83 - Temporal Profile of Mitochondrial Calcium Homeostasis, Membrane Integrity and Apoptosis Markers in a Preclinical Model of Severe Penetrating TBI

Abstract Text:

Background:
Traumatic Brain Injury (TBI) is a major health concern within both the military and civilian sectors worldwide. Combat-related head injury has been one of the leading causes of military death and disability. During the post-injury phase of moderate to severe TBI, the most common mitochondria-centered cellular responses involved are calcium (Ca2+) overload, oxidative stress, and impaired energy homeostasis. Their imbalance subsequently may prompt the downstream cellular processes such as apoptotic / necrotic neuronal death and ultimately alters behavior outcomes after TBI. Although, mitochondrial dysfunction is a pivotal target for neuroprotection strategies for TBI, the comprehensive time-course evaluation of excitotoxic responses of mitochondrial dysfunction such as Ca2+ homeostasis, membrane integrity and cell death, are lacking in the pre-clinical penetrating TBI (PTBI). The recently published mitochondrial bioenergetics time-course analyses in the PTBI model revealed that the injury core and perilesional regions (i.e., frontal cortex + striatum) displayed biphasic energy crisis response, which initiated immediately at 30 min after trauma, and extended up to 2 weeks post-injury period (J. Neurotrauma 2021, PMID: 33544034). In the interest of future evaluation of mitochondria targeted neuroprotection drugs using mitochondria mediated excitotoxicity responses, we conducted a full time-course evaluation of mitochondrial Ca2+ homeostasis, membrane integrity and cell death markers following PTBI in rats.

Methods:
Anesthetized adult male Sprague-Dawley rats (280–350 g) were subjected to either 10% unilateral PTBI or Sham craniectomy. Time-course examination of Ca2+ dynamics, membrane integrity and apoptosis markers evaluated at 30 min, 3h, 6h, 24h, 3d, 7d and 14d (n=6-10 animals/group X 7 time-points). At each time-point, animals were euthanized, and mitochondria were isolated from the injury core and perilesional area of the brain. Mitochondrial samples were evaluated immediately or stored at -800C for Western blot analysis. Mitochondrial Ca2+ dynamics parameters such as Ca2+ buffering capacity and membrane permeability transition pore (mPTP) opening time were assessed in a real-time condition using a spectrophotofluorometer. In addition, membrane integrity markers such as cytochrome C (Cyt C) and voltage-dependent anion channel (VDAC), and apoptotic indicator glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured using Western blot.

Results:
PTBI group mitochondrial Ca2+ dynamics parameters resulted in a “U-shaped pattern” of significant loss in Ca2+ buffering capacity (12-76%, vs. Sham) during the 2 weeks post-injury period. The peak loss of Ca2+ buffering capacity was noted at 24h post-PTBI. The time-course evaluation of membrane integrity markers such as Cyt-C and VDAC, both protein markers revealed significant loss of expression (20-50% vs. Sham) during the 2 weeks post-injury period. Additionally, the GAPDH expression was increased significantly by ~2-4 fold in the PTBI group.

Conclusion:
Following PTBI, mitochondria displayed a significant loss of Ca2+ buffering capacity and early activation of mPTP. In parallel, PTBI mitochondria showed loss of membrane integrity responses as evidenced by a decrease in Cyt C and VDAC markers, together with significantly elevated apoptosis markers. Our results demonstrated that early intervention is necessary to mitigate mitochondria targeted excitotoxic responses to achieve neuroprotection following PTBI.

Support: US_Army_CCCRP_H_001_2018_WRAIR.

Keywords: Penetrating Traumatic Brain Injury, Mitochondria, Time-course, Calcium Homeostasis, Mitochondrial Permeability Transition Pore, Membrane Integrity