Hyperbaric Oxygen Therapy Alleviates Memory and Motor Impairments Following Traumatic Brain Injury via the Modulation of Mitochondrial-Dysfunction-Induced Neuronal Apoptosis in Rats

Abstract

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in young adults, characterized by primary and secondary injury. Primary injury is the immediate mechanical damage, while secondary injury results from delayed neuronal death, often linked to mitochondrial damage accumulation. Hyperbaric oxygen therapy (HBOT) has been proposed as a potential treatment for modulating secondary post-traumatic neuronal death. However, the specific molecular mechanism by which HBOT modulates secondary brain damage through mitochondrial protection remains unclear. Spatial learning, reference memory, and motor performance were measured in rats before and after Controlled Cortical Impact (CCI) injury. The HBOT (2.5 ATA) was performed 4 h following the CCI and twice daily (12 h intervals) for four consecutive days. Mitochondrial functions were assessed via high-resolution respirometry on day 5 following CCI. Moreover, IHC was performed at the end of the experiment to evaluate cortical apoptosis, neuronal survival, and glial activation. The current result indicates that HBOT exhibits a multi-level neuroprotective effect. Thus, we found that HBOT prevents cortical neuronal loss, reduces the apoptosis marker (cleaved-Caspase3), and modulates glial cell proliferation. Furthermore, HBO treatment prevents the reduction in mitochondrial respiration, including non-phosphorylation state, oxidative phosphorylation, and electron transfer capacity. Additionally, a superior motor and spatial learning performance level was observed in the CCI group treated with HBO compared to the CCI group. In conclusion, our findings demonstrate that HBOT during the critical period following the TBI improves cognitive and motor damage via regulating glial proliferation apoptosis and protecting mitochondrial function, consequently preventing cortex neuronal loss.

Keywords: apoptosis; hyperbaric oxygen therapy (HBOT); mitochondria respiration; secondary brain injury; traumatic brain injury.

Figure 1

The early HBO treatment modulates memory and motor dysfunction following CCI. (A) The experimental timeline. Spatial memory and motor performance were measured before and after the CCI. After CCI, animals were randomly assigned to one of the experiment groups: CCI group. CCI-HBOT group. The HBO treatment was performed after 4 h of the CCI and twice a day to day 4 after the CCI. The animals were sacrificed at the end of the study. (B) The Morris water maze testing established similar learning curves in the two groups before the injury. Following CCI, treatment with HBO led to a significant improvement in the learning curve, expressed by a reduced latency to reach the platform. (C) The sensorimotor recovery following CCI and CCI-HBOT were tested using the rotarod test. Motor performance is expressed by latency to fall (Second). Treatment with HBO significantly improved motor performance compared to the CCI group. Mixed Model ANOVA; Student t-test; Paired sample t-test. Mean ± SEM. *** p < 0.001 Compared to the 1ST test session following the CCI; ^# p < 0.05, ^#### p < 0.001 Difference between groups.

Figure 2

HBOT prevents cortical neuronal loss following CCI. (A) Cortical neuronal staining. Representative images of brain neuronal staining with anti-NeuN in CCI and CCI-HBOT in the injured (upper panel) and uninjured hemisphere (lower panel). Scale bar: 100 µm. (B) The number of positive NeuN-labeled cells. NeuN expression was significantly reduced in the injured hemisphere compared with the uninjured hemisphere in both groups. A significantly lower expression of NeuN (injured hemisphere) was noted in the CCI group compared to the CCI-HBOT group. Mean ± SEM. One-way Anova followed with Tukey’s test. *** p < 0.0001 Difference between groups; ^### p < 0.0001 Difference between injured and uninjured hemispheres of the same group.

Figure 3

CCI induces a robust level of cortical apoptosis. (A) Cortical activated caspase-3 staining. Representative images of cortical apoptosis staining with anti-Caspase3 in CCI and CCI-HBOT in the injured (upper panel) and uninjured hemisphere (lower panel). Scale bar: 100 µm. (B) Number of positive caspase-3 labeled cells. Caspase-3 expression was significantly increased in the injured hemisphere compared with the uninjured hemisphere in both groups. A significantly lower expression of caspase-3 (injured hemisphere) was noted in the CCI-HBOT group compared to the CCI group. Mean ± SEM. One-way Anova followed with Tukey’s test. *** p < 0.0001 Difference between groups; ^### p < 0.0001 Difference between injured and uninjured hemispheres of the same group.

Figure 4

The early HBO treatment modulates cortical glial proliferation following CCI (A) Glial cell staining. Representative images of cortical GFAP staining in CCI and CCI-HBOT in the injured (upper panel) and uninjured hemisphere (lower panel). Scale bar: 100 µm. (B) Number of positive GFAP-labeled cells. GFAP expression was significantly increased in the injured hemisphere compared with the uninjured hemisphere in both groups. A significantly higher expression of GFAP (injured hemisphere) was noted in the CCI group compared to the CCI-HBOT group. Mean ± SEM. One-way Anova followed with Tukey’s test. *** p < 0.0001 Difference between groups; ^# p < 0.05, ^### p < 0.0001 Difference between injured and uninjured hemispheres of the same group.

Figure 5

The effect of CCI on mitochondrial oxygen consumption. (A) Respiratory SUIT protocol. Different steps and respiratory states were tested by adding various substrates, uncouplers, and inhibitors. Providing a comprehensive overview of the experimental design and methods used to investigate mitochondrial function following CCI injury and HBOT treatment. (B) An illustration of the experiment is when oxygen consumption rates are measured in response to adding pyruvate–malate, glutamate, ADP, succinate rotenone, oligomycin, CCCP, and antimycin. Blue-Thin line represents the oxygen concentration of the chamber (left y axis) and the oxygen flux per mass (right y axis). (C) Basal mitochondrial respiration in the CCI and CCI group treated with HBO for 4 days. (D) Mitochondrial respiration in the non-phosphorylation resting states-LEAK. (E) Mitochondrial oxygen consumption in the OXPHOS states coupled to ADP phosphorylation for maximal ATP production. (F) Mitochondrial oxygen consumption in the ET capacity state. (N = 6) (Respiratory flux is expressed in pmol O₂ per second per milligram wet tissue mass-pmol O₂/s/mg). Mean ± SEM. One-way Anova followed with Tukey’s test. * p < 0.05, ** p < 0.001, *** p < 0.0001.