Oxidative energy metabolism is depressed after mild/moderate traumatic brain injury (TBI) during early development, accompanied by behavioral debilitation and secondary neuronal death. A TBI metabolome analysis revealed broad effects with a striking impact on energy metabolism. Our studies on mitochondrial modulators and their effects on brain function have shown that kaempferol, a stimulator of the mitochondrial Ca²⁺ uniporter channel (mCU), enhanced neural and neurovascular activity in the normal brain and improved stimulus-induced brain activation and behavior after TBI during early development. Because kaempferol enhances mitochondrial Ca²⁺ uptake and cycling, with protective effects after TBI, we tested the hypothesis that kaempferol treatment during the acute/subacute stage after TBI (0-72 h) acted on mitochondria in improving TBI outcome. Developmental age rats (P31) underwent TBI and were treated with vehicle or kaempferol (1 mg/kg intraperitoneally) in three doses at 1, 24, and 48 h after TBI. Brains were harvested at 72 h and subjected to liquid chromatography mass spectrometric measurements. Decrease in pyruvate and tricarboxylic acid (TCA) cycle flux were observed in the untreated and vehicle-treated group, consistent with previously established energy metabolic decline after TBI. Kaempferol improved TCA cycle flux, maintained mitochondrial functional integrity as observed by decreased acyl carnitines, improved neural viability as evidenced by higher N-acetyl aspartate levels. The positive outcomes of kaempferol on metabolic profile corresponded with improved sensorimotor behavior.

Ru360, a mitochondrial Ca²⁺ uptake inhibitor, was tested in a unilateral fluid percussion TBI model in developing rats (P31). Vehicle and Ru360 treated TBI rats underwent sensorimotor behavioral monitoring between 24 and 72 h, thereafter which 185 brain metabolites were analyzed postmortem using LC/MS. Ru360 treatment after TBI improved sensorimotor behavioral recovery, upregulated glycolytic and pentose phosphate pathways, mitigated oxidative stress and prevented NAD⁺ depletion across both hemispheres. While neural viability improved ipsilaterally, it reduced contralaterally. Ru360 treatment, overall, had a global impact with most benefit near the strongest injury impact areas, while perturbing mitochondrial oxidative energetics in the milder TBI impact areas.

Pathophysiology of developmental traumatic brain injury (TBI) is unique due to intrinsic differences in the developing brain. Energy metabolic studies of the brain during early development (P13 to P30) have indicated acute oxidative energy metabolic decreases below 24 h after TBI, which generally recovered by 48 h. However, marked neurodegeneration and altered neural functional connectivity have been observed at later stages into adolescence. As secondary neurodegeneration is most prominent during the first week after TBI in the rat model, we hypothesized that the subacute TBI-metabolome may contain predictive markers of neurodegeneration. Sham and TBI metabolomes were examined at 72 h after a mild to moderate intensity TBI in male Sprague-Dawley rats aged P31. Sensorimotor behavior was assessed at 24, 48 and 72 h after injury, followed by 72-hour postmortem brain removal for metabolomics using Liquid Chromatography/Mass Spectrometry (LC-MS) measurement. Broad TBI-induced metabolomic shifts occurred with relatively higher intensity in the injury-lateralized (ipsilateral) hemisphere. Intensity of metabolomic perturbation correlated with the extent of sensorimotor behavioral deficit. N-acetyl-aspartate (NAA) levels at 72 h after TBI, predicted the extent of neurodegeneration assessed histochemically 7-days post TBI. Results from the multivariate untargeted approach clearly distinguished metabolomic shifts induced by TBI. Several pathways including amino acid, fatty acid and energy metabolism continued to be affected at 72 h after TBI, whose collective effects may determine the overall pathological response after TBI in early development including neurodegeneration.

Traumatic brain injury (TBI) in general has varied neuropathological consequences depending upon the intensity and biomechanics of the injury. Furthermore, in pediatric TBI, intrinsic developmental changes add further complexity, necessitating a biochemical dimension for improved TBI characterization. In our earlier study investigating the subacute stage TBI metabolome (72 h post-injury) in a developmental rat model, significant ipsilateral brain biochemical changes occurred across 25 metabolite sets as determined by metabolite set enrichment analysis (MSEA). The broad metabolic perturbation was accompanied by behavioral deficits and neuronal loss across the ipsilateral hemisphere containing the injury epicenter. In order to obtain a consolidated biochemical profile of the TBI assessment, a subgrouping of the 190 identified brain metabolites was performed. Metabolites were divided into seven major subgroups: oxidative energy/mitochondrial, glycolysis/pentose phosphate pathway, fatty acid, amino acid, neurotransmitters/neuromodulators, one-carbon/folate and other metabolites. Subgroups were based on the chemical nature and association with critically altered biochemical pathways after TBI as obtained from our earlier untargeted analysis. Each metabolite subgroup extracted from the ipsilateral sham and TBI brains were modeled using multivariate partial least square discriminant analysis (PLS-DA) with the model accuracy used as a measurable index of TBI neurochemical impact. Volcano plots of each subgroup, corrected for multiple comparisons, determined the TBI neurochemical specificity. The results provide a ranked biochemical profile along with specificity of changes after developmental TBI, enabling a consolidated biochemical template for future classification of different TBI intensities and injury types in animal models.

Targeting mitochondrial ion homeostasis using Kaempferol, a mitochondrial Ca²⁺ uniporter channel activator, improves energy metabolism and behavior soon after a traumatic brain injury (TBI) in developing rats. Because of broad TBI pathophysiology and brain mitochondrial heterogeneity, Kaempferol-mediated early-stage behavioral and brain metabolic benefits may accrue from diverse sources within the brain. We hypothesized that Kaempferol influences TBI outcome by differentially impacting the neural, vascular, and synaptic/axonal compartments. After TBI at early development (P31), functional magnetic resonance imaging and diffusion tensor imaging (DTI) were applied to determine imaging outcomes at adolescence (2 months post-injury). Vehicle and Kaempferol treatments were made at 1, 24, and 48 h post-TBI, and their effects were assessed at adolescence. A significant increase in neural connectivity was observed after Kaempferol treatment as assessed by the spatial extent and strength of the somatosensory cortical and hippocampal resting-state functional connectivity (RSFC) networks. However, no significant RSFC changes were observed in the thalamus. DTI measures of fractional anisotropy (FA) and apparent diffusion coefficient, representing synaptic/axonal and microstructural integrity, showed significant improvements after Kaempferol treatment, with highest changes in the frontal and parietal cortices and hippocampus. Kaempferol treatment also increased corpus callosal FA, indicating measurable improvement in the interhemispheric structural connectivity. TBI prognosis was significantly altered at adolescence by early Kaempferol treatment, with improved neural connectivity, neurovascular coupling, and parenchymal microstructure in select brain regions. However, Kaempferol failed to improve vasomotive function across the whole brain, as measured by cerebrovascular reactivity. The differential effects of Kaempferol treatment on various brain functional compartments support diverse cellular-level mitochondrial functional outcomes in vivo.