (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We investigated NOS expression and NO synthesis in the lumbar enlargement of rats after sciatic nerve transection at P2 and treatment with the antioxidant melatonin (sc; 1 mg/kg). At time points ranging from P2 to P7, expression of each isoform was assessed by RT-PCR and immunohistochemistry; catalytic rates of calcium-dependent (nNOS, eNOS) and independent (iNOS) NOS were measured by the conversion of [3H]L-arginine to [3H]L-citrulline. All NOS isoforms were expressed and active in unlesioned animals. nNOS and iNOS were detected in some small cells in the parenchyma. Only endothelial cells were positive for eNOS. No NOS isoform was detected in motoneurons. Axotomy did not change these immunohistochemical findings, nNOS and iNOS mRNA expression and calcium-independent activity at all survival times. However, sciatic nerve transection reduced eNOS mRNA levels at P7 and increased calcium-dependent activity at 1 and 6 h. Melatonin did not alter NOS expression. Despite having no action on NOS activity in unlesioned controls the neurohormone enhanced calcium-dependent activity at 1 and 72 h and reduced calcium-independent catalysis at 72 h in lesioned rats. These results suggest that NOS isoforms are constitutive in the neonatal lumbar enlargement and are not overexpressed after sciatic axotomy. Changes in NO synthesis induced by axotomy and melatonin administration in the current model are discussed considering some beneficial and deleterious effects that NO may have.

Superoxide dismutase isoforms 1 and 2 in lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin treatment.

Oxidative stress has been implicated in motoneuron death secondary to axotomy in the neonatal period. We studied the effect of sciatic transection at P2 on the motoneuron population in the lumbar enlargement of newborn rats looking for a protective role of daily doses of the antioxidant melatonin. The animals were allowed to survive from P2 to P7, and the spinal cords were processed for immunohistochemistry for superoxide dismutase (SOD) isoforms 1 and 2 and nitric oxide synthase (nNOS) (at 2, 3, 5, and 7 days post-natum), histological neuron counting and immunoblotting for the SOD isoforms (both at 2, 3, and 7 days post-natum). Melatonin reduced by 75% motoneuron loss due to axotomy at P3 and P7. Neither sciatic transection nor melatonin induced any detectable changes in the immunoreactivity patterns of the enzymes. SOD1 was expressed diffusely in the cytoplasm of neurons and ependyma and in the nuclei of presumed glial cells from P2 to P7. SOD2 was detected in neurons and ependyma and its expression was similar to SOD1 at P2 but decreased later to a spotty cytoplasmic pattern in motoneurons. nNOS was localized to the cytoplasm of a few small cells in the ventral and dorsal horns and around the central canal. Immunoblotting at 1 day postaxotomy detected a significant increase in SOD1 expression in melatonin-treated axotomized rats and a decrease in controls after axotomy and vehicle. Blotting for SOD2 did not show significant changes between groups at any time. This study provides the first evidence that SOD2 immunostaining pattern varies during motoneuron postnatal development and that melatonin alters the expression of SOD1 in the present model of peripheral nerve injury.

Evaluating motor neuron death in neonatal rats subjected to sciatic nerve lesion.

Neonatal sciatic nerve lesion is a useful experimental model for the study of neuronal cell death. Sciatic nerve transection or crush is the most frequently used approach to evaluate motoneuron loss in the lumbar enlargement of the spinal cord. Here we describe and illustrate the surgical procedures performed in our laboratory to assess motoneuron cell death and the related cellular mechanisms.