Exposure to an enriched environment modulates the synaptic vesicle cycle in a mouse spinal cord injury model.

Spinal cord injury (SCI) leads to motor and sensory impairment below the site of injury, thereby necessitating rehabilitation. An enriched environment (EE) increases social interaction and locomotor activity in a mouse model, similar to human rehabilitation. However, the impact of EE on presynaptic plasticity in gene expression levels remains unclear. Hence, this study aimed to investigate the therapeutic potential of EE in an SCI mouse model. Mice with spinal cord contusion were divided into two groups: those housed in standard cages (control) and those in EE conditions (EE). Each group was housed separately for either 2- or 8-weeks post-injury, after which RNA sequencing was performed and compared to a sham group (receiving only a dorsal laminectomy). The synaptic vesicle cycle (SVC) pathway and related genes showed significant downregulation after SCI at both time points. Subsequently, we investigated whether exposure to EE for 2- and 8-weeks post-SCI could modulate the SVC pathway and its related genes. Notably, exposure to EE for 8 weeks resulted in a marked reversal effect of SVC-related gene expression, along with stimulation of axon regeneration and mitigation of locomotor activity loss. Thus, prolonged exposure to EE increased presynaptic activity, fostering axon regeneration and functional improvement by modulating the SVC in the SCI mouse model. These findings suggest that EE exposure proves effective in inducing activity-dependent plasticity, offering a promising therapeutic approach akin to rehabilitation training in patients with SCI.

3'-Sialyllactose alleviates bone loss by regulating bone homeostasis.

Osteoporosis is a common skeletal disease that results in an increased risk of fractures. However, there is no definitive cure, warranting the development of potential therapeutic agents. 3'-Sialyllactose (3'-SL) in human milk regulates many biological functions. However, its effect on bone metabolism remains unknown. This study aimed to investigate the molecular mechanisms underlying the effect of 3'-SL on bone homeostasis. Treatment of human bone marrow stromal cells (hBMSCs) with 3'-SL enhanced osteogenic differentiation and inhibited adipogenic differentiation of hBMSCs. RNA sequencing showed that 3'-SL enhanced laminin subunit gamma-2 expression and promoted osteogenic differentiation via the phosphatidylinositol 3­kinase/protein kinase B signaling pathway. Furthermore, 3'-SL inhibited the receptor activator of nuclear factor κB ligand-induced osteoclast differentiation of bone marrow-derived macrophages through the nuclear factor κB and mitogen­activated protein kinase signaling pathway, ameliorated osteoporosis in ovariectomized mice, and positively regulated bone remodeling. Our findings suggest 3'-SL as a potential drug for osteoporosis.

Therapeutic effects of polydeoxyribonucleotide in an in vitro neuronal model of ischemia/reperfusion injury.

Polydeoxyribonucleotide (PDRN) is an agonist that selectively stimulates adenosine A2A receptor (ADORA2A), which suppresses inflammatory responses. Ischemia/reperfusion (I/R) injury plays a major role in the pathogenesis of ischemic stroke by inducing neuroinflammation. Therefore, this study aimed to investigate the therapeutic effects of PDRN in an in vitro I/R injury model. The in vitro model was established with differentiated Neuro-2a cells under oxygen and glucose deprivation condition. The cells were treated with PDRN for 24 h under reoxygenation condition. As the results of RNA-seq transcriptome analysis, CSF1, IL-6, PTPN6, RAC2, and STAT1 were identified of its relation to the effect of PDRN on inflammatory responses in the model. To further investigate therapeutic effects of PDRN, RT-qPCR, western blotting, LDH assay, and TUNEL assay were performed. PDRN significantly reversed the expression of genes and proteins related to inflammatory responses. The elevated ADORA2A expression by PDRN treatment downregulated JAK/STAT pathway in the model. Furthermore, PDRN inhibited neuronal cell death in the model. Consequently, our results suggested that PDRN alleviated inflammatory responses through inhibition of JAK/STAT pathway by mediating ADORA2A expression and inhibited neuronal cell death in the model. These results provide significant insights into potential therapeutic approaches involving PDRN treatment for I/R injury.

Low-frequency repetitive magnetic stimulation suppresses neuroblastoma progression by downregulating the Wnt/ß-catenin signaling pathway.

Repetitive magnetic stimulation (rMS) has been suggested as a non-invasive treatment for various neurological or psychiatric diseases. Contrary to the application previously used, the purpose of the present study was to elucidate whether low-frequency rMS could suppress tumor progression in in vitro and in vivo neuroblastoma models, and to explore the underlying mechanisms. The results demonstrated that low-frequency rMS treatment significantly suppressed cell proliferation and tumor progression in the models. Moreover, low-frequency rMS treatment downregulated the Wnt/ß-catenin signaling pathway and induced apoptosis. The Wnt/ß-catenin signaling pathway activator, Wnt agonist, was found to counteract the effect of low-frequency rMS treatment, while the Wnt/ß-catenin signaling pathway inhibitor, Wnt antagonist, exhibited a tumor suppression effect, similar to the effect of low-frequency rMS treatment. Taken together, our data demonstrated that low-frequency rMS treatment suppressed neuroblastoma progression by downregulating the Wnt/ß-catenin signaling pathway, suggesting that low-frequency rMS treatment may be a potential therapeutic strategy for the tumor suppression.

Randomized, triple-blind, placebo-controlled study to evaluate the safety of 6'-Sialyllactose in healthy adults.

Sialyllactoses (SL) are an abundant component of human milk. There have been many studies on the biological effects of SL in humans. SL can be produced using an economical method of enzyme synthesis. Although the European Food Safety Authority has published the human safety and appropriate intake dose of 6'-SL sodium salt as a novel food, it has suggested that the appropriate dose for particular medical purposes be judged on a case-by-case basis. Also, as revealed in the same report, there are no data on toxicity when 6'-SL is used in human intervention. However, clinical studies have only confirmed the safety of 3'-SL for therapeutic intervention in humans, and the safety for therapeutic use of 6'-SL, which is more abundant than 3'-SL in human milk, has not been confirmed. In this study, to determine the safety of 6'-SL use in humans, participants were randomly assigned to the placebo (maltodextrin) and 6'-SL groups, and then 3 g of powder was orally administered twice a day for 12 weeks. There were no serious adverse reactions, such as life-threatening complications requiring hospitalization, causing disability, or causing deformity during the use of 6'-SL. There were no clinically significant differences among the baseline, sixth, and twelfth week clinical chemistry tests, such as aspartate aminotransferase, alanine aminotransferase, and creatinine. Most of the adverse reactions were gastrointestinal problems such as diarrhea, abdominal discomfort, and bloating, with no significant difference in the proportions between the placebo and 6'-SL groups. These results support the safety of the 6'-SL for human use.

Very early environmental enrichment protects against apoptosis and improves functional recovery from hypoxic-ischemic brain injury.

Appropriate rehabilitation of stroke patients at a very early phase results in favorable outcomes. However, the optimal strategy for very early rehabilitation is at present unclear due to the limited knowledge on the effects of very early initiation of rehabilitation based on voluntary exercise (VE). Environmental enrichment (EE) is a therapeutic paradigm for laboratory animals that involves complex combinations of physical, cognitive, and social stimuli, as well as VE. Few studies delineated the effect of EE on apoptosis in very early stroke in an experimental model. Although a minimal benefit of early rehabilitation in stroke models has been claimed in previous studies, these were based on a forced exercise paradigm. The aim of this study is to determine whether very early exposure to EE can effectively regulate Fas/FasL-mediated apoptosis following hypoxic-ischemic (HI) brain injury and improve neurobehavioral function. C57Bl/6 mice were housed for 2 weeks in either cages with EE or standard cages (SC) 3 h or 72 h after HI brain injury. Very early exposure to EE was associated with greater improvement in motor function and cognitive ability, reduced volume of the infarcted area, decreased mitochondria-mediated apoptosis, and decreased oxidative stress. Very early exposure to EE significantly downregulated Fas/FasL-mediated apoptosis, decreased expression of Fas, Fas-associated death domain, cleaved caspase-8/caspase-8, cleaved caspase-3/caspase-3, as well as Bax and Bcl-2, in the cerebral cortex and the hippocampus. Delayed exposure to EE, on the other hand, failed to inhibit the extrinsic pathway of apoptosis. This study demonstrates that very early exposure to EE is a potentially useful therapeutic translation for stroke rehabilitation through effective inhibition of the extrinsic and intrinsic apoptotic pathways.

In vivo gene editing via homology-independent targeted integration for adrenoleukodystrophy treatment.

Adrenoleukodystrophy (ALD) is caused by various pathogenic mutations in the X-linked ABCD1 gene, which lead to metabolically abnormal accumulations of very long-chain fatty acids in many organs. However, curative treatment of ALD has not yet been achieved. To treat ALD, we applied two different gene-editing strategies, base editing and homology-independent targeted integration (HITI), in ALD patient-derived fibroblasts. Next, we performed in vivo HITI-mediated gene editing using AAV9 vectors delivered via intravenous administration in the ALD model mice. We found that the ABCD1 mRNA level was significantly increased in HITI-treated mice, and the plasma levels of C24:0-LysoPC (lysophosphatidylcholine) and C26:0-LysoPC, sensitive diagnostic markers for ALD, were significantly reduced. These results suggest that HITI-mediated mutant gene rescue could be a promising therapeutic strategy for human ALD treatment.

Macrocyclic Immunoproteasome Inhibitors as a Potential Therapy for Alzheimer's Disease.

Previously, we reported that immunoproteasome (iP)-targeting linear peptide epoxyketones improve cognitive function in mouse models of Alzheimer's disease (AD) in a manner independent of amyloid ß. However, these compounds' clinical prospect for AD is limited due to potential issues, such as poor brain penetration and metabolic instability. Here, we report the development of iP-selective macrocyclic peptide epoxyketones prepared by a ring-closing metathesis reaction between two terminal alkenes attached at the P2 and P3/P4 positions of linear counterparts. We show that a lead macrocyclic compound DB-60 (20) effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC50: 105 nM) and has metabolic stability superior to its linear counterpart. DB-60 (20) also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts and offer promising potential as an AD drug candidate.

Environmental Enrichment Enhances Cav 2.1 Channel-Mediated Presynaptic Plasticity in Hypoxic-Ischemic Encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a devastating neonatal brain condition caused by lack of oxygen and limited blood flow. Environmental enrichment (EE) is a classic paradigm with a complex stimulation of physical, cognitive, and social components. EE can exert neuroplasticity and neuroprotective effects in immature brains. However, the exact mechanism of EE on the chronic condition of HIE remains unclear. HIE was induced by a permanent ligation of the right carotid artery, followed by an 8% O2 hypoxic condition for 1 h. At 6 weeks of age, HIE mice were randomly assigned to either standard cages or EE cages. In the behavioral assessments, EE mice showed significantly improved motor performances in rotarod tests, ladder walking tests, and hanging wire tests, compared with HIE control mice. EE mice also significantly enhanced cognitive performances in Y-maze tests. Particularly, EE mice showed a significant increase in Cav 2.1 (P/Q type) and presynaptic proteins by molecular assessments, and a significant increase of Cav 2.1 in histological assessments of the cerebral cortex and hippocampus. These results indicate that EE can upregulate the expression of the Cav 2.1 channel and presynaptic proteins related to the synaptic vesicle cycle and neurotransmitter release, which may be responsible for motor and cognitive improvements in HIE.

3'-Sialyllactose Protects SW1353 Chondrocytic Cells From Interleukin-1ß-Induced Oxidative Stress and Inflammation.

Osteoarthritis (OA) is a major degenerative joint disease. Oxidative stress and inflammation play key roles in the pathogenesis of OA. 3'-Sialyllactose (3'-SL) is derived from human milk and is known to regulate a variety of biological functions related to immune homeostasis. This study aimed to investigate the therapeutic mechanisms of 3'-SL in interleukin-1ß (IL-1ß)-treated SW1353 chondrocytic cells. 3'-SL potently suppressed IL-1ß-induced oxidative stress by increasing the levels of enzymatic antioxidants. 3'-SL significantly reversed the IL-1ß mediated expression levels of reactive oxygen species in IL-1ß-stimulated chondrocytic cells. In addition, 3'-SL could reverse the increased levels of inflammatory markers such as nitrite, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, IL-1ß, and IL-6 in IL-1ß-stimulated chondrocytic cells. Moreover, 3'-SL significantly inhibited the apoptotic process, as indicated by the downregulation of the pro-apoptotic protein Bax, upregulation of the anti-apoptotic protein Bcl-2 expression, and significant reduction in the number of TUNEL-positive cells in the IL-1ß-treated chondrocytic cells. Furthermore, 3'-SL reversed cartilage destruction by decreasing the release of matrix metalloproteinases (MMP), such as MMP1, MMP3, and MMP13. In contrast, 3'-SL significantly increased the expression levels of matrix synthesis proteins, such as collagen II and aggrecan, in IL-1ß-treated chondrocytic cells. 3'-SL dramatically suppressed the activation of mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways, which are related to the pathogenesis of OA. Taken together, our data suggest that 3'-SL alleviates IL-1ß-induced OA pathogenesis via inhibition of activated MAPK and PI3K/AKT/NF-κB signaling cascades with the downregulation of oxidative stress and inflammation. Therefore, 3'-SL has the potential to be used as a natural compound for OA therapy owing to its ability to activate the antioxidant defense system and suppress inflammatory responses.

Parasympathetic Effect Induces Cell Cycle Activation in Upper Limbs of Paraplegic Patients with Spinal Cord Injury.

The present study aimed to investigate gene expression changes related to cell cycle activation in patients with spinal cord injury (SCI) and to further evaluate the difference between the upper and lower limbs of SCI patients. Fibroblasts were obtained from the upper and lower limbs of SCI patients and healthy subjects. To investigate gene expression profiling in the fibroblasts from SCI patients compared to the healthy subjects, RNA-Seq transcriptome analysis was performed. To validate the parasympathetic effects on cell cycle activation, fibroblasts from upper or lower limbs of SCI patients were treated with the anticholinergic agents tiotropium or acetylcholine, and quantitative RT-PCR and Western blot were conducted. Cell proliferation was significantly increased in the upper limbs of SCI patients compared with the lower limbs of SCI patients and healthy subjects. The pathway and genes involved in cell cycle were identified by RNA-Seq transcriptome analysis. Expression of cell-cycle-related genes CCNB1, CCNB2, PLK1, BUB1, and CDC20 were significantly higher in the upper limbs of SCI patients compared with the lower limbs of SCI patients and healthy subjects. When the fibroblasts were treated with tiotropium the upper limbs and acetylcholine in the lower limbs, the expression of cell-cycle-related genes and cell proliferation were significantly modulated. This study provided the insight that cell proliferation and cell cycle activation were observed to be significantly increased in the upper limbs of SCI patients via the parasympathetic effect.

Effect of Polydeoxyribonucleotide on Angiogenesis and Wound Healing in an In Vitro Model of Osteoarthritis.

Osteoarthritis (OA) is degenerative disease, leading to pain and functional disability. It is reported that polydeoxyribonucleotide (PDRN) is a suitable therapy for OA. However, the therapeutic mechanisms of PDRN in OA are not fully understood. To investigate the effect of PDRN in an in vitro model of OA, interleukin (IL)-1ß or phosphate-buffered saline (PBS) was used to treat a human chondrocytic cell line in hypoxic conditions for 24 h (IL-1ß group or control group). PDRN was then used to treat IL-1ß group cells for 24 h (PDRN group). By Label-Based Human Antibody Array 1000, angiopoietin-2 (ANG-2), platelet-derived growth factor (PDGF), angiostatin, and endostatin, which were related to angiogenesis, were chosen for further validation studies. Quantitative real-time reverse transcription polymerase chain reaction and western blot analysis validated that the levels of PDGF and ANG-2, which were related to pro-angiogenesis, were significantly increased in the PDRN group compared with those in the control group or the IL-1ß group. However, the levels of endostatin and angiostatin, which were related in anti-angiogenesis, were significantly decreased in the PDRN group compared with those in the control group or the IL-1ß group. In the same manner, vascular endothelial growth factor, which was a mediator of angiogenesis, was significantly increased in the PDRN group compared with those in the control group or the IL-1ß group. Furthermore, wound closure was significantly increased in the PDRN group compared with the control group or the IL-1ß group by in vitro scratch assay. Moreover, PDRN decreased expression of metalloproteinase 13, as a catabolic factor for OA, but increased expression of aggrecan, which was an anabolic factor for OA. These data suggest that PDRN may promote angiogenesis and wound healing via down-regulation of catabolism and up-regulation of anabolism in an in vitro model of OA.

Environmental Enrichment Upregulates Striatal Synaptic Vesicle-Associated Proteins and Improves Motor Function.

Environmental enrichment (EE) is a therapeutic paradigm that consists of complex combinations of physical, cognitive, and social stimuli. The mechanisms underlying EE-mediated synaptic plasticity have yet to be fully elucidated. In this study, we investigated the effects of EE on synaptic vesicle-associated proteins and whether the expression of these proteins is related to behavioral outcomes. A total of 44 CD-1® (ICR) mice aged 6 weeks were randomly assigned to either standard cages or EE (N = 22 each). Rotarod and ladder walking tests were then performed to evaluate motor function. To identify the molecular mechanisms underlying the effects of EE, we assessed differentially expressed proteins (DEPs) in the striatum by proteomic analysis. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry were conducted to validate the expressions of these proteins. In the behavioral assessment, EE significantly enhanced performance on the rotarod and ladder walking tests. A total of 116 DEPs (54 upregulated and 62 downregulated proteins) were identified in mice exposed to EE. Gene ontology (GO) analysis demonstrated that the upregulated proteins in EE mice were primarily related to biological processes of synaptic vesicle transport and exocytosis. The GO terms for these biological processes commonly included Synaptic vesicle glycoprotein 2B (SV2B), Rabphilin-3A, and Piccolo. The qRT-PCR and western blot analyses revealed that EE increased the expression of SV2B, Rabphilin-3A and Piccolo in the striatum compared to the control group. Immunohistochemistry showed that the density of Piccolo in the vicinity of the subventricular zone was significantly increased in the EE mice compared with control mice. In conclusion, EE upregulates proteins associated with synaptic vesicle transport and exocytosis such as SV2B, Rabphilin-3A and Piccolo in the striatum. These upregulated proteins may be responsible for locomotor performance improvement, as shown in rotarod and ladder walking tests. Elucidation of these changes in synaptic protein expression provides new insights into the mechanism and potential role of EE.

High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+-Calmodulin-Dependent Protein Kinase II-cAMP-Response Element-Binding Protein Pathway.

Repetitive transcranial magnetic stimulation (rTMS) can be used in various neurological disorders. However, neurobiological mechanism of rTMS is not well known. Therefore, in this study, we examined the global gene expression patterns depending on different frequencies of repetitive magnetic stimulation (rMS) in both undifferentiated and differentiated Neuro-2a cells to generate a comprehensive view of the biological mechanisms. The Neuro-2a cells were randomly divided into three groups-the sham (no active stimulation) group, the low-frequency (0.5 Hz stimulation) group, and high-frequency (10 Hz stimulation) group-and were stimulated 10 min for 3 days. The low- and high-frequency groups of rMS on Neuro-2a cells were characterized by transcriptome array. Differentially expressed genes were analyzed using the Database of Annotation Visualization and Integrated Discovery program, which yielded a Kyoto Encyclopedia of Genes and Genomes pathway. Amphetamine addiction pathway, circadian entrainment pathway, long-term potentiation (LTP) pathway, neurotrophin signaling pathway, prolactin signaling pathway, and cholinergic synapse pathway were significantly enriched in high-frequency group compared with low-frequency group. Among these pathways, LTP pathway is relevant to rMS, thus the genes that were involved in LTP pathway were validated by quantitative real-time polymerase chain reaction and western blotting. The expression of glutamate ionotropic receptor N-methyl d-aspartate 1, calmodulin-dependent protein kinase II (CaMKII) δ, and CaMKIIα was increased, and the expression of CaMKIIγ was decreased in high-frequency group. These genes can activate the calcium (Ca2+)-CaMKII-cAMP-response element-binding protein (CREB) pathway. Furthermore, high-frequency rMS induced phosphorylation of CREB, brain-derived neurotrophic factor (BDNF) transcription via activation of Ca2+-CaMKII-CREB pathway. In conclusion, high-frequency rMS enhances the expression of BDNF by activating Ca2+-CaMKII-CREB pathway in the Neuro-2a cells. These findings may help clarify further therapeutic mechanisms of rTMS.

The Differential Effects of Repetitive Magnetic Stimulation in an In Vitro Neuronal Model of Ischemia/Reperfusion Injury.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive therapy that has been implicated in treatment of serious neurological disorders. However, the neurobiological mechanisms underlying the effects of rTMS remain unclear. Therefore, this study examined the differential effects of repetitive magnetic stimulation (rMS) in an in vitro neuronal model of ischemia/reperfusion (I/R) injury, depending on low and high frequency. Neuro-2a cells were differentiated with retinoic acid and established for in vitro neuronal model of I/R injury under a subsequent 3 h of oxygen and glucose deprivation/reoxygenation (OGD/R) condition. After the I/R injury, the differentiated neuronal cells were stimulated with rMS on day 1 and randomly divided into three groups: OGD/R+sham, OGD/R+low-frequency, and OGD/R+high-frequency groups. High-frequency rMS increases cell proliferation through activation of extracellular signal-regulated kinases and AKT-signaling pathway and inhibits apoptosis in OGD/R-injured cells. Furthermore, high-frequency rMS increases Ca2+-calmodulin-dependent protein kinase II (CaMKII)-cAMP-response element binding protein (CREB) signaling pathway, further leading to alternation of brain-derived neurotrophic factor expression and synaptic plasticity in OGD/R injured cells. These results verified the neurobiological mechanisms of frequency-dependent rMS in I/R injury-treated neuronal cells. These mechanisms will help develop more powerful and credible rTMS stimulation treatment protocols.

Anti-inflammatory Effect of DNA Polymeric Molecules in a Cell Model of Osteoarthritis.

The DNA polymeric molecules polydeoxynucleotide (PDRN) and polynucleotide (PN) can be used as new alternative treatment for osteoarthritis (OA); however, the underlying mechanisms are not fully understood. In this study, we investigated the effect of PDRN and PN on gene-expression profiles in a cell model of OA using transcriptome analysis. Under hypoxic conditions, human chondrosarcoma cells were stressed for 24 h in the presence of interleukin (IL)-1ß and subsequently treated with PDRN, PN, or hyaluronic acid (HA) for another 24 h, followed by transcriptome analysis. The results of the transcriptome study comprising differentially expressed genes were analyzed using the Database of Annotation Visualization and Integrated Discovery program, which yielded Kyoto Encyclopedia of Genes and Genomes pathways. Toll-like receptor (TLR)- and nucleotide-binding oligomerization domain-like receptor (NLR)-signaling pathways were related between the IL-1ß group and the group treated with DNA polymeric molecules. The genes involved in the TLR- and NLR-signaling pathways were validated using real-time quantitative polymerase chain reaction and western blot. Among these genes, IL-6, IL-1ß, IL-8, and chemokine (C-C motif) ligand 3 were dramatically upregulated in the IL-1ß group, but significantly downregulated in the group treated with DNA polymeric molecules. Specifically, PN treatment resulted in a greater decrease in the expression of these genes as compared with PDRN treatment. Both PDRN and PN treatments were involved in the anti-inflammatory response associated with OA progression, with PN treatment exhibiting additional anti-inflammatory properties relative to PDRN treatment. These results provide insight into potential therapeutic approaches involving PDRN and PN treatment of OA.

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci's method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence ("no cortical injury" group) or presence ("cortical injury" group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

Elucidation of Gene Expression Patterns in the Brain after Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating neurological disease. The pathophysiological mechanisms of SCI have been reported to be relevant to central nervous system injury such as brain injury. In this study, gene expression of the brain after SCI was elucidated using transcriptome analysis to characterize the temporal changes in global gene expression patterns in a SCI mouse model. Subjects were randomly classified into 3 groups: sham control, acute (3 h post-injury), and subacute (2 wk post-injury) groups. We sought to confirm the genes differentially expressed between post-injured groups and sham control group. Therefore, we performed transcriptome analysis to investigate the enriched pathways associated with pathophysiology of the brain after SCI using Database for Annotation Visualization, and Integrated Discovery (DAVID), which yielded Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Following enriched pathways were found in the brain: oxidative phosphorylation pathway; inflammatory response pathways-cytokine-cytokine receptor interaction and chemokine signaling pathway; and endoplasmic reticulum (ER) stress-related pathways-antigen processing and presentation and mitogen-activated protein kinase signaling pathway. Oxidative phosphorylation pathway was identified at acute phase, while inflammation response and ER stress-related pathways were identified at subacute phase. Since the following pathways-oxidative phosphorylation pathway, inflammatory response pathways, and ER stress-related pathways-have been well known in the SCI, we suggested a link between SCI and brain injury. These mechanisms provide valuable reference data for better understanding pathophysiological processes in the brain after SCI.

The Modulation of Neurotrophin and Epigenetic Regulators: Implication for Astrocyte Proliferation and Neuronal Cell Apoptosis After Spinal Cord Injury.

OBJECTIVE: To investigate alterations in the expression of the main regulators of neuronal survival and death related to astrocytes and neuronal cells in the brain in a mouse model of spinal cord injury (SCI). METHODS: Eight-week-old male imprinting control region mice (n=36; 30-35 g) were used in this study and randomly assigned to two groups: the naïve control group (n=18) and SCI group (n=18). The mice in both groups were randomly allocated to the following three time points: 3 days, 1 week, and 2 weeks (n=6 each). The expression levels of regulators such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), histone deacetylase 1 (HDAC1), and methyl-CpG-binding protein 2 (MeCP 2) in the brain were evaluated following thoracic contusive SCI. In addition, the number of neuronal cells in the motor cortex (M1 and M2 areas) and the number of astrocytes in the hippocampus were determined by immunohistochemistry. RESULTS: BDNF expression was significantly elevated at 2 weeks after injury (p=0.024). The GDNF level was significantly elevated at 3 days (p=0.042). The expression of HDAC1 was significantly elevated at 1 week (p=0.026). Following SCI, compared with the control the number of NeuN-positive cells in the M1 and M2 areas gradually and consistently decreased at 2 weeks after injury. In contrast, the number of astrocytes was significantly increased at 1 week (p=0.029). CONCLUSION: These results demonstrate that the upregulation of BDNF, GDNF and HDAC1 might play on important role in brain reorganization after SCI.