Sex-Specific Alterations in Inflammatory MicroRNAs in Mouse Brain and Bone Marrow CD11b+ Cells Following Traumatic Brain Injury.

Sex is a key biological variable in traumatic brain injury (TBI) and plays a significant role in neuroinflammatory responses. However, the molecular mechanisms contributing to this sexually dimorphic neuroinflammatory response remain elusive. Here we describe a significant and previously unreported tissue enrichment and sex-specific alteration of a set of inflammatory microRNAs (miRNAs) in CD11b+ cells of brain and bone marrow isolated from naïve mice as well as mice subjected to TBI. Our data from naïve mice demonstrated that expression levels of miR-146a-5p and miR-150-5p were relatively higher in brain CD11b+ cells, and that miR-155-5p and miR-223-3p were highly enriched in bone marrow CD11b+ cells. Furthermore, while miR-150-5p and miR-155-5p levels were higher in male brain CD11b+ cells, no significant sexual difference was observed for miR-146a-5p and miR-223-3p. However, TBI resulted in sex-specific differential responses of these miRNAs in brain CD11b+ cells. Specifically, miR-223-3p levels in brain CD11b+ cells were markedly elevated in both sexes in response to TBI at 3 and 24 h, with levels in females being significantly higher than males at 24 h. We then focused on analyzing several miR-223-3p targets and inflammation-related marker genes following injury. Corresponding to the greater elevation of miR-223-3p in females, the miR-223-3p targets, TRAF6 and FBXW7 were significantly reduced in females compared to males. Interestingly, anti-inflammatory genes ARG1 and IL4 were higher in females after TBI than in males. These observations suggest miR-223-3p and other inflammatory responsive miRNAs may play a key role in sex-specific neuroinflammatory response following TBI.

MicroRNAs as Biomarkers for Predicting Complications following Aneurysmal Subarachnoid Hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is a high mortality hemorrhagic stroke that affects nearly 30,000 patients annually in the United States. Approximately 30% of aSAH patients die during initial hospitalization and those who survive often carry poor prognosis with one in five having permanent physical and/or cognitive disabilities. The poor outcome of aSAH can be the result of the initial catastrophic event or due to the many acute or delayed neurological complications, such as cerebral ischemia, hydrocephalus, and re-bleeding. Unfortunately, no effective biomarker exists to predict or diagnose these complications at a clinically relevant time point when neurologic injury can be effectively treated and managed. Recently, a number of studies have demonstrated that microRNAs (miRNAs) in extracellular biofluids are highly associated with aSAH and complications. Here we provide an overview of the current research on relevant human studies examining the correlation between miRNAs and aSAH complications and discuss the potential application of using miRNAs as biomarkers in aSAH management.

Enforced lysosomal biogenesis rescues erythromycin- and clindamycin-induced mitochondria-mediated cell death in human cells.

Antibiotics are the front-line treatment against many bacterial infectious diseases in human. The excessive and long-term use of antibiotics in human cause several side effects. It is important to understand the underlying molecular mechanisms of action of antibiotics in the host cell to avoid the side effects due to the prevalent uses. In the current study, we investigated the crosstalk between mitochondria and lysosomes in the presence of widely used antibiotics: erythromycin (ERM) and clindamycin (CLDM), which target the 50S subunit of bacterial ribosomes. We report here that both ERM and CLDM induced caspase activation and cell death in several different human cell lines. The activity of the mitochondrial respiratory chain was compromised in the presence of ERM and CLDM leading to bioenergetic crisis and generation of reactive oxygen species. Antibiotics treatment impaired autophagy flux and lysosome numbers, resulting in decreased removal of damaged mitochondria through mitophagy, hence accumulation of defective mitochondria. We further show that over-expression of transcription factor EB (TFEB) increased the lysosome number, restored mitochondrial function and rescued ERM- and CLDM-induced cell death. These studies indicate that antibiotics alter mitochondria and lysosome interactions leading to apoptotsis and may develop a novel approach for targeting inter-organelle crosstalk to limit deleterious antibiotic-induced side effects.

Altered Cerebellar Circuitry following Thoracic Spinal Cord Injury in Adult Rats.

Cerebellar function is critical for coordinating movement and motor learning. However, events occurring in the cerebellum following spinal cord injury (SCI) have not been investigated in detail. We provide evidence of SCI-induced cerebellar synaptic changes involving a loss of granule cell parallel fiber input to distal regions of the Purkinje cell dendritic tree. This is accompanied by an apparent increase in synaptic contacts to Purkinje cell proximal dendrites, presumably from climbing fibers originating in the inferior olive. We also observed an early stage injury-induced decrease in the levels of cerebellin-1, a synaptic organizing molecule that is critical for establishing and maintaining parallel fiber-Purkinje cell synaptic integrity. Interestingly, this transsynaptic reorganizational pattern is consistent with that reported during development and in certain transgenic mouse models. To our knowledge, such a reorganizational event has not been described in response to SCI in adult rats. Regardless, the novel results of this study are important for understanding SCI-induced synaptic changes in the cerebellum, which may prove critical for strategies focusing on promoting functional recovery.

Increased Incidence of Spinal Abscess and Substance Abuse after Implementation of State Mandated Prescription Drug Legislation.

OBJECTIVES: To investigate the incidence of spinal abscess and substance abuse in a tertiary care hospital after state legislation titled "House Bill 1" (HB1) mandated stricter regulation of prescription drugs of abuse in Kentucky in 2012. DESIGN: A retrospective case series study design was used to review the incidence of spinal abscess and drug abuse diagnoses admissions from 2010 to 2014. Variances in the incidence of spinal abscess and substance abuse were plotted across this time frame. RESULTS: The incidence of intraspinal abscess increased 1.56-fold in 2011 (n = 26) and 2012 (n = 25) relative to 2010 (n = 16). However, in 2013, the year following implementation of HB1 legislation, the incidence of intraspinal abscess increased 2.38-fold (n = 38) and then 4.19-fold (n = 67) in 2014. The incidence of intraspinal abscess in subjects with drug abuse diagnosis remained constant between 2010 (n = 3) and 2012 (n = 3). However, it increased twofold (n = 7) in 2013 and then ninefold (n = 27) in 2014. A correlation coefficient (rSAD ) of 0.775 revealed a strong association between the increase incidence of intraspinal abscess and diagnosis of drug abuse. CONCLUSIONS: The results of this retrospective study demonstrate an increased incidence of intraspinal abscess associated with drug abuse after passage of HB1 legislation regulating prescriptions of controlled medications in Kentucky. This increased incidence may be related to individuals relying on nonprescription drugs of abuse due to more highly regulated access to controlled prescription medications. However, additional factors unrelated to HB1 legislation must be taken into account.

Surgical intervention combined with weight-bearing walking training promotes recovery in patients with chronic spinal cord injury: a randomized controlled study.

JOURNAL/nrgr/04.03/01300535-202412000-00032/figure1/v/2024-04-08T165401Z/r/image-tiff For patients with chronic spinal cord injury, the conventional treatment is rehabilitation and treatment of spinal cord injury complications such as urinary tract infection, pressure sores, osteoporosis, and deep vein thrombosis. Surgery is rarely performed on spinal cord injury in the chronic phase, and few treatments have been proven effective in chronic spinal cord injury patients. Development of effective therapies for chronic spinal cord injury patients is needed. We conducted a randomized controlled clinical trial in patients with chronic complete thoracic spinal cord injury to compare intensive rehabilitation (weight-bearing walking training) alone with surgical intervention plus intensive rehabilitation. This clinical trial was registered at ClinicalTrials.gov (NCT02663310). The goal of surgical intervention was spinal cord detethering, restoration of cerebrospinal fluid flow, and elimination of residual spinal cord compression. We found that surgical intervention plus weight-bearing walking training was associated with a higher incidence of American Spinal Injury Association Impairment Scale improvement, reduced spasticity, and more rapid bowel and bladder functional recovery than weight-bearing walking training alone. Overall, the surgical procedures and intensive rehabilitation were safe. American Spinal Injury Association Impairment Scale improvement was more common in T7-T11 injuries than in T2-T6 injuries. Surgery combined with rehabilitation appears to have a role in treatment of chronic spinal cord injury patients.

Differential proteomic analysis of acute contusive spinal cord injury in rats using iTRAQ reagent labeling and LC-MS/MS.

In this experimental study, differential labeling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) proteomic approach was used to investigate differences in the proteome of rat spinal cord at 24 h following a moderate contusion injury. Spinal cord protein samples from the injury epicenter (or from sham controls) were trypsinized and differentially labeled with iTRAQ isotopic reagents. The differentially labeled samples were then combined into one sample mixture, separated by LC, and analyzed using MS/MS. Proteins were quantified by comparing the peak areas of iTRAQ reporter fragment ions in MS/MS spectra. The outcome of this analysis revealed that proteins involved in ubiquitination, endocytosis and exocytosis, energy metabolism, inflammatory response, oxidative stress, cytoskeletal disruption, and vascular damage were significantly altered at 24 h following spinal cord injury (SCI). This study demonstrates the utility of the iTRAQ method in proteomic studies and provides further insights into secondary events that occur during acute times following SCI.

Concurrent blockade of free radical and microsomal prostaglandin E synthase-1-mediated PGE2 production improves safety and efficacy in a mouse model of amyotrophic lateral sclerosis.

While free radicals and inflammation constitute major routes of neuronal injury occurring in amyotrophic lateral sclerosis (ALS), neither antioxidants nor non-steroidal anti-inflammatory drugs have shown significant efficacy in human clinical trials. We examined the possibility that concurrent blockade of free radicals and prostaglandin E(2) (PGE(2))-mediated inflammation might constitute a safe and effective therapeutic approach to ALS. We have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50 nM as a potent spin-trapping molecule and inhibited microsomal PGE(2) synthase-1 (mPGES-1) activity in response to both lipopolysaccharide-treated BV2 cell with IC(50) of 230 nM and recombinant human mPGES-1 protein with IC(50) of 249 nM in vitro. In superoxide dismutase 1(G93A) transgenic mouse model of ALS, AAD-2004 blocked free radical production, PGE(2) formation, and microglial activation in the spinal cords. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to riluzole or ibuprofen. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in superoxide dismutase 1(G93A). Targeting both mPGES-1-mediated PGE(2) and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases.

Functional recovery outcomes following acute stroke is associated with abundance of gut microbiota related to inflammation, butyrate and secondary bile acid.

Accumulating evidence suggests that gut microbes modulate brain plasticity via the bidirectional gut-brain axis and play a role in stroke rehabilitation. However, the microbial species alterations associated with stroke and their correlation with functional outcome measures following acute stroke remain unknown. Here we measure post-stroke gut dysbiosis and how it correlates with gut permeability and cognitive functions in 12 stroke participants, 18 controls with risk factors for stroke, and 12 controls without risk factors. Stool samples were used to measure the microbiome with whole genome shotgun sequencing and leaky gut markers. We genotyped APOE status and measured diet composition and motor, cognitive, and emotional status using NIH Toolbox. We used linear regression methods to identify gut microbial associations with cognitive and emotional assessments. We did not find significance differences between the two control groups. In contrast, the bacteria populations of the Stroke group were statistically dissimilar from the control groups. Relative abundance analysis revealed notable decreases in butyrate-producing microbial taxa, secondary bile acid-producing taxa, and equol-producing taxa. The Stroke group had higher levels of the leaky gut marker alpha-1-antitrypsin in the stool than either of the groups and several taxa including Roseburia species (a butyrate producer) were negatively correlated with alpha-1-antitrypsin. Stroke participants scored lower on memory testing than those in the two control groups. Stroke participants with more Roseburia performed better on the picture vocabulary task; more Bacteroides uniformis (a butyrate producer) and less Escherichia coli (a pro-inflammatory species) reported higher levels of self-efficacy. Intakes of fiber, fruit and vegetable were lower, but sweetened beverages were higher, in the Stroke group compared with controls. Vegetable consumption was correlated with many bacterial changes among the participants, but only the species Clostridium bolteae, a pro-inflammatory species, was significantly associated with stroke. Our findings indicate that stroke is associated with a higher abundance of proinflammatory species and a lower abundance of butyrate producers and secondary bile acid producers. These altered microbial communities are associated with poorer functional performances. Future studies targeting the gut microbiome should be developed to elucidate whether its manipulation could optimize rehabilitation and boost recovery.

Blood-derived iron mediates free radical production and neuronal death in the hippocampal CA1 area following transient forebrain ischemia in rat.

Abnormal brain iron homeostasis has been proposed as a pathological event leading to oxidative stress and neuronal injury under pathological conditions. We examined the possibility that neuronal iron overload would mediate free radical production and delayed neuronal death (DND) in hippocampal CA1 area after transient forebrain ischemia (TFI). Mitochondrial free radicals (MFR) were biphasically generated in CA1 neurons 0.5-8 and 48-60 h after TFI. Treatment with Neu2000, a potent spin trapping molecule, as well as trolox, a vitamin E analogue, blocked the biphasic MFR production and attenuated DND in the CA1, regardless of whether it was administered immediately or even 24 h after reperfusion. The late increase in MFR was accompanied by iron accumulation and blocked by the administration of deferoxamine-an iron chelator. Iron accumulation was attributable to prolonged upregulation of the transferrin receptor and to increased uptake of peripheral iron through a leaky blood-brain barrier. Infiltration of iron-containing cells and iron accumulation were attenuated by depletion of circulating blood cells through X-ray irradiation of the whole body except the head. The present findings suggest that excessive iron transported from blood mediates slowly evolving oxidative stress and neuronal death in CA1 after TFI, and that targeting iron-mediated oxidative stress holds extended therapeutic time window against an ischemic event.

Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine.

An expanded polyglutamine domain in huntingtin underlies the pathogenic events in Huntington disease (HD), characterized by chorea, dementia and severe weight loss, culminating in death. Transglutaminase (TGase) may be critical in the pathogenesis, via cross-linking huntingtin. Administration of the TGase competitive inhibitor, cystamine, to transgenic mice expressing exon 1 of huntingtin containing an expanded polyglutamine repeat, altered the course of their HD-like disease. Cystamine given intraperitoneally entered brain where it inhibited TGase activity. When treatment began after the appearance of abnormal movements, cystamine extended survival, reduced associated tremor and abnormal movements and ameliorated weight loss. Treatment did not influence the appearance or frequency of neuronal nuclear inclusions. Unexpectedly, cystamine treatment increased transcription of one of the two genes shown to be neuroprotective for polyglutamine toxicity in Drosophila, dnaj (also known as HDJ1 and Hsp40 in humans and mice, respectively). Inhibition of TGase provides a new treatment strategy for HD and other polyglutamine diseases.

A Highly Predictive MicroRNA Panel for Determining Delayed Cerebral Vasospasm Risk Following Aneurysmal Subarachnoid Hemorrhage.

Approximately one-third of aneurysmal subarachnoid hemorrhage (aSAH) patients develop delayed cerebral vasospasm (DCV) 3-10 days after aneurysm rupture resulting in additional, permanent neurologic disability. Currently, no validated biomarker is available to determine the risk of DCV in aSAH patients. MicroRNAs (miRNAs) have been implicated in virtually all human diseases, including aSAH, and are found in extracellular biofluids including plasma and cerebrospinal fluid (CSF). We used a custom designed TaqMan Low Density Array miRNA panel to examine the levels of 47 selected brain and vasculature injury related miRNAs in CSF and plasma specimens collected from 31 patients with or without DCV at 3 and 7 days after aSAH, as well as from eight healthy controls. The analysis of the first 18-patient cohort revealed a striking differential expression pattern of the selected miRNAs in CSF and plasma of aSAH patients with DCV from those without DCV. Importantly, this differential expression was observed at the early time point (3 days after aSAH), before DCV event occurs. Seven miRNAs were identified as reliable DCV risk predictors along with a prediction model constructed based on an array of additional 19 miRNAs on the panel. These chosen miRNAs were then used to predict the risk of DCV in a separate, testing cohort of 15 patients. The accuracy of DCV risk prediction in the testing cohort reached 87%. The study demonstrates that our novel designed miRNA panel is an effective predictor of DCV risk and has strong applications in clinical management of aSAH patients.

Temporal changes in inflammatory mitochondria-enriched microRNAs following traumatic brain injury and effects of miR-146a nanoparticle delivery.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function. We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation (i.e., miR-142-3p, miR-142-5p, and miR-146a) are altered in the hippocampus at 3-12 hours following a severe traumatic brain injury. In the present study, we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats. In addition, several inflammatory genes were analyzed in the cytosol fractions. The analysis showed that while elevated levels were observed in cytoplasm, the mitochondria-enriched miRNAs, miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury. Although not statistically significant, miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury, and recovered by 7 days. In contrast, miRNAs that are not enriched in mitochondria, including miR-124a, miR-150, miR-19b, miR-155, and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days. The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury. No major alterations were observed in sham operated animals. This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers. These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling. To test this, we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappaB inflammatory modulators in the injured brain. Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), two important modulators of the nuclear factor-kappaB (NF-κB) pro-inflammatory pathway. Moreover, injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels. Taken together, our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain. All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee (IACUC protocol # 2014-1300) on August 17, 2017.

The Mitochondria-Associated ER Membranes Are Novel Subcellular Locations Enriched for Inflammatory-Responsive MicroRNAs.

The mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are specific ER domains that contact the mitochondria and function to facilitate communication between ER and mitochondria. Disruption of contact between the mitochondria and ER is associated with a variety of pathophysiological conditions including neurodegenerative diseases. Considering the many cellular functions of MAMs, we hypothesized that MAMs play an important role in regulating microRNA (miRNA) activity linked to its unique location between mitochondria and ER. Here we present new findings from human and rat brains indicating that the MAMs are subcellular sites enriched for specific miRNAs. We employed subcellular fractionation and TaqMan® RT-qPCR miRNA analysis to quantify miRNA levels in subcellular fractions isolated from male rat brains and six human brain samples. We found that MAMs contain a substantial number of miRNAs and the profile differs significantly from that of cytosolic, mitochondria, or ER. Interestingly, MAMs are particularly enriched in inflammatory-responsive miRNAs, including miR-146a, miR-142-3p, and miR-142-5p in both human and rat brains; miR-223 MAM enrichment was observed only in human brain samples. Further, mitochondrial uncoupling or traumatic brain injury in male rats resulted in the alteration of inflammatory miRNA enrichment in the isolated subcellular fractions. These observations demonstrate that miRNAs are distributed differentially in organelles and may re-distribute between organelles and the cytosol in response to cellular stress and metabolic demands.

Methodology for Subcellular Fractionation and MicroRNA Examination of Mitochondria, Mitochondria Associated ER Membrane (MAM), ER, and Cytosol from Human Brain.

Eukaryotic cell organelles exert unique functions individually but also interact with each other for essential cellular functions. This physical interface between the organelles serves as an important platform for biomolecule trafficking and signaling. Mitochondria are membrane-bound organelles and form a dynamic contact with other organelles. The interactions and communication between mitochondria and endoplasmic reticulum (ER) are facilitated by an ER specific domain, named mitochondria associated ER membrane (MAM). Due to its unique location, the MAM is a "hotspot" for important cell signaling and biochemical processes including calcium homeostasis, lipid synthesis/exchange, inflammasome and autophagosome formation, and mitochondria fission/fusion. Although techniques are available for isolation of organelle fractions including MAM, most utilize animal tissues and cell lines. Here we describe a protocol that is tailored to the isolation of highly purified MAM, mitochondria, ER, and cytosol from human brain. In addition, we include a protocol for the isolation of total RNA and subsequent analysis of microRNAs from these highly purified organelle fractions. Finally, we include a panel of protein markers that are useful for validating the enrichment and purity of each subcellular fraction.

Neuroproteomic methods in spinal cord injury.

Spinal cord injury (SCI) is a major public health problem with no known effective treatment. Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult leading to neuronal dysfunction and death; yet, the molecular mechanisms underlying its dysfunction are poorly understood. Furthermore, while use of imaging methods (e.g., computed tomography scans and magnetic resonance imaging) may help define injury severity and location, they do not elucidate biological mechanisms of SCI progression. The lack of comparable biomarkers for monitoring SCI makes accurate diagnosis and evaluation of SCI progression difficult. Spinal cord contusion is an extensively used SCI model in rats that best represents the etiology of SCI in humans. In this chapter, we describe a two-dimensional (2D) gel electrophoresis-based proteomic approach to investigate the injury-related differences in the proteome and phosphoproteome of spinal cord lesion epicenter at 24 h after spinal cord contusion in rats. The purpose of this study is to elucidate the mechanisms of acute spinal cord dysfunction, as well as discover novel biomarker candidates to evaluate the biological mechanisms of SCI progression and the injury severity.

Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue.

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

Focal cerebral ischemia in the TNFalpha-transgenic rat.

BACKGROUND: To determine if chronic elevation of the inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), will affect infarct volume or cortical perfusion after focal cerebral ischemia. METHODS: Transgenic (TNFalpha-Tg) rats overexpressing the murine TNFalpha gene in brain were prepared by injection of mouse DNA into rat oocytes. Brain levels of TNFalpha mRNA and protein were measured and compared between TNFalpha-Tg and non-transgenic (non-Tg) littermates. Mean infarct volume was calculated 24 hours or 7 days after one hour of reversible middle cerebral artery occlusion (MCAO). Cortical perfusion was monitored by laser-Doppler flowmetry (LDF) during MCAO. Cortical vascular density was quantified by stereology. Post-ischemic cell death was assessed by immunohistochemistry and regional measurement of caspase-3 activity or DNA fragmentation. Unpaired t tests or analysis of variance with post hoc tests were used for comparison of group means. RESULTS: In TNFalpha-Tg rat brain, the aggregate mouse and rat TNFalpha mRNA level was fourfold higher than in non-Tg littermates and the corresponding TNFalpha protein level was increased fivefold (p

Targeting the mitochondrial permeability transition pore in traumatic central nervous system injury.

The mitochondrion serves many functions in the central nervous system (CNS) and other organs beyond the well-recognized role of adenosine triphosphate (ATP) production. This includes calcium-dependent cell signaling, regulation of gene expression, synthesis and release of cytotoxic reactive oxygen species, and the release of cytochrome c and other apoptotic cell death factors. Traumatic injury to the CNS results in a rapid and, in some cases, sustained loss of mitochondrial function. One consequence of compromised mitochondrial function is induction of the mitochondrial permeability transition (mPT) state due to formation of the cyclosporine A sensitive permeability transition pore (mPTP). In this mini-review, we summarize evidence supporting the involvement of the mPTP as a mediator of mitochondrial and cellular demise following CNS traumatic injury and discuss the beneficial effects and limitations of the current ex-perimental strategies targeting the mPTP.

Post-Injury Treatment with NIM811 Promotes Recovery of Function in Adult Female Rats after Spinal Cord Contusion: A Dose-Response Study.

Mitochondrial homeostasis is essential for maintaining cellular function and survival in the central nervous system (CNS). Mitochondrial function is significantly compromised after spinal cord injury (SCI) and is associated with accumulation of high levels of calcium, increased production of free radicals, oxidative damage, and eventually mitochondrial permeability transition (mPT). The formation of the mPT pore (mPTP) and subsequent mPT state are considered to be end stage events in the decline of mitochondrial integrity, and strategies that inhibit mPT can limit mitochondrial demise. Cyclosporine A (CsA) is thought to inhibit mPT by binding to cyclophilin D and has been shown to be effective in models of CNS injury. CsA, however, also inhibits calcineurin, which is responsible for its immunosuppressive properties. In the present study, we conducted a dose-response examination of NIM811, a nonimmunosuppressive CsA analog, on recovery of function and tissue sparing in a rat model of moderate to severe SCI. The results of our experiments revealed that NIM811 (10 mg/kg) significantly improved open field locomotor performance, while the two higher doses tested (20 and 40 mg/kg) significantly improved return of reflexive bladder control and significantly decreased the rostral-caudal extent of the lesion. Taken together, these results demonstrate the ability of NIM811 to improve recovery of function in SCI and support the role of protecting mitochondrial function as a potential therapeutic target.