Early mitochondrial dysfunction after cortical contusion injury

Abstract

Following traumatic brain injury, mitochondria sustain structural and functional impairment, which contributes to secondary damage that can continue for days after the initial injury. The present study investigated mitochondrial bioenergetic changes in the rat neocortex at 1 and 3 h after mild, moderate, and severe injuries. Brains from young adult Sprague-Dawley rats were harvested from the injured and contralateral cortex to assess possible changes in mitochondrial respiration abilities following a unilateral cortical contusion injury. Differential centrifugation was used to isolate synaptic and extrasynaptic mitochondria from cortical tissue. Bioenergetics was assessed using a Clark-type electrode and results were graphed as a function of injury severity and time post-injury. Respiration was significantly affected by all injury severity levels compared to uninjured tissue. Complex 1- and complex 2-driven respirations were affected proportionally to the severity of the injury, indicating that damage to mitochondria may occur on a gradient. Total oxygen utilization, respiratory control ratio, ATP production, and maximal respiration capabilities were all significantly decreased in the injured cortex at both 1 and 3 h post-trauma. Although mitochondria displayed bioenergetic deficits at 1 h following injury, damage was not exacerbated by 3 h. This study stresses the importance of early therapeutic intervention and suggests a window of approximately 1-3 h before greater dysfunction occurs.

FIG. 1.

Typical oxygen utilization trace of healthy mitochondria taken from a sham animal. State I: no substrates for respiration have been added; no respiration is apparent. State II: addition of P/M, with basal rate of respiration. State III: addition of ADP completes the necessary substrates needed for operation of the electron transport chain (ETC); the high level of oxygen utilization indicates that ADP is getting converted into ATP. State IV: addition of oligomycin; return to basal rate of respiration since the ATP synthase is shut down and no electrons are allowed to return to the matrix. The ETC continues only to maintain mitochondrial membrane potential due to loss of protons back into the matrix. State V: addition of FCCP; this represents the maximum rate of respiration, causing uncoupling of the ETC to ATP synthesis, and allows protons to rush back into the matrix. Rotenone is then added to shut down complex 1–driven respiration. State V (succinate): addition of succinate; this is the maximum rate of respiration via complex 2, since FCCP is present in the system.

FIG. 2.

Overall oxygen utilization rate. As injury severity increases the overall oxygen utilization significantly drops, indicating that the mitochondria are not responding to the additions of substrates as robustly following injury. Bars represent group means ± SD (*p < 0.01 compared to sham; ^†p < 0.05 compared to mild, ^#p < 0.01 compared to moderate).

FIG. 3.

ADP rates. The ability of mitochondria to phosphorylate ADP significantly drops with injury severity. Bars represent group means ± SD (*p < 0.05 compared to sham; ^†p < 0.05 compared to mild; ^#p < 0.05 compared to moderate).

FIG. 4.

Oligomycin rates. The inner membrane becomes increasingly damaged, causing more of the proton to be lost back into the matrix, resulting in less of the proton gradient being coupled with ATP production. Bars represent group means ± SD (*p < 0.05 compared sham/mild; ^#p < 0.01 compared to sham/mild).

FIG. 5.

Respiratory control ratio (RCR). RCR is an index of how coupled respiration is to phosphorylating ADP. A RCR of 5 indicates well-coupled mitochondria. RCRs significantly drop with increasing injury severity. The data suggest that mitochondria are displaying an uncoupling of respiration from ATP production, meaning a decreased ability of mitochondria to produce ATP. Bars represent group means ± SD (*p < 0.05 compared to sham; ^†p < 0.05 compared to mild injury; ^#p < 0.05 compared to moderate injury).

FIG. 6.

FCCP rates. Maximal respiration capability drops with increasing injury severity. Bars represent group means ± SD (*p < 0.05 compared to sham; ^†p < 0.05 compared to mild injury; ^#p < 0.05 compared to moderate injury).

FIG. 7.

Succinate rates. Not only is Complex 1-driven respiration affected by injury, but Complex 2 as well. Bars are group means ± SD (*p < 0.05 compared to sham; ^†p < 0.05 compared to mild injury; ^#p < 0.05 compared to moderate injury).