Enriched environment prevents hypobaric hypoxia induced neurodegeneration and is independent of antioxidant signaling

Abstract

Hypobaric hypoxia (HH) induced neurodegeneration has been attributed to several factors including increased oxidative stress, glutamate excitotoxicity, decreased growth factors, apoptosis, etc. Though enriched environment (EE) has been known to have beneficial effects in various neurological disorders, its effect on HH mediated neurodegeneration remains to be studied. Therefore, the present study was conducted to explore the effect of EE on HH induced neurodegeneration. Male Sprague-Dawley rats were placed in enriched and standard conditions during exposure to HH (7 days) equivalent to an altitude of 25,000 ft. The effect of EE on oxidative stress markers, apoptosis, and corticosterone level in hippocampus was investigated. EE during exposure to HH was found to decrease neurodegeneration as evident from decreased caspase 3 expression and LDH leakage. However, no significant changes were observed in ROS, MDA, and antioxidant status of hippocampus. HH elevates corticosterone level and affected the diurnal corticoid rhythm which may contribute to neurodegeneration, whereas EE ameliorate this effect. Because of the association of neurotrophins and stress and/or corticosterone the BDNF and NGF levels were also examined and it was found that HH decreases their level but concurrent exposure to EE maintains their level. Moreover, inhibition of Tyrosine kinase receptor (Trk) with K252a nullifies the protective effect of EE, whereas Trk activation with agonist, amitriptyline showed protective effect similar to EE. Taken together, we conclude that EE has a potential to ameliorate HH mediated neuronal degeneration which may act through antioxidant independent pathway by modulation of neurotrophins.

Fig. 1

Represents percentage change in ROS (a), lipid peroxidation (b), and LDH release (c) in hippocampus. Data represents mean ± SEM with n = 6. EE exposure fails to ameliorate HH mediated upregulation in ROS and MDA level, whereas it shows promising effect in reducing LDH leakage during hypobaric hypoxic exposure. * Represents p < 0.001 when compared to control group, whereas ** represents p < 0.05 when compared to hypoxia group

Fig. 2

Corticosterone level averaged across group exposed to hypoxia with or without EE. Hypoxia leads to elevation in corticosterone level in plasma (c) when compared to control (a), whereas in presence of EE hypoxia fails to upregulate corticosterone level (d). No significant changes was found in control group housed in EE (b) and percentage change in corticosterone level in all groups (e). * Represents p < 0.001 when compared to control group, whereas ** represents p < 0.01 when compared to hypoxia group

Fig. 3

Western blot analysis of caspase 3. Lanes from left to right shows Normoxia, Hypoxia, Hypoxia + EE, and Normoxia + EE. Corresponding graphs denote mean ± SEM of percentage change in optical density of three individual blots. * Represents p < 0.001 when compared to control group, whereas ** represents p < 0.05 when compared to hypoxia group

Fig. 4

Western blot analysis of BDNF (a) and NGF (b). Lanes from left to right shows Normoxia, Hypoxia, Hypoxia + EE, and Normoxia + EE. Corresponding graphs denote mean ± SEM of percentage change in optical density of three individual blots. * Represents p < 0.001 when compared to control group whereas ** represents p < 0.01 when compared to hypoxia group

Fig. 5

EE mediated neuroprotection against HH induced neuronal degeneration in CA1 regions of hippocampus, is neurotrophins dependent as evident from increase in caspase 3 immunoreactivity on inhibiting Trk by K252a. a Normoxia, b Hypoxia, c Hypoxia + EE, d Hypoxia + EE + K252a. Total magnification 400×, bar indicate 25 μm. Graph showing percentage increase in caspase 3 positive neurons. Bars represent mean ± SEM of average of six fields in six individuals of each group. *p < 0.001 and **p < 0.05 when compared to normoxic group

Fig. 6

Percentage change in caspase 3 activity using DEVD-AMC cleavage assay in different groups. EE decreased caspase 3 activity equivalent to positive control i.e., Trk agonist (Amitriptyline). This shows that EE work through neurotrophins regulated Trk activation. Bars represent mean ± SEM of six individuals. Asterisk and equal and parallel to symbol denotes p < 0.001 and p < 0.005, respectively, when compared to control group, whereas double asterisk represents p < 0.005 when compared to hypoxic group