Enhancement of motor functional recovery in thoracic spinal cord injury: voluntary wheel running versus forced treadmill exercise.

JOURNAL/nrgr/04.03/01300535-202503000-00028/figure1/v/2024-06-17T092413Z/r/image-tiff Spinal cord injury necessitates effective rehabilitation strategies, with exercise therapies showing promise in promoting recovery. This study investigated the impact of rehabilitation exercise on functional recovery and morphological changes following thoracic contusive spinal cord injury. After a 7-day recovery period after spinal cord injury, mice were assigned to either a trained group (10 weeks of voluntary running wheel or forced treadmill exercise) or an untrained group. Bi-weekly assessments revealed that the exercise-trained group, particularly the voluntary wheel exercise subgroup, displayed significantly improved locomotor recovery, more plasticity of dopaminergic and serotonin modulation compared with the untrained group. Additionally, exercise interventions led to gait pattern restoration and enhanced transcranial magnetic motor-evoked potentials. Despite consistent injury areas across groups, exercise training promoted terminal innervation of descending axons. In summary, voluntary wheel exercise shows promise for enhancing outcomes after thoracic contusive spinal cord injury, emphasizing the role of exercise modality in promoting recovery and morphological changes in spinal cord injuries. Our findings will influence future strategies for rehabilitation exercises, restoring functional movement after spinal cord injury.

Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition.

As an enveloped virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delivers its viral genome into host cells via fusion of the viral and cell membranes. Here, we show that ANO6/TMEM16F-mediated cell surface exposure of phosphatidylserine is critical for SARS-CoV-2 entry and that ANO6-selective inhibitors are effective against SARS-CoV-2 infections. Application of the SARS-CoV-2 Spike pseudotyped virus (SARS2-PsV) evokes a cytosolic Ca2+ elevation and ANO6-dependent phosphatidylserine externalization in ACE2/TMPRSS2-positive mammalian cells. A high-throughput screening of drug-like chemical libraries identifies three different structural classes of chemicals showing ANO6 inhibitory effects. Among them, A6-001 displays the highest potency and ANO6 selectivity and it inhibits the single-round infection of SARS2-PsV in ACE2/TMPRSS2-positive HEK 293T cells. More importantly, A6-001 strongly inhibits authentic SARS-CoV-2-induced phosphatidylserine scrambling and SARS-CoV-2 viral replications in Vero, Calu-3, and primarily cultured human nasal epithelial cells. These results provide mechanistic insights into the viral entry process and offer a potential target for pharmacological intervention to protect against coronavirus disease 2019 (COVID-19).

Deletion of the Alzheimer's disease risk gene Abi3 locus results in obesity and systemic metabolic disruption in mice.

Alzheimer's disease (AD) genetics studies have identified a coding variant within ABI3 gene that increases the risk of developing AD. Recently, we demonstrated that deletion of the Abi3 gene locus dramatically exacerbates AD neuropathology in a transgenic mouse model of amyloidosis. In the course of this AD project, we unexpectedly found that deletion of the Abi3 gene locus resulted in a dramatic obese phenotype in non-transgenic mice. Here, we report our investigation into this serendipitous metabolic finding. Specifically, we demonstrate that mice with deletion of the Abi3 gene locus (Abi3-/- ) have dramatically increased body weight and body fat. Further, we determined that Abi3-/- mice have impaired energy expenditure. Additionally, we found that deletion of the Abi3 gene locus altered gene expression within the hypothalamus, particularly within immune-related pathways. Subsequent immunohistological analysis of the central nervous system (CNS) revealed that microglia number and area were decreased specifically within the mediobasal hypothalamus of Abi3-/- mice. Altogether, this investigation establishes the functional importance of the Abi3 gene locus in the regulation of systemic metabolism and maintenance of healthy body weight. While our previous findings indicated the importance of Abi3 in neurodegeneration, this study indicates that Abi3 related functions are also essential for metabolic regulation.

Critical regulation of follicular helper T cell differentiation and function by Gα13 signaling.

GPCR-Gα protein-mediated signal transduction contributes to spatiotemporal interactions between immune cells to fine-tune and facilitate the process of inflammation and host protection. Beyond this, however, how Gα proteins contribute to the helper T cell subset differentiation and adaptive response have been underappreciated. Here, we found that Gα13 signaling in T cells plays a crucial role in inducing follicular helper T (Tfh) cell differentiation in vivo. T cell-specific Gα13-deficient mice have diminished Tfh cell responses in a cell-intrinsic manner in response to immunization, lymphocytic choriomeningitis virus infection, and allergen challenges. Moreover, Gα13-deficient Tfh cells express reduced levels of Bcl-6 and CXCR5 and are functionally impaired in their ability to adhere to and stimulate B cells. Mechanistically, Gα13-deficient Tfh cells harbor defective Rho-ROCK2 activation, and Rho agonist treatment recuperates Tfh cell differentiation and expression of Bcl-6 and CXCR5 in Tfh cells of T cell-specific Gα13-deficient mice. Conversely, ROCK inhibitor treatment hampers Tfh cell differentiation in wild-type mice. These findings unveil a crucial regulatory role of Gα13-Rho-ROCK axis in optimal Tfh cell differentiation and function, which might be a promising target for pharmacologic intervention in vaccine development as well as antibody-mediated immune disorders.

Cross-Protection against MERS-CoV by Prime-Boost Vaccination Using Viral Spike DNA and Protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

Acoustofluidic assembly of 3D neurospheroids to model Alzheimer's disease.

Neuroinflammation plays a central role in the progression of many neurodegenerative diseases such as Alzheimer's disease, and challenges remain in modeling the complex pathological or physiological processes. Here, we report an acoustofluidic method that can rapidly construct 3D neurospheroids and inflammatory microenvironments for modeling microglia-mediated neuroinflammation in Alzheimer's disease. By incorporating a unique contactless and label-free acoustic assembly, this cell culture platform can assemble dissociated embryonic mouse brain cells into hundreds of uniform 3D neurospheroids with controlled cell numbers, composition (e.g. neurons, astrocytes, and microglia), and environmental components (e.g. amyloid-ß aggregates) in hydrogel within minutes. Moreover, this platform can maintain and monitor the interaction among neurons, astrocytes, microglia, and amyloid-ß aggregates in real-time for several days to weeks, after the integration of a high-throughput, time-lapse cell imaging approach. We demonstrated that our engineered 3D neurospheroids can represent the amyloid-ß neurotoxicity, which is one of the main pathological features of Alzheimer's disease. Using this method, we also investigated the microglia migratory behaviors and activation in the engineered 3D inflammatory microenvironment at a high throughput manner, which is not easy to achieve in 2D neuronal cultures or animal models. Along with the simple fabrication and setup, the acoustofluidic technology is compatible with conventional Petri dishes and well-plates, supports the fine-tuning of the cellular and environmental components of 3D neurospheroids, and enables the high-throughput cellular interaction investigation. We believe our technology may be widely used to facilitate 3D in vitro brain models for modeling neurodegenerative diseases, discovering new drugs, and testing neurotoxicity.

Cross-Protective Shigella Whole-Cell Vaccine With a Truncated O-Polysaccharide Chain.

Shigella is a highly prevalent bacterium causing acute diarrhea and dysentery in developing countries. Shigella infections are treated with antibiotics but Shigellae are increasingly resistant to these drugs. Vaccination can be a countermeasure against emerging antibiotic-resistant shigellosis. Because of the structural variability in Shigellae O-antigen polysaccharides (Oag), cross-protective Shigella vaccines cannot be derived from single serotype-specific Oag. We created an attenuated Shigella flexneri 2a strain with one rather than multiple Oag units by disrupting the Oag polymerase gene (Δwzy), which broadened protective immunogenicity by exposing conserved surface proteins. Inactivated Δwzy mutant cells combined with Escherichia coli double mutant LT(R192G/L211A) as adjuvant, induced potent antibody responses to outer membrane protein PSSP-1, and type III secretion system proteins IpaB and IpaC. Intranasal immunization with the vaccine preparation elicited cross-protective immunity against S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and Shigella sonnei in a mouse pneumonia model. Thus, S. flexneri 2a Δwzy represents a promising candidate strain for a universal Shigella vaccine.

Riociguat prevents hyperoxia-induced lung injury and pulmonary hypertension in neonatal rats without effects on long bone growth.

Bronchopulmonary dysplasia (BPD) remains the most common and serious chronic lung disease of premature infants. Severe BPD complicated with pulmonary hypertension (PH) increases the mortality of these infants. Riociguat is an allosteric soluble guanylate cyclase stimulator and is approved by the FDA for treating PH in adults. However, it has not been approved for use in neonates due to concern for adverse effects on long bone growth. To address this concern we investigated if administration of riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without side effects on long bone growth in newborn rats. Newborn rats were randomized to normoxia (21% O2) or hyperoxia (85% O2) exposure groups within 24 hours of birth, and received riociguat or placebo by once daily intraperitoneal injections during continuous normoxia or hyperoxia exposure for 9 days. In the hyperoxia control group, radial alveolar count, mean linear intercept and vascular density were significantly decreased, the pathological hallmarks of BPD, and these were accompanied by an increased inflammatory response. There was also significantly elevated vascular muscularization of peripheral pulmonary vessels, right ventricular systolic pressure and right ventricular hypertrophy indicating PH. However, administration of riociguat significantly decreased lung inflammation, improved alveolar and vascular development, and decreased PH during hyperoxia by inducing cGMP production. Additionally, riociguat did not affect long bone growth or structure. These data indicate that riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without affecting long bone growth and structure and hence, suggests riociguat may be a potential novel agent for preventing BPD and PH in neonates.

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms.

AIM: Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSµ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. RESULTS: GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSµ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1-/- muscle. Functional analyses of GC1-/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1-/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. INNOVATION: GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. CONCLUSIONS: These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

GSNOR Deficiency Enhances In Situ Skeletal Muscle Strength, Fatigue Resistance, and RyR1 S-Nitrosylation Without Impacting Mitochondrial Content and Activity.

AIM: Nitric oxide (NO) plays important, but incompletely defined roles in skeletal muscle. NO exerts its regulatory effects partly though S-nitrosylation, which is balanced by denitrosylation by enzymes such as S-nitrosoglutathione reductase (GSNOR), whose functions in skeletal muscle remain to be fully deciphered. RESULTS: GSNOR null (GSNOR-/-) tibialis anterior (TA) muscles showed normal growth and were stronger and more fatigue resistant than controls in situ. However, GSNOR-/- lumbrical muscles showed normal contractility and Ca2+ handling in vitro, suggesting important differences in GSNOR function between muscles or between in vitro and in situ environments. GSNOR-/- TA muscles exhibited normal mitochondrial content, and capillary densities, but reduced type IIA fiber content. GSNOR inhibition did not impact mitochondrial respiratory complex I, III, or IV activities. These findings argue that enhanced GSNOR-/- TA contractility is not driven by changes in mitochondrial content or activity, fiber type, or blood vessel density. However, loss of GSNOR led to RyR1 hypernitrosylation, which is believed to increase muscle force output under physiological conditions. cGMP synthesis by soluble guanylate cyclase (sGC) was decreased in resting GSNOR-/- muscle and was more responsive to agonist (DETANO, BAY 41, and BAY 58) stimulation, suggesting that GSNOR modulates cGMP production in skeletal muscle. INNOVATION: GSNOR may act as a "brake" on skeletal muscle contractile performance under physiological conditions by modulating nitrosylation/denitrosylation balance. CONCLUSIONS: GSNOR may play important roles in skeletal muscle contractility, RyR1 S-nitrosylation, fiber type specification, and sGC activity. Antioxid. Redox Signal. 26, 165-181.

Bcl-2 knockdown accelerates T cell receptor-triggered activation-induced cell death in jurkat T cells.

Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-κB) translocation.

Protective functions of peroxiredoxin-1 against cytokine-induced MIN6 pancreatic ß-cell line death.

Pancreatic ß-cells play a crucial role in glucose homeostasis, and the failure of these cells to function results in the development of type 1 diabetes (T1D). The MIN6 cell line, which closely resembles pancreatic ß-cells, was used to unravel the relationship between pancreatic ß-cell function and the antioxidant enzyme PRX-1. PRX-1 was knocked down in MIN6 cells using a shPRX-1 lentiviral construct, and a mixture of inflammatory cytokines was administered to challenge the MIN6 cells. Nitric oxide (NO) production and inducible NO synthase (iNOS) expression were elevated in shPRX-1 compared with the control. Also, shPRX-1 transduced cells showed higher levels of NF-κB nuclear translocation, suggesting that PRX-1 has a regulatory role in NF-κB nuclear translocation and iNOS expression. In correlation with NO levels, decreased anti-apoptotic gene Bcl-xl level and elevated pro-apoptotic gene Bim levels were observed in shPRX-1 cells compared with scramble, and cell viability decreased accordingly. A rescue experiment was performed subsequently using an iNOS inhibitor to confirm NO as the cause of cell death. Overall, the results of this study suggest possible protective roles of the antioxidant enzyme PRX-1 in the insulinoma cell line MIN6 and possibly in pancreatic ß-cells under T1D conditions.

Function of ezrin-radixin-moesin proteins in migration of subventricular zone-derived neuroblasts following traumatic brain injury.

Throughout life, newly generated neuroblasts from the subventricular zone migrate toward the olfactory bulb through the rostral migratory stream. Upon brain injury, these migrating neuroblasts change their route and begin to migrate toward injured regions, which is one of the regenerative responses after brain damage. This injury-induced migration is triggered by stromal cell-derived factor 1 (SDF1) released from microglia near the damaged site; however, it is still unclear how these cells transduce SDF1 signals and change their direction. In this study, we found that SDF1 promotes the phosphorylation of ezrin-radixin-moesin (ERM) proteins, which are key molecules in organizing cell membrane and linking signals from the extracellular environment to the intracellular actin cytoskeleton. Blockade of ERM activation by overexpressing dominant-negative ERM (DN-ERM) efficiently perturbed the migration of neuroblasts. Considering that DN-ERM-expressing neuroblasts failed to maintain proper migratory cell morphology, it appears that ERM-dependent regulation of cell shape is required for the efficient migration of neuroblasts. These results suggest that ERM activation is an important step in the directional migration of neuroblasts in response to SDF1-CXCR4 signaling following brain injury.

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice.

Ezrin is a member of the ezrin-radixin-moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell-matrix recognition, cell-cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.

Characterization of embryonic cortical neuron death in prolonged cell suspension.

Cell transplantation may be an effective therapeutic strategy for many neurodegenerative diseases. However, difficulty in obtaining a sufficient amount of donor cells and low graft survival are two major limiting factors. Dissociation of cells from tissues or culture is an inevitable step for cell transplantation, and cell viability in suspension may influence the outcome of the cell therapy. To this end, we asked whether the suspension time of freshly dissociated neurons in vitro affects their viability. Following 4-24h cell suspension, primary cortical neurons underwent cell death. Interestingly, the neurons exhibited only marginal caspase-3 immunoreactivity with very few sub-G1 apoptotic cell proportions in flow cytometry. In addition, the suppression of caspase-3 or Bax action failed to prevent cell death of primary cortical neurons, indicating minimal apoptotic cell death. On the other hand, there was a marked increase in the TdT-mediated dUTP nick end labeling-positive and propidium iodide-labeled necrotic cells (∼50%) with enhanced poly [ADP-ribose] polymerase-1 activity. Therefore, prevention against necrosis rather than apoptosis may be required for optimal benefits in cell transplantation.

Regional difference of reactive astrogliosis following traumatic brain injury revealed by hGFAP-GFP transgenic mice.

Reactive astrocytes greatly influence the wound healing and neuronal regeneration processes following brain injury. However, the origin and fate of reactive astrocytes appear to be different depending on the type, severity and duration of brain injury. Using the cryogenic traumatic brain injury model, here we comprehensively addressed the regional differences of reactive astrocytes in the injured cortex. In the proximal region of injury site, NG2-expressing and cytoplasmic Olig2-labeled cells were densely localized 3 days after the injury. Next to this proximal layer, most of reactive astrocytes did not express NG2 but exhibited radial glia-like shape with elongated processes. Accordingly, they expressed the progenitor or radial glial markers, such as vimentin, brain lipid binding protein (BLBP) and the green fluorescent protein (GFP) under the control of the human GFAP (hGFAP) promoter. However, only few glial fibrillary acidic protein (GFAP) expressing astrocytes were found in this layer. Distal to the injury site, most of astrocytes strongly expressed GFAP with hypertonic morphology. At day 15 after injury, all layers expressing GFAP and other marker expressions disappeared, indicating the termination of reactive astrogliosis. Taken together, our data suggest that reactive astrogliosis occurs in a regionally segregated manner in the early phase of brain injury.

Dissociation of progressive dopaminergic neuronal death and behavioral impairments by Bax deletion in a mouse model of Parkinson's diseases.

Parkinson's disease (PD) is a common, late-onset movement disorder with selective degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Although the neurotoxin 6-hydroxydopamine (6-OHDA) has been used to induce progressive degeneration of DA neurons in various animal models of PD, the precise molecular pathway and the impact of anti-apoptotic treatment on this neurodegeneration are less understood. Following a striatal injection of 6-OHDA, we observed atrophy and progressive death of DA neurons in wild-type mice. These degenerating DA neurons never exhibited signs of apoptosis (i.e., caspase-3 activation and cytoplasmic release of cytochrome C), but rather show nuclear translocation of apoptosis-inducing factor (AIF), a hallmark of regulated necrosis. However, mice with genetic deletion of the proapoptotic gene Bax (Bax-KO) exhibited a complete absence of 6-OHDA-induced DA neuron death and nuclear translocation of AIF, indicating that 6-OHDA-induced DA neuronal death is mediated by Bax-dependent AIF activation. On the other hand, DA neurons that survived in Bax-KO mice exhibited marked neuronal atrophy, without significant improvement of PD-related behavioral deficits. These findings suggest that anti-apoptotic therapy may not be sufficient for PD treatment, and the prevention of Bax-independent neuronal atrophy may be an important therapeutic target.

Induction of ezrin-radixin-moesin molecules after cryogenic traumatic brain injury of the mouse cortex.

Traumatic brain injury promotes rapid induction of microglial cells and infiltration of peripheral macrophages to the injury sites. Such inflammatory responses are mediated by the activation and migration of immune cells, which are influenced by the actin cytoskeleton remodeling. In this study, we observed that the phosphorylation and expressions of ezrin-radixin-moesin (ERM) proteins, which are linkers for cell surface with actin cytoskeleton, are induced in the activated microglia/macrophages, whereas ERM molecules are only marginally expressed in quiescent microglia in the normal brain. These results suggest that ERM activation in the injury penumbra is implicated in the inflammatory immune responses after traumatic brain injury.

Induction of Per1 expression following an experimentally induced epilepsy in the mouse hippocampus.

The Period1 (Per1) is a clock-oscillating gene product that plays an essential role in the generation and modulation of circadian rhythm in the suprachiasmatic nucleus (SCN) of hypothalamus. However, Per1 is also expressed in many other brain regions including cerebral cortex, hippocampus, and amygdala, suggesting that Per1 may be involved in the broader cellular functions in addition to the rhythm regulation. In this study, we found that chemical or electrical seizure-inducing stimulations regulate Per1 expression. Treatments with electric convulsive shock (ECS) or kainic acid (KA) robustly up-regulated the expressions of per1 mRNA and protein in the hippocampal formation and cerebral cortex. In consistent, we found that neuronal depolarization or KA treatment increased per1 mRNA expression in cultured primary cortical neurons. Because it has been demonstrated that Per family molecules contribute to the regulation of stress-induced cell death, we also explored the effect of Per1 overexpression on the survival of cultured neurons. However, neither basal, staurosporine- nor KA-induced neuronal death was affected by forced expression of Per1. Collectively, these results suggest that the Per1 expression is neuronal activity- and epileptogen-dependent, although its functional significance is remained to be explored.

Expression of connexin29 and 32 in the penumbra region after traumatic brain injury of mice.

Connexins (Cx) are transmembrane proteins forming vertebrate gap junction channels for direct cell-cell communication. We found that the expressions of two Cx family members, Cx29 and Cx32, were progressively increased in the sharp border of injury penumbra regions after cryotraumatic brain injury. Although these two Cxs are expressed exclusively in the oligodendrocytes in the normal cerebral cortex, their expressions were increased in the astrocytes and microglia localized in the injury border. Highly selective induction of Cxs in the injury border suggests that altered Cxs may contribute to the propagations of injury-related and/or regeneration signals after acute brain injury.