Kaempferol Treatment after Traumatic Brain Injury during Early Development Mitigates Brain Parenchymal Microstructure and Neural Functional Connectivity Deterioration at Adolescence.

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

Prediction of Task-Related BOLD fMRI with Amplitude Signatures of Resting-State fMRI

Blood oxygen contrast-functional magnetic resonance imaging (fMRI) signals are a convolution of neural and vascular components. Several studies indicate that task-related (T-fMRI) or resting-state (R-fMRI) responses linearly relate to hypercapnic task responses. Based on the linearity of R-fMRI and T-fMRI with hypercapnia demonstrated by different groups using different study designs, we hypothesized that R-fMRI and T-fMRI signals are governed by a common physiological mechanism and that resting-state fluctuation of amplitude (RSFA) should be linearly related to T-fMRI responses. We tested this prediction in a group of healthy younger humans where R-fMRI, T-fMRI, and hypercapnic (breath hold, BH) task measures were obtained form the same scan session during resting state and during performance of motor and BH tasks. Within individual subjects, significant linear correlations were observed between motor and BH task responses across voxels. When averaged over the whole brain, the subject-wise correlation between the motor and BH tasks showed a similar linear relationship within the group. Likewise, a significant linear correlation was observed between motor-task activity and RSFA across voxels and subjects. The linear rest–task (R–T) relationship between motor activity and RSFA suggested that R-fMRI and T-fMRI responses are governed by similar physiological mechanisms. A practical use of the R–T relationship is its potential to estimate T-fMRI responses in special populations unable to perform tasks during fMRI scanning. Using the R–T relationship determined from the first group of 12 healthy subjects, we predicted the T-fMRI responses in a second group of 7 healthy subjects. RSFA in both the lower and higher frequency ranges robustly predicted the magnitude of T-fMRI responses at the subject and voxel levels. We propose that T-fMRI responses are reliably predictable to the voxel level in situations where only R-fMRI measures are possible, and may be useful for assessing neural activity in task non-compliant clinical populations

Consolidated Biochemical Profile of Subacute Stage Traumatic Brain Injury in Early Development

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

SMF-1, SMF-2 and SMF-3 DMT1 Orthologues Regulate and Are Regulated Differentially by Manganese Levels in C. elegans

Manganese (Mn) is an essential metal that can exert toxic effects at high concentrations, eventually leading to Parkinsonism. A major transporter of Mn in mammals is the divalent-metal transporter (DMT1). We characterize here DMT1-like proteins in the nematode C. elegans, which regulate and are regulated by Mn and iron (Fe) content. We identified three new DMT1-like genes in C. elegans: smf-1, smf-2 and smf-3. All three can functionally substitute for loss of their yeast orthologues in S. cerevisiae. In the worm, deletion of smf-1 or smf-3 led to an increased Mn tolerance, while loss of smf-2 led to increased Mn sensitivity. smf mRNA levels measured by QRT-PCR were up-regulated upon low Mn and down-regulated upon high Mn exposures. Translational GFP-fusions revealed that SMF-1 and SMF-3 strongly localize to partially overlapping apical regions of the gut epithelium, suggesting a differential role for SMF-1 and SMF-3 in Mn nutritional intake. Conversely, SMF-2 was detected in the marginal pharyngeal epithelium, possibly involved in metal-sensing. Analysis of metal content upon Mn exposure in smf mutants revealed that SMF-3 is required for normal Mn uptake, while smf-1 was dispensable. Higher smf-2 mRNA levels correlated with higher Fe content, supporting a role for SMF-2 in Fe uptake. In smf-1 and smf-3 but not in smf-2 mutants, increased Mn exposure led to decreased Fe levels, suggesting that both metals compete for transport by SMF-2. Finally, SMF-3 was post-translationally and reversibly down-regulated following Mn-exposure. In sum, we unraveled a complex interplay of transcriptional and post-translational regulations of 3 DMT1-like transporters in two adjacent tissues, which regulate metal-content in C. elegans

Spontaneous Brain Activity in the Default Mode Network Is Sensitive to Different Resting-State Conditions with Limited Cognitive Load

BACKGROUND: Recent functional MRI (fMRI) studies have demonstrated that there is an intrinsically organized default mode network (DMN) in the resting brain, primarily made up of the posterior cingulate cortex (PCC) and the medial prefrontal cortex (MPFC). Several previous studies have found that the DMN is minimally disturbed during different resting-state conditions with limited cognitive demand. However, this conclusion was drawn from the visual inspection of the functional connectivity patterns within the DMN and no statistical comparison was performed. METHODOLOGY/PRINCIPAL FINDINGS: Four resting-state fMRI sessions were acquired: 1) eyes-closed (EC) (used to generate the DMN mask); 2) EC; 3) eyes-open with no fixation (EO); and 4) eyes-open with a fixation (EO-F). The 2-4 sessions were counterbalanced across participants (n = 20, 10 males). We examined the statistical differences in both functional connectivity and regional amplitude of low frequency fluctuation (ALFF) within the DMN among the 2-4 resting-state conditions (i.e., EC, EO, and EO-F). Although the connectivity patterns of the DMN were visually similar across these three different conditions, we observed significantly higher functional connectivity and ALFF in both the EO and the EO-F conditions as compared to the EC condition. In addition, the first and second resting EC conditions showed significant differences within the DMN, suggesting an order effect on the DMN activity. CONCLUSIONS/SIGNIFICANCE: Our findings of the higher DMN connectivity and regional spontaneous activities in the resting state with the eyes open suggest that the participants might have more non-specific or non-goal-directed visual information gathering and evaluation, and mind wandering or daydreaming during the resting state with the eyes open as compared to that with the eyes closed, thus providing insights into the understanding of unconstrained mental activity within the DMN. Our results also suggest that it should be cautious when choosing the type of a resting condition and designating the order of the resting condition in multiple scanning sessions in experimental design

Functional Connectivity fMRI of the Rodent Brain: Comparison of Functional Connectivity Networks in Rat and Mouse

At present, resting state functional MRI (rsfMRI) is increasingly used in human neuropathological research. The present study aims at implementing rsfMRI in mice, a species that holds the widest variety of neurological disease models. Moreover, by acquiring rsfMRI data with a comparable protocol for anesthesia, scanning and analysis, in both rats and mice we were able to compare findings obtained in both species. The outcome of rsfMRI is different for rats and mice and depends strongly on the applied number of components in the Independent Component Analysis (ICA). The most important difference was the appearance of unilateral cortical components for the mouse resting state data compared to bilateral rat cortical networks. Furthermore, a higher number of components was needed for the ICA analysis to separate different cortical regions in mice as compared to rats

Robust Reproducible Resting State Networks in the Awake Rodent Brain

Resting state networks (RSNs) have been studied extensively with functional MRI in humans in health and disease to reflect brain function in the un-stimulated state as well as reveal how the brain is altered with disease. Rodent models of disease have been used comprehensively to understand the biology of the disease as well as in the development of new therapies. RSN reported studies in rodents, however, are few, and most studies are performed with anesthetized rodents that might alter networks and differ from their non-anesthetized state. Acquiring RSN data in the awake rodent avoids the issues of anesthesia effects on brain function. Using high field fMRI we determined RSNs in awake rats using an independent component analysis (ICA) approach, however, ICA analysis can produce a large number of components, some with biological relevance (networks). We further have applied a novel method to determine networks that are robust and reproducible among all the components found with ICA. This analysis indicates that 7 networks are robust and reproducible in the rat and their putative role is discussed

Beneficial Effects of Kaempferol after Developmental Traumatic Brain Injury Is through Protection of Mitochondrial Function, Oxidative Metabolism, and Neural Viability

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

Facilitating Mitochondrial Calcium Uptake Improves Activation-Induced Cerebral Blood Flow and Behavior after mTBI.

Mild to moderate traumatic brain injury (mTBI) leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca(2+) overload. However, in the surviving cellular population, mitochondrial Ca(2+) influx, and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca(2+) uptake in a rat model of mTBI. In developing rats (P25-P26) sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF) in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI) at adulthood (P67-P73). Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca(2+) uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca(2+) uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity following kaempferol treatment suggests that facilitation of mitochondrial Ca(2+) uptake in the early window after injury may sustain optimal neural activity and metabolism and contribute to improved function of the surviving cellular populations after mTBI