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Excessive production of nitric oxide (NO) might have detrimental effects on the hypoxia-related neuropathology. This study aimed to test if mild hypoxic preconditioning (MHPC) would attenuate the pathological changes in the brainstem motoneurons having a different functional component after peripheral nerve crush injury (PNCI). Prior to PNCI treatment, young adult rats were caged in the mild hypoxic altitude chamber with 79Torr of the partial oxygen concentration ( pO(2)) (i.e., 0.5atm at 5500m in height) for 4 weeks to adapt the environmental changes. After that, all the animals having successfully crushed both the hypoglossal and vagus nerves (left-side) were allowed to survive for 3, 7, 14, 30 and 60 successive days in normoxic condition. Nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry revealed that MHPC reduces NADPH-d/nNOS expression in the hypoglossal nucleus (HN) and the dorsal motor nucleus of the vagus (DMN) at different time points after PNCI. The morphological findings were further ascertained by Western blot analysis of nNOS and nitrite assay for NO production. Both the morphological and quantitative results peaked at 7 days in HN, whereas for those in DMN were progressively increased up to 60 days following PNCI. The staining intensity of NADPH-d/nNOS(+) neurons, expression of nNOS protein, NO production levels as well as the neuronal loss in HN and DMN of MHPC rats following PNCI were attenuated, especially for those having a longer survival period over 14 days. The MHPC treatment might induce minute amounts of NO to alter the state of milieu of the experimental animals to protect against the PNCI.

The PI3 (phosphatidylinositol-3) kinase/Akt (protein kinase B) signal pathway is involved in the molecular signaling that regulates retrograde axonal transport of neurotrophins in the nervous system. Previous work showed that a reduced retrograde axonal transport of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of diabetic rats occurred in the presence of normal production of neurotrophins and neurotrophin receptors. To assess the potential involvement of an impaired PI3 kinase/Akt signal pathway in the diabetes-induced reduction in retrograde axonal transport of neurotrophins in the vagus nerve, we characterized diabetes-induced changes in the PI3 kinase/Akt signal pathway in the vagus nerve and vagal afferent neurons. Control and streptozotocin (STZ)-induced diabetic rats with a duration of 16 weeks, kinase assays, Western blotting, and immunocytochemistry were used to show that diabetes resulted in alterations in activity and protein expression of the PI3 kinase/Akt signal pathway in the vagus nerve and vagal afferent neurons. Diabetes caused a significant decrease in enzymatic activity of PI3 kinase and Akt (52 and 36% of control, respectively) in the vagus nerve. The reduced enzymatic activity was not associated with decreased protein expression of the p85 subunit of PI3 kinase, Akt and phosphorylation of Akt (ser473). In contrast, there was a significant increase in the phosphorylation of p70s6 kinase (thr421/ser424) along with a normal protein expression of p70s6 kinase in the vagus nerve of diabetic rats. However, diabetes induced an overall decrease in immunoreactivity of the p85 subunit of PI3 kinase, phospho-Akt (ser473) and phospho-p70s6/p85s6 kinase (thr421/ser424) in vagal afferent neurons. Thus, impaired PI3 kinase/Akt signal pathway may partly account for the reduced retrograde axonal transport of neurotrophins in the vagus nerve of STZ-induced diabetic rats.