Nitrous Oxide Impairs Axon Regeneration after Nervous System Injury in Male Rats.

BACKGROUND: Nitrous oxide can induce neurotoxicity. The authors hypothesized that exposure to nitrous oxide impairs axonal regeneration and functional recovery after central nervous system injury. METHODS: The consequences of single and serial in vivo nitrous oxide exposures on axon regeneration in four experimental male rat models of nervous system injury were measured: in vitro axon regeneration in cell culture after in vivo nitrous oxide administration, in vivo axon regeneration after sharp spinal cord injury, in vivo axon regeneration after sharp optic nerve injury, and in vivo functional recovery after blunt contusion spinal cord injury. RESULTS: In vitro axon regeneration 48 h after a single in vivo 70% N2O exposure is less than half that in the absence of nitrous oxide (mean ± SD, 478 ± 275 um; n = 48) versus 210 ± 152 um (n = 48; P < 0.0001). A single exposure to 80% N2O inhibits the beneficial effects of folic acid on in vivo axonal regeneration after sharp spinal cord injury (13.4 ± 7.1% regenerating neurons [n = 12] vs. 0.6 ± 0.7% regenerating neurons [n = 4], P = 0.004). Serial 80% N2O administration reverses the benefit of folic acid on in vivo retinal ganglion cell axon regeneration after sharp optic nerve injury (1277 ± 180 regenerating retinal ganglion cells [n = 7] vs. 895 ± 164 regenerating retinal ganglion cells [n = 7], P = 0.005). Serial 80% N2O exposures reverses the benefit of folic acid on in vivo functional recovery after blunt spinal cord contusion (estimate for fixed effects ± standard error of the estimate: folic acid 5.60 ± 0.54 [n = 9] vs. folic acid + 80% N2O 5.19 ± 0.62 [n = 7], P < 0.0001). CONCLUSIONS: These data indicate that nitrous oxide can impair the ability of central nervous system neurons to regenerate axons after sharp and blunt trauma.

Protein kinase C gamma-mediated phosphorylation of GluA1 in the postsynaptic density of spinal dorsal horn neurons accompanies neuropathic pain, and dephosphorylation by calcineurin is associated with prolonged analgesia.

Loss of calcineurin (protein phosphatase 3) activity and protein content in the postsynaptic density (PSD) of spinal dorsal horn neurons was associated with pain behavior after chronic constriction injury (CCI) of the rat sciatic nerve, and intrathecal administration of the phosphatase provided prolonged analgesia (Miletic et al. 2013). In this study, we examined whether one consequence of the loss of calcineurin was the persistent phosphorylation of the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPAR) receptors in the PSD. This would allow continual activation of AMPAR receptors at the synapse to help maintain a long-lasting enhancement of synaptic function, ie, neuropathic pain. We also investigated if the phosphorylation was mediated by protein kinase A (PKA), protein kinase C gamma (PKCγ), or calcium-calmodulin dependent kinase II (CaMKII), and if the prolonged calcineurin analgesia was associated with GluA1 dephosphorylation. Mechanical thresholds and thermal latencies were obtained before CCI. Seven days later, the behavioral testing was repeated before saline, calcineurin, or the specific peptide inhibitors of PKA (PKI-tide), PKCγ (PKC 19-31), or CaMKII (autocamtide-2-related inhibitory peptide) were injected intrathecally. The behavior was retested before the animals were euthanized and their PSD isolated. All CCI animals developed mechanical and thermal hypersensitivity. This was associated with phosphorylation of GluA1 in the ipsilateral PSD at Ser831 (but not Ser845) by PKCγ and not by PKA or CaMKII. Intrathecal treatment with calcineurin provided prolonged analgesia, and this was accompanied by GluA1 dephosphorylation. Therapy with calcineurin may prove useful in the prolonged clinical management of well-established neuropathic pain.

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.