Table 1.
Shows experimental design describing groups, number of individuals, exposure duration and interventions.
Figure 1.
Behavior changes was studied using Morris Water Maze test and representative tack sheet showing memory test of Control (I), Hypoxia (II), Enriched environment with Hypoxia (III) and Enriched environment without hypoxia (IV) exposure.
Latency (A) and Pathlength (B) were increased on HH exposure whereas EE revert back these effects to normal. In contrast HH leads to lessen the number of platform crossing (C) and time spent in target quadrant (D) and EE again showed promising effect by increasing their value. Data represents Mean ± SEM. “*” and “#” represents p<0.001 when compared to control and hypoxic group whereas “**” represents p<0.05 when compared to hypoxic group.
Figure 2.
Fluoro Jade (FJ) and Cresyl violet staining, was assessed to study neurodegeneration and cell morphology.
It was observed that Hypobaric hypoxia leads to neurodegeneration and cell pyknosis as evident from increased number of FJ positive cells and pyknotic cells whereas enriched environment decreases number when compared to hypobaric hypoxia alone group. Data represents Mean ± SEM. “*” and “**” represents p<0.001 when compared to control and hypoxic group.
Figure 3.
Effect of Trk antagonist on Behavior was studied using Morris Water Maze test and representative tack sheet showing memory test of Normoxia (I), Hypoxia (II), Enriched environment + Hypoxia (III) and Enriched environment + hypoxia + K252a (IV) exposure.
Inhibition of TrkB with K252a nullifies the protective effect of Enriched environment on hypobaric hypoxia induced memory impairment as evident from increased pathlength (A) and latency (B) and decreased No. of platform crossing (C) and time spent in target quadrant (D). Data represents Mean ± SEM. “*” and “#” represents p<0.001 when compared to control and hypoxic group whereas “**” represents p<0.05 when compared to hypoxic group.
Figure 4.
Role of neurotrophins was assessed using gene silencing technique for TrkA and TrkB receptors.
Results showed that knockdown of TrkB significantly increased caspase3 activity even in presence of enriched environment whereas blocking of TrkA has diminutive effect on caspase3 activity in comparison to TrkB inhibition. To further explore the signaling cascade, ERK and PI3K pathway was blocked with U0216 and Wortmannin respectively and it has been observed that inhibition of PI3K pathway significantly increases caspase3 activity (B) whereas inhibiting ERK pathway have no significant effect in group exposed to hypobaric hypoxia in enriched cage (C). Data represents Mean ± SEM. In “*” and “#” represents p<0.001 when compared to control and hypoxic group while “?”represents p<0.05 when compared to control group.
Figure 5.
Downstream cascade was further explored and it was shown that hypobaric hypoxia reduces GSK3ß phosphorylation (A) and hence its inactivation whereas enriched environment significantly ameliorate this effect.
In presence of PI3K inhibitor (Wortmannin) enriched environment have little effect on GSK3ß phosphorylation. Inhibition of ERK pathway with U0216 showed no significant effect on GSK3ß phosphorylation. Moreover hypobaric hypoxia also decreases the CREB phosphorylation (B) whereas exposing them with enriched environment normalizes this decreased level of CREB phosphorylation. Similarly inhibition of GSK3ß with AR-A014418 elevates the CREB phosphorylation equivalent to group exposed to enriched environment during hypobaric hypoxia. Data represents Mean ± SEM. In “*” and “#” represents p<0.001 when compared to control and hypoxic group while “?”represents p<0.05 when compared to control group.
Figure 6.
Schematic diagram of signaling events in enriched environment mediated neuroprotection during hypobaric hypoxia.