Age-related mitochondrial changes after traumatic brain injury

Abstract

Mitochondrial dysfunction is known to occur following traumatic brain injury (TBI) and has been well characterized. This study assessed possible age-related changes in the cortical mitochondrial bioenergetics following TBI. Three hours following a moderate TBI, tissue from the ipsilateral hemisphere (site of impact and penumbra) and the corresponding contralateral region were harvested from young (3- to 5-month-old) and aged (22- to 24-month-old) Fischer 344 rats. Synaptic and extrasynaptic mitochondria were isolated using a Ficoll gradient, and several bioenergetic parameters were examined using a Clark-type electrode. Injury-related respiration deficits were observed in both young and aged rats. Synaptic mitochondria showed an age-related decline in the rate of ATP production, and a decline in respiratory control ratios (RCR), which were not apparent in the extrasynaptic fraction. Following respiration analysis, mitochondrial samples were probed for oxidative damage (3-nitrotyrosine [3-NT], 4-hydroxynonenal [4-HNE], and protein carbonyls [PC]). All markers of oxidative damage were elevated with injury and age in the synaptic fraction, but only with injury in the extrasynaptic fraction. Synaptic mitochondria displayed the highest levels of oxidative damage and may contribute to the synaptic bioenergetic deficits seen following injury. Data indicate that cortical synaptic mitochondria appear to have an increased susceptibility to perturbation with age, suggesting that the increased mitochondrial dysfunction observed following injury may impede recovery in aged animals.

FIG. 1.

Quantification of all states of respiration for both (A) young and (B) aged rats for both the extrasynaptic and synaptic mitochondrial fractions. No significant age-related changes were apparent in any respiration parameter in extrasynaptic mitochondria, but all respiration states were significantly higher than the synaptic fraction. The range of the respiration responses in the extrasynaptic fraction are significantly higher than those in the synaptic fraction, indicating a baseline shift in respiration between these two fractions [bars represent group means ± standard deviation; *p < 0.05 compared to extrasynaptic values; ^#p < 0.05; ^##p < 0.01 compared to contralateral values; P/M, pyruvate/malate; ADP, adenosine diphosphate; Oligo, oligomycin; FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone].

FIG. 2.

Mitochondrial bioenergetic assessment in cortical samples ipsilateral to the injury. (A) Overall oxygen utilization rate. The dashed line represents the overall oxygen utilization rates from the contralateral hemisphere. No significant age-related changes were apparent in this respiration parameter in either mitochondrial fraction. A significant difference between these two mitochondrial fractions did exist. The subtle decline seen in the synaptic fraction of aged animals may be indicative of changes in only certain states of respiration. (B) Respiratory control ratio (RCR). RCR values are used as a general index of how coupled respiration is to ATP production. The dashed line indicates the RCR value in the contralateral hemisphere. Mitochondria are severely dysfunctional and respiration becomes uncoupled from ATP production when RCR values drop below 5. RCR values declined significantly as a result of the injury in both mitochondrial fractions, but only synaptic mitochondria became severely dysfunctional. These data suggest that extrasynaptic mitochondria are able retain their ability to efficiently produce ATP for at least 3 h post-injury, while synaptic mitochondria have already become dysfunctional. Percentages of contralateral respiration rates for synaptic and extrasynaptic fractions are shown in: (C) state III–ADP, (D) state IV–oligomycin, (E) state V–FCCP, and (F) state V–succinate (bars represent group means ± standard deviation; *p < 0.05 compared to young values).

FIG. 3.

Changes in measures of oxidative stress following injury. The levels of oxidized proteins, as measured by levels of protein carbonyls (A), 4-hydroxynonenal (4-HNE)-protein adducts (B), and 3-nitrotyrosine (3-NT), a biomarker of reactive nitrogen species formation (C) were determined in purified synaptic and extrasynaptic mitochondrial fractions (bars represent group means ± standard deviation; *p < 0.05; **p < 0.01; ^#p < 0.001 compared to young animals).