Peripheral Blood Mitochondrial DNA as a Biomarker of Cerebral Mitochondrial Dysfunction following Traumatic Brain Injury in a Porcine Model

Abstract

Background: Traumatic brain injury (TBI) has been shown to activate the peripheral innate immune system and systemic inflammatory response, possibly through the central release of damage associated molecular patterns (DAMPs). Our main purpose was to gain an initial understanding of the peripheral mitochondrial response following TBI, and how this response could be utilized to determine cerebral mitochondrial bioenergetics. We hypothesized that TBI would increase peripheral whole blood relative mtDNA copy number, and that these alterations would be associated with cerebral mitochondrial bioenergetics triggered by TBI.

Methodology: Blood samples were obtained before, 6 h after, and 25 h after focal (controlled cortical impact injury: CCI) and diffuse (rapid non-impact rotational injury: RNR) TBI. PCR primers, unique to mtDNA, were identified by aligning segments of nuclear DNA (nDNA) to mtDNA, normalizing values to nuclear 16S rRNA, for a relative mtDNA copy number. Three unique mtDNA regions were selected, and PCR primers were designed within those regions, limited to 25-30 base pairs to further ensure sequence specificity, and measured utilizing qRT-PCR.

Results: Mean relative mtDNA copy numbers increased significantly at 6 and 25 hrs after following both focal and diffuse traumatic brain injury. Specifically, the mean relative mtDNA copy number from three mitochondrial-specific regions pre-injury was 0.84 ± 0.05. At 6 and 25 h after diffuse non-impact TBI, mean mtDNA copy number was significantly higher: 2.07 ± 0.19 (P < 0.0001) and 2.37 ± 0.42 (P < 0.001), respectively. Following focal impact TBI, relative mtDNA copy number was also significantly higher, 1.35 ± 0.12 (P < 0.0001) at 25 hours. Alterations in mitochondrial respiration in the hippocampus and cortex post-TBI correlated with changes in the relative mtDNA copy number measured in peripheral blood.

Conclusions: Alterations in peripheral blood relative mtDNA copy numbers may be a novel biosignature of cerebral mitochondrial bioenergetics with exciting translational potential for non-invasive diagnostic and interventional studies.

Fig 1. Relative quantification values of mtDNA concentrations for each gene measured from pre-injury to 6 hours and 25 hours post-injury in diffuse, rapid non-impact rotational (RNR) traumatic brain injury.

Box plot analysis illustrating the difference in relative quantification values of mtDNA concentrations measured in peripheral whole blood for each gene measured from pre-injury to 6 and 25 hours post-RNR. Increases in all three genes measured were similar following RNR at both time points. Nicotinamide adenine dinuclueotide dehydrogenase (NADH) subunit 4mitochondrial (ND4-1). Cytochrome c oxidase subunit (COI-1) and I-A (COI-1A). All values are mean ± SEM.

Fig 2. Relative quantification values of mtDNA concentrations measured in peripheral whole blood for each gene measured from pre-injury to post-injury in focal, controlled cortical impact (CCI) traumatic brain injury.

Box plot analysis illustrating the difference in relative quantification values of mtDNA concentrations measured in peripheral whole blood for each gene measured from pre-injury to 25 hours post-CCI. Increases in all three genes measured were similar following CCI at 25 hours. Nicotinamide adenine dinuclueotide dehydrogenase (NADH) subunit 4mitochondrial (ND4-1). Cytochrome c oxidase subunit (COI-1) and I-A (COI-1A). All values are mean ± SEM.

Fig 3. Peripheral blood mtDNA copy number in rapid non-impact rotational (RNR) traumatic brain injury.

RNR traumatic brain injury resulted in significant increases in relative quantification mitochondrial copy number (RQ) measured in peripheral whole blood at 6 hours and 25 hours post-RNR. All values are the mean of three specific mitochondrial genes measured (ND4-1, COI-1, and COI-1A) and pooled together ± SEM. Nicotinamide adenine dinuclueotide dehydrogenase (NADH) subunit 4mitochondrial (ND4-1). Cytochrome c oxidase subunit (COI-1) and I-A (COI-1A). *P<0.001 vs. Pre-RNR.

Fig 4. Peripheral blood mtDNA copy number in focal, controlled cortical impact (CCI) traumatic brain injury.

CCI traumatic brain injury resulted in significant increases in relative quantification mitochondrial copy number (RQ) measured in peripheral whole blood at 25 hours post-RNR. All values are the mean of three specific mitochondrial genes measured (ND4-1, COI-1, and COI-1A) pooled together ± SEM. Nicotinamide adenine dinuclueotide dehydrogenase (NADH) subunit 4mitochondrial (ND4-1). Cytochrome c oxidase subunit (COI-1) and I-A (COI-1A). *P<0.0001 vs. Pre-CCI.

Fig 5. Association between peripheral blood mtDNA copy number and cerebral bioenergetics 6 hours and 25 hours after rapid, non-impact rotational (RNR) traumatic brain injury.

(A) There was a significant correlation between the increase in peripheral blood relative mtDNA quantification copy number (RQ), from pre- to post-injury and the decrease in hippocampal maximal oxidative phosphorylation 6 hours post-RNR from sham respiration values. RNR: rapid non-impact rotational traumatic brain injury. (B) There was a significant correlation between the increase in peripheral blood relative mtDNA quantification copy number (RQ), from pre- to post-injury and the decrease in hippocampal maximal oxidative phosphorylation (OXPHOS_CI+CII) 25 hours post-RNR from sham respiration. RNR: rapid non-impact rotational traumatic brain injury.

Fig 6. Association between peripheral blood mtDNA copy number and cerebral bioenergetics 25 hours after controlled cortical impact (CCI) traumatic brain injury.

There was a significant correlation between the increase in peripheral blood relative mtDNA quantification copy number (RQ), from pre- to post-injury and the decrease in maximal oxidative phosphorylation (OXPHOS_CI+CII) in peri-contusional CCI tissue 25 hours post-CCI from sham respiration. CCI: controlled cortical impact traumatic brain injury.