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

Kilbaugh TJ, Lvova M, Karlsson M, Zhang Z, Leipzig J, Wallace DC, Margulies SS - PLoS ONE (2015)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0130927.g001: 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.

Mentions: Mean peripheral blood mtDNA copy number was significantly increased after focal (25 hours) and diffuse (6 hours and 25 hours) traumatic brain injury compared to pre-injury levels. Using relative quantitative PCR techniques to measure relative mtDNA copy number, for 2 genes devoid of NUMT determined by bioinformatics analysis, we measured the relative content determined by the ratio of mtDNA/16S rRNA with respect to the COI and ND4-1 genes in peripheral blood, following TBI from diffuse (Table 1) and focal injuries (Table 2). To determine the pre-injury average relative mtDNA copy number across all animals, we first determined that there was no statistical difference between pre-injury relative mtDNA copy number across all genes measured in RNR animals (n = 24) and CCI animals (n = 15) (P = 0.29, Mann Whitney U test). In addition, there was no difference in the delta change between pre- and post- relative mtDNA copy number across the 3 genes at either 6 (P = 0.86) or 25 hours (P = 0.95), or between the two time points (P = 0.94, repeated-measures ANOVA) for diffuse RNR injuries (Fig 1). Similarly, there were no differences in the delta changes across all three genes 25 hours after focal CCI (P = 0.98) (Fig 2). Therefore, we defined the pre-injury relative mtDNA copy number by pooling together the RQ of all genes obtained from all animals to determine the average pre-injury mean, 0.84 ± 0.05 (n = 39). Next, we pooled the post-injury relative mtDNA copy number across all genes for each injury group and time point, and then compared these values to the pooled pre-injury relative mtDNA copy number. We found that there was a significant increase in relative mtDNA copy number 6 hours after diffuse RNR TBI (n = 12), 2.07 ± 0.19 (P < 0.0001, Wilcoxon signed rank test) (Fig 3). Furthermore, 25 hours post-RNR the relative mtDNA copy number (n = 12) 2.37 ± 0.42 (P = 0.0005) was significantly increased compared to pre-injury levels. There was no significant difference in relative mtDNA copy number between time points measured at 6 and 25 hours post-RNR (P = 0.62, Mann-Whitney U test) (Fig 3). Similarly, the relative mtDNA copy number (n = 15) 25 hours post-injury was significantly increased, 1.35 ± 0.12 (P < 0.0001) compared to pre-injury relative mtDNA copy number (Fig 4).


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

Kilbaugh TJ, Lvova M, Karlsson M, Zhang Z, Leipzig J, Wallace DC, Margulies SS - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476697&req=5

pone.0130927.g001: 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.
Mentions: Mean peripheral blood mtDNA copy number was significantly increased after focal (25 hours) and diffuse (6 hours and 25 hours) traumatic brain injury compared to pre-injury levels. Using relative quantitative PCR techniques to measure relative mtDNA copy number, for 2 genes devoid of NUMT determined by bioinformatics analysis, we measured the relative content determined by the ratio of mtDNA/16S rRNA with respect to the COI and ND4-1 genes in peripheral blood, following TBI from diffuse (Table 1) and focal injuries (Table 2). To determine the pre-injury average relative mtDNA copy number across all animals, we first determined that there was no statistical difference between pre-injury relative mtDNA copy number across all genes measured in RNR animals (n = 24) and CCI animals (n = 15) (P = 0.29, Mann Whitney U test). In addition, there was no difference in the delta change between pre- and post- relative mtDNA copy number across the 3 genes at either 6 (P = 0.86) or 25 hours (P = 0.95), or between the two time points (P = 0.94, repeated-measures ANOVA) for diffuse RNR injuries (Fig 1). Similarly, there were no differences in the delta changes across all three genes 25 hours after focal CCI (P = 0.98) (Fig 2). Therefore, we defined the pre-injury relative mtDNA copy number by pooling together the RQ of all genes obtained from all animals to determine the average pre-injury mean, 0.84 ± 0.05 (n = 39). Next, we pooled the post-injury relative mtDNA copy number across all genes for each injury group and time point, and then compared these values to the pooled pre-injury relative mtDNA copy number. We found that there was a significant increase in relative mtDNA copy number 6 hours after diffuse RNR TBI (n = 12), 2.07 ± 0.19 (P < 0.0001, Wilcoxon signed rank test) (Fig 3). Furthermore, 25 hours post-RNR the relative mtDNA copy number (n = 12) 2.37 ± 0.42 (P = 0.0005) was significantly increased compared to pre-injury levels. There was no significant difference in relative mtDNA copy number between time points measured at 6 and 25 hours post-RNR (P = 0.62, Mann-Whitney U test) (Fig 3). Similarly, the relative mtDNA copy number (n = 15) 25 hours post-injury was significantly increased, 1.35 ± 0.12 (P < 0.0001) compared to pre-injury relative mtDNA copy number (Fig 4).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus