Leveraging publicly available coronavirus data to identify new therapeutic targets for COVID-19.

Many important questions remain regarding severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the viral pathogen responsible for COVID-19. These questions include the mechanisms explaining the high percentage of asymptomatic but highly infectious individuals, the wide variability in disease susceptibility, and the mechanisms of long-lasting debilitating effects. Bioinformatic analysis of four coronavirus datasets representing previous outbreaks (SARS-CoV-1 and MERS-CoV), as well as SARS-CoV-2, revealed evidence of diverse host factors that appear to be coopted to facilitate virus-induced suppression of interferon-induced innate immunity, promotion of viral replication and subversion and/or evasion of antiviral immune surveillance. These host factors merit further study given their postulated roles in COVID-19-induced loss of smell and brain, heart, vascular, lung, liver, and gut dysfunction.

Principal component analysis of blood microRNA datasets facilitates diagnosis of diverse diseases.

Early, ideally pre-symptomatic, recognition of common diseases (e.g., heart disease, cancer, diabetes, Alzheimer's disease) facilitates early treatment or lifestyle modifications, such as diet and exercise. Sensitive, specific identification of diseases using blood samples would facilitate early recognition. We explored the potential of disease identification in high dimensional blood microRNA (miRNA) datasets using a powerful data reduction method: principal component analysis (PCA). Using Qlucore Omics Explorer (QOE), a dynamic, interactive visualization-guided bioinformatics program with a built-in statistical platform, we analyzed publicly available blood miRNA datasets from the Gene Expression Omnibus (GEO) maintained at the National Center for Biotechnology Information at the National Institutes of Health (NIH). The miRNA expression profiles were generated from real time PCR arrays, microarrays or next generation sequencing of biologic materials (e.g., blood, serum or blood components such as platelets). PCA identified the top three principal components that distinguished cohorts of patients with specific diseases (e.g., heart disease, stroke, hypertension, sepsis, diabetes, specific types of cancer, HIV, hemophilia, subtypes of meningitis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, mild cognitive impairment, aging, and autism), from healthy subjects. Literature searches verified the functional relevance of the discriminating miRNAs. Our goal is to assemble PCA and heatmap analyses of existing and future blood miRNA datasets into a clinical reference database to facilitate the diagnosis of diseases using routine blood draws.

MicroRNA sequencing of rat hippocampus and human biofluids identifies acute, chronic, focal and diffuse traumatic brain injuries.

High-throughput sequencing technologies could improve diagnosis and classification of TBI subgroups. Because recent studies showed that circulating microRNAs (miRNAs) may serve as noninvasive markers of TBI, we performed miRNA-seq to study TBI-induced changes in rat hippocampal miRNAs up to one year post-injury. We used miRNA PCR arrays to interrogate differences in serum miRNAs using two rat models of TBI (controlled cortical impact [CCI] and fluid percussion injury [FPI]). The translational potential of our results was evaluated by miRNA-seq analysis of human control and TBI (acute and chronic) serum samples. Bioinformatic analyses were performed using Ingenuity Pathway Analysis, miRDB, and Qlucore Omics Explorer. Rat miRNA profiles identified TBI across all acute and chronic intervals. Rat CCI and FPI displayed distinct serum miRNA profiles. Human miRNA profiles identified TBI across all acute and chronic time points and, at 24 hours, discriminated between focal and diffuse injuries. In both species, predicted gene targets of differentially expressed miRNAs are involved in neuroplasticity, immune function and neurorestoration. Chronically dysregulated miRNAs (miR-451a, miR-30d-5p, miR-145-5p, miR-204-5p) are linked to psychiatric and neurodegenerative disorders. These data suggest that circulating miRNAs in biofluids can be used as "molecular fingerprints" to identify acute, chronic, focal or diffuse TBI and potentially, presence of neurodegenerative sequelae.

Non-Invasive Transcranial Nano-Pulsed Laser Therapy Ameliorates Cognitive Function and Prevents Aberrant Migration of Neural Progenitor Cells in the Hippocampus of Rats Subjected to Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to chronic diseases, including neurodegenerative disorders and epilepsy. The hippocampus, one of the most affected brain region after TBI, plays a critical role in learning and memory and is one of the only two regions in the brain in which new neurons are generated throughout life from neural stem cells (NSC) in the dentate gyrus (DG). These cells migrate into the granular layer where they integrate into the hippocampus circuitry. While increased proliferation of NSC in the hippocampus is known to occur shortly after injury, reduced neuronal maturation and aberrant migration of progenitor cells in the hilus contribute to cognitive and neurological dysfunctions, including epilepsy. Here, we tested the ability of a novel, proprietary non-invasive nano-pulsed laser therapy (NPLT), that combines near-infrared laser light (808 nm) and laser-generated, low-energy optoacoustic waves, to mitigate TBI-driven impairments in neurogenesis and cognitive function in the rat fluid percussion injury model. We show that injured rats treated with NPLT performed significantly better in a hippocampus-dependent cognitive test than did sham rats. In the DG, NPLT significantly decreased TBI-dependent impaired maturation and aberrant migration of neural progenitors, while preventing TBI-induced upregulation of specific microRNAs (miRNAs) in NSC. NPLT did not significantly reduce TBI-induced microglia activation in the hippocampus. Our data strongly suggest that NPLT has the potential to be an effective therapeutic tool for the treatment of TBI-induced cognitive dysfunction and dysregulation of neurogenesis, and point to modulation of miRNAs as a possible mechanism mediating its neuroprotective effects.

MicroRNA profiling identifies a novel compound with antidepressant properties.

Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17ß-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties.

Estradiol-sertraline synergy in ovariectomized rats.

This study investigated estradiol (E(2)) modulation of the antidepressant effects of a selective serotonin (5-HT) reuptake inhibitor (SSRI; sertraline) and a tricyclic antidepressant (imipramine) as measured by the forced swim test (FST) followed by assessment of gene and protein expression for the 5-HT transporter (SERT) and multiple 5-HT receptors. Female Sprague-Dawley rats were ovariectomized (OVX) and two-thirds of the rats received E(2) implants (OVE). 4 weeks later, implants were withdrawn in half of the OVE rats (OVW) to capture a time point when E(2) levels were rapidly declining. Rats in each hormone group were treated with vehicle, sertraline (10 mg/kg) or imipramine (10 mg/kg), 24, 5 and 1h before the FST. Immediately after the FST, midbrain, hippocampus and prefrontal cortex tissue was removed and frozen for analysis of gene expression via quantitative real-time PCR (midbrain tissue) and protein expression via Western blot (prefrontal cortex and hippocampal tissue). In the FST, sertraline decreased immobility and increased swimming in OVE rats, as well as increased swimming in OVW rats. In contrast, no sertraline effect was observed in OVX rats. Rats treated with imipramine showed increased climbing but no changes in immobility or swimming. No changes in protein expression were detected in any treatment group. However, in vehicle-treated rats, E(2) increased midbrain SERT mRNA expression, with no effect on midbrain mRNA for the 5-HT receptors. In sertraline-treated rats, E(2) decreased 5-HT(2A) receptor mRNA, and E(2)-withdrawal increased 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptor mRNA. In imipramine-treated rats, E(2) (and E(2)-withdrawal) did not affect mRNA expression for any of the target genes. Thus, E(2) synergized behaviorally and neurochemically with an SSRI but not a tricyclic antidepressant.

Hypertonic resuscitation improves neuronal and behavioral outcomes after traumatic brain injury plus hemorrhage.

BACKGROUND: : Resuscitation with hypertonic saline or hypertonic saline plus l-arginine acutely improves cerebral blood flow after traumatic brain injury (TBI) followed by hemorrhagic hypotension. The authors investigated whether hypertonic saline or hypertonic l-arginine would improve long-term neuronal survival and behavioral outcomes 15 days after TBI and hemorrhagic hypotension. METHODS: : Mean arterial pressure, arterial blood gases, pH, plasma glucose, hematocrit, and hemoglobin were measured in male Sprague-Dawley rats before and after moderate (2.0 atm) fluid percussion TBI. Rats were assigned to one of six groups: (1) sham TBI, (2) hemorrhage only, (3) TBI only, (4) TBI plus hemorrhage and resuscitation with 0.9% saline, (5) TBI plus hemorrhage and resuscitation with hypertonic saline (7.5%), or (6) TBI plus hemorrhage and resuscitation with l-arginine (100 mg/kg) in hypertonic saline. On postinjury days 1-5, vestibulomotor function was assessed using beam balance and beam walking tasks. On postinjury days 11-15, spatial memory function was assessed using the Morris water maze. After behavioral testing, neuronal counting was performed bilaterally on specific hippocampal regions. RESULTS: : Groups receiving hypertonic saline (P < 0.05, day 15 vs. day 11) or hypertonic l-arginine (P < 0.05, days 13-15 vs. day 11) showed improved performance over time on the Morris water maze, as well as significantly improved neuronal survival in the contralateral hippocampus (P < 0.05, hypertonic saline or hypertonic l-arginine vs. normal saline) compared with untreated TBI or normal saline-treated TBI plus hemorrhage groups. CONCLUSIONS: : Hypertonic saline and hypertonic l-arginine were both effective at promoting long-term neuronal survival and behavioral recovery. The slightly earlier improvement in Morris water maze performance in the hypertonic l-arginine group warrants further studies to determine whether higher doses of l-arginine provide additional improvement. This study supports the therapeutic benefits of hypertonic resuscitation after TBI plus hemorrhagic hypotension.

Laser Capture Microdissection in Traumatic Brain Injury Research: Obtaining Hippocampal Subregions and Pools of Injured Neurons for Genomic Analyses.

The methods presented here are based on our laboratory's 15 years of experience using laser capture microdissection to obtain samples for the study of gene expression after traumatic brain injury (TBI) using a well-established rat model of experimental TBI. Here, we describe how to use the ArcturusXT laser capture microdissection system to capture swaths of specific regions of the rat hippocampus as well as specific neuronal populations defined by Fluoro-Jade C staining. Staining with Fluoro-Jade C identifies a neuron that is in the process of degeneration. We have optimized our protocols for Fluoro-Jade C tissue staining and laser capture microdissection to maintain RNA integrity which is essential for a variety of downstream applications, such as microarray, PCR array, and quantitative real-time PCR analyses.

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

Nano-Pulsed Laser Therapy Is Neuroprotective in a Rat Model of Blast-Induced Neurotrauma.

We have developed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines the benefits of near-infrared laser light (808 nm) and ultrasound (optoacoustic) waves, which are generated with each short laser pulse within the tissue. We tested NPLT in a rat model of blast-induced neurotrauma (BINT) to determine whether transcranial application of NPLT provides neuroprotective effects. The laser pulses were applied on the intact rat head 1 h after injury using a specially developed fiber-optic system. Vestibulomotor function was assessed on post-injury days (PIDs) 1-3 on the beam balance and beam walking tasks. Cognitive function was assessed on PIDs 6-10 using a working memory Morris water maze (MWM) test. BDNF and caspase-3 messenger RNA (mRNA) expression was measured by quantitative real-time PCR (qRT-PCR) in laser-captured cortical neurons. Microglia activation and neuronal injury were assessed in brain sections by immunofluorescence using specific antibodies against CD68 and active caspase-3, respectively. In the vestibulomotor and cognitive (MWM) tests, NPLT-treated animals performed significantly better than the untreated blast group and similarly to sham animals. NPLT upregulated mRNA encoding BDNF and downregulated the pro-apoptotic protein caspase-3 in cortical neurons. Immunofluorescence demonstrated that NPLT inhibited microglia activation and reduced the number of cortical neurons expressing activated caspase-3. NPLT also increased expression of BDNF in the hippocampus and the number of proliferating progenitor cells in the dentate gyrus. Our data demonstrate a neuroprotective effect of NPLT and prompt further studies aimed to develop NPLT as a therapeutic intervention after traumatic brain injury (TBI).

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

Persistent Behavioral Deficits in Rats after Parasagittal Fluid Percussion Injury.

Although traumatic brain injury (TBI) is now considered a chronic disease, few studies have investigated the long-term behavioral deficits elicited by a well-established rodent model of injury. Here we evaluate behavioral measures, commonly used in TBI research, to determine which tests are useful for studying long-term effects of brain injury in rats. Male Sprague-Dawley rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid-percussion injury (FPI), sham injury, or maintenance as naïve cohorts. Rats underwent neuroscore, beam-balance, and beam-walk tests for 3 days after injury. Subsequently, in separate groups at 3, 6, or 12 months, they were re-tested on the same tasks followed by a working memory version of the Morris water maze. On post-injury days (PIDs) 1-3, significant effects of injury on neuroscore, beam-balance, and beam-walk were observed. Differences in the beam-walk task were not detectable at any of the later time-points. However, deficits persisted in beam-balance out to 3 months and neuroscore out to 6 months. Working memory deficits persisted out to 12 months, at which time a reference memory deficit was also evident. These data suggest that balance and motor coordination recovered more quickly than neurological deficits. Furthermore, while deficits in working memory remained stable over the 12-month period, the late onset of the reference memory deficit points to the progressive nature of the injury, or an age/TBI interaction. In conclusion, standard behavioral tests are useful measures of persistent behavioral deficits after parasagittal FPI and provide evidence that TBI is a chronic condition that can change over time and worsen with age.

Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

Estrous cycle influence on individual differences in the response to novelty and cocaine in female rats.

In rats, individual differences in vulnerability to self-administration of drugs of abuse can be predicted by individual locomotor responses to a novel environment. This phenomenon has been well described for male rats, however very little information is available with regard to female rats and the added complication of estrous cycle hormone changes influencing activity levels. This study was designed to explore the relationship between individual responsiveness to exposure to a novel environment, the estrous cycle, and the response to cocaine in intact, cycling female rats. Locomotor activity of naive female rats was measured upon exposure to a novel environment followed by determination of estrous cycle stage and level of circulating estradiol. Rats were identified as high-responder (HR; 15% most active) or low-responder (LR; 15% least active) rats based on the locomotor response. Hyperactivity in response to cocaine was greater in HR than in LR rats. These data in combination with evaluation of the stage of estrous suggest that the estrous cycle interacts with individual phenotypic characteristics to modify the sensitivity to cocaine in female animals.

Influence of estrous cycle and estradiol on behavioral sensitization to cocaine in female rats.

The hypotheses that the estrous cycle and estradiol modulate behavioral sensitization to cocaine in female rats were assessed. In an analysis of sensitization across the estrous cycle, female rats were administered saline or cocaine (15 mg/kg) twice daily for 5 days. Sensitization developed in the intact female rats as measured by the significant increase in stimulant behaviors seen between day 1 and day 5 of treatment. Rats were challenged with cocaine (5 mg/kg) at 3 days following discontinuation of drug treatment. The expression of sensitization as measured between cocaine and saline-treated rats was evident only in female rats in diestus at the time of the challenge test with cocaine. To explore the role of estradiol in sensitization, female rats were ovariectomized or ovariectomized and implanted with estradiol for two weeks prior to treatment with cocaine (15 mg/kg) twice daily for 5 days. Sensitization developed in both ovariectomized and ovariectomized+estradiol rats treated with cocaine as measured by the significant increase in stimulant-like behaviors seen between day 1 and day 5 of treatment. Rats were challenged with 5 mg/kg of cocaine at 3, 13 and 34 days following discontinuation of drug treatment. While neither hormone treatment group exposed to the cocaine regimen expressed sensitization at 3 days of withdrawal, both groups exhibited sensitization at 13 and 34 days following discontinuation of cocaine treatment. The estradiol-treated groups exhibited higher levels of activity relative to their untreated cohorts in both saline or cocaine treatment groups. These results suggest that detection of sensitization in female rats is not only influenced by injection regimen and length of abstinence but also by the presence of estrogens which effectively enhance the response to an acute cocaine challenge in the presence or absence of prior cocaine exposure.

Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function. Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al., has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types. We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 °C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury.

Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11ß hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects.

Traumatic brain injury-induced dysregulation of the circadian clock.

Circadian rhythm disturbances are frequently reported in patients recovering from traumatic brain injury (TBI). Since circadian clock output is mediated by some of the same molecular signaling cascades that regulate memory formation (cAMP/MAPK/CREB), cognitive problems reported by TBI survivors may be related to injury-induced dysregulation of the circadian clock. In laboratory animals, aberrant circadian rhythms in the hippocampus have been linked to cognitive and memory dysfunction. Here, we addressed the hypothesis that circadian rhythm disruption after TBI is mediated by changes in expression of clock genes in the suprachiasmatic nuclei (SCN) and hippocampus. After fluid-percussion TBI or sham surgery, male Sprague-Dawley rats were euthanized at 4 h intervals, over a 48 h period for tissue collection. Expression of circadian clock genes was measured using quantitative real-time PCR in the SCN and hippocampus obtained by laser capture and manual microdissection respectively. Immunofluorescence and Western blot analysis were used to correlate TBI-induced changes in circadian gene expression with changes in protein expression. In separate groups of rats, locomotor activity was monitored for 48 h. TBI altered circadian gene expression patterns in both the SCN and the hippocampus. Dysregulated expression of key circadian clock genes, such as Bmal1 and Cry1, was detected, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing. In fact, disruption of circadian locomotor activity rhythms in injured animals occurred concurrently. These results provide an explanation for how TBI causes disruption of circadian rhythms as well as a rationale for the consideration of drugs with chronobiotic properties as part of a treatment strategy for TBI.

Cerebrovascular connexin expression: effects of traumatic brain injury.

Traumatic brain injury (TBI) results in dysfunction of the cerebrovasculature. Gap junctions coordinate vasomotor responses and evidence suggests that they are involved in cerebrovascular dysfunction after TBI. Gap junctions are comprised of connexin proteins (Cxs), of which Cx37, Cx40, Cx43, and Cx45 are expressed in vascular tissue. This study tests the hypothesis that TBI alters Cx mRNA and protein expression in cerebral vascular smooth muscle and endothelial cells. Anesthetized (1.5% isoflurane) male Sprague-Dawley rats received sham or fluid-percussion TBI. Two, 6, and 24 h after, cerebral arteries were harvested, fresh-frozen for RNA isolation, or homogenized for Western blot analysis. Cerebral vascular endothelial and smooth muscle cells were selected from frozen sections using laser capture microdissection. RNA was quantified by ribonuclease protection assay. The mRNA for all four Cx genes showed greater expression in the smooth muscle layer compared to the endothelial layer. Smooth muscle Cx43 mRNA expression was reduced 2 h and endothelial Cx45 mRNA expression was reduced 24 h after injury. Western blot analysis revealed that Cx40 protein expression increased, while Cx45 protein expression decreased 24 h after injury. These studies revealed significant changes in the mRNA and protein expression of specific vascular Cxs after TBI. This is the first demonstration of cell type-related differential expression of Cx mRNA in cerebral arteries, and is a first step in evaluating the effects of TBI on gap junction communication in the cerebrovasculature.

L-Arginine decreases fluid-percussion injury-induced neuronal nitrotyrosine immunoreactivity in rats.

Peroxynitrite is a powerful oxidant capable of nitrating phenolic moieties, such as tyrosine or tyrosine residues in proteins and increases after traumatic brain injury (TBI). First, we tested the hypothesis that TBI increases nitrotyrosine (NT) immunoreactivity in the brain by measuring the number of NT-immunoreactive neurons in the cerebral cortex and hippocampus of rats subjected to parasagittal fluid-percussion TBI. Second, we tested the hypothesis that treatment with L-arginine, a substrate for nitric oxide synthase, further increases NT immunoreactivity over TBI alone. Rats were anesthetized with isoflurane and subjected to TBI, sham TBI, or TBI followed by treatment with L-arginine (100 mg/kg). Twelve, 24, or 72 h after TBI, brains were harvested. Coronal sections (10 microm) were incubated overnight with rabbit polyclonal anti-NT antibody, rinsed, and incubated with a biotinylated secondary antibody. The antigen-antibody complex was visualized using a peroxidase-conjugated system with diaminobenzidine as the chromagen. The number of NT-positive cortical and hippocampal neurons increased significantly in both ipsilateral and contralateral hemispheres up to 72 h after TBI compared with the sham-injured group. Remarkably, treatment with L-arginine reduced the number of NT-positive neurons after TBI in both cortex and hippocampus. Our results indicate that L-arginine actually prevents TBI-induced increases in NT immunoreactivity.