Identification of key molecules in the formation of portal vein tumor thrombus in hepatocellular carcinoma based on single cell transcriptomics and in vitro experiments.

Background: The presence of portal vein tumor thrombus (PVTT) is a significant indicator of advanced-stage hepatocellular carcinoma (HCC). Unfortunately, the prediction of PVTT occurrence remains challenging, and there is a lack of comprehensive research exploring the underlying mechanisms of PVTT formation and its association with immune infiltration. Methods: Our approach involved analyzing single-cell sequencing data, applying high dimensional weighted gene co-expression network analysis (hdWGCNA), and identifying key genes associated with PVTT development. Furthermore, we constructed competing endogenous RNA (ceRNA) networks and employed weighted gene co-expression network analysis (WGCNA), as well as three machine-learning techniques, to identify the upstream regulatory microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) of the crucial mRNAs. We employed fuzzy clustering of time series gene expression data (Mfuzz), gene set variation analysis (GSVA), and cell communication analysis to uncover significant signaling pathways involved in the activation of these important mRNAs during PVTT development. In addition, we conducted immune infiltration analysis, survival typing, and drug sensitivity analysis using The Cancer Genome Atlas (TCGA) cohort to gain insights into the two patient groups under study. Results: Through the implementation of hdWGCNA, we identified 110 genes that was closely associated with PVTT. Among these genes, TMEM165 emerged as a crucial candidate, and we further investigated its significance using COX regression analysis. Furthermore, through machine learning techniques and survival analysis, we successfully identified the upstream regulatory miRNA (hsa-miR-148a) and lncRNA (LINC00909) that targeted TMEM165. These findings shed light on the complex regulatory network surrounding TMEM165 in the context of PVTT. Moreover, we conducted CIBERSORT analysis, which unveiled correlations between TMEM165 and immune infiltration in HCC patients. Specifically, TMEM165 exhibited associations with various immune cell populations, including memory B cells and CD8+ T cells. Additionally, we observed implications for immune function, particularly in relation to immune checkpoints, within the context of HCC. Conclusions: The regulatory axis involving TMEM165, hsa-miR-148a, and LINC00909 emerges as a crucial determinant in the development of PVTT in HCC patients, and it holds significant implications for prognosis. Furthermore, alterations in the TMEM165/hsa-miR-148a/LINC00909 regulatory axis exhibit a strong correlation with immune infiltration within the HCC tumor microenvironment, leading to immune dysfunction and potential failure of immunotherapy.

Inhibition of TREM-1 alleviates neuroinflammation by modulating microglial polarization via SYK/p38MAPK signaling pathway after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI), as a major public health problem, is characterized by high incidence rate, disability rate, and mortality rate. Neuroinflammation plays a crucial role in the pathogenesis of TBI. Triggering receptor expressed on myeloid cells-1 (TREM-1) is recognized as an amplifier of the inflammation in diseases of the central nervous system (CNS). However, the function of TREM-1 remains unclear post-TBI. This study aimed to investigate the function of TREM-1 in neuroinflammation induced by TBI. METHODS: Brain water content (BWC), modified neurological severity score (mNSS), and Morris Water Maze (MWM) were measured to evaluate the effect of TREM-1 inhibition on nervous system function and outcome after TBI. TREM-1 expression in vivo was evaluated by Western blotting. The cellular localization of TREM-1 in the damaged region was observed via immunofluorescence staining. We also conducted Western blotting to examine expression of SYK, p-SYK and other downstream proteins. RESULTS: We found that inhibition of TREM-1 reduced brain edema, decreased mNSS and improved neurobehavioral outcomes after TBI. It was further determined that TREM-1 was expressed on microglia and modulated subtype transition of microglia. Inhibition of TREM-1 alleviated neuroinflammation, which was associated with SYK/p38MAPK signaling pathway. CONCLUSIONS: These findings suggest that TREM-1 can be a potential clinical therapeutic target for alleviating neuroinflammation after TBI.

Phosphorylation of 4.1N by CaMKII Regulates the Trafficking of GluA1-containing AMPA Receptors During Long-term Potentiation in Acute Rat Hippocampal Brain Slices.

OBJECTIVE: GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) inserted into postsynaptic membranes are key to the process of long-term potentiation (LTP). Some evidence has shown that 4.1N plays a critical role in the membrane trafficking of AMPARs. However, the underlying mechanism behind this is still unclear. We investigated the role of 4.1N-mediated membrane trafficking of AMPARs during theta-burst stimulation long-term potentiation (TBS-LTP), to illustrate the molecular mechanism behind LTP. METHODS: LTP was induced by TBS in rat hippocampal CA1 neuron. Tat-GluA1 (MPR), which disrupts the association of 4.1N-GluA1, and autocamtide-2-inhibitory peptide, myristoylated (Myr-AIP), a CaMKII antagonist, were used to explore the role of 4.1N in the AMPARs trafficking during TBS-induced LTP. Immunoprecipitation (IP) and immunoblotting (IB)were used to detect protein expression, phosphorylation, and the interaction of p-CaMKII-4.1N-GluA1. RESULTS: We found that Myr-AIP attenuated increases of p-CaMKII (T286), p-GluA1 (ser831), and 4.1N phosphorylation after TBS-LTP, and decreased the association of p-CaMKII-4.1N-GluA1, along with the expression of GluA1, at postsynaptic densities during TBS-LTP. We also designed interfering peptides to disrupt the interaction between 4.1N and GluA1, which showed that Tat-GluA1 (MPR) or Myr-AIP inhibited TBS-LTP and attenuated increases of GluA1 at postsynaptic sites, while Tat-GluA1 (MPR) or Myr-AIP had no effects on miniature excitatory postsynaptic currents (mEPSCs) in non-stimulated hippocampal CA1 neurons. CONCLUSION: Active CaMKII enhanced the phosphorylation of 4.1N and facilitated the association of p-CaMKII with 4.1N-GluA1, which in turn resulted in GluA1 trafficking during TBS-LTP. The association of 4.1N-GluA1 is required for LTP, but not for basal synaptic transmission.

Nrf2/HO-1 blocks TXNIP/NLRP3 interaction via elimination of ROS in oxygen-glucose deprivation-induced neuronal necroptosis.

Acute ischemic stroke (AIS) is known to trigger a cascade of inflammatory events that induces secondary tissue damages. As a type of regulated inflammatory cell death, necroptosis is associated with AIS, whilst its regulation during neuroinflammation is not well understood. In particular, the actual function of NOD-like-receptor family pyrin domain-containing-3(NLRP3) inflammasome in cortical neuronal necroptosis still not clear. Herein, we explored the function of nuclear factor erythroid-2 related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) in oxygen-glucose deprivation (OGD) induced neuronal necroptosis and its underlying mechanism. To establish an in vitro model of neuronal necrosis, we used OGD/caspase-8 inhibitors (Q-VD-OPh, QVD) to treat rat primary cortical neurons (PCNs) after reoxygenation, wherein we found that the model cause an elevated ROS levels by mediating TXNIP/NLRP3 interactions, which in turn activated the NLRP3 inflammasome. Also, we observed that regulation of nuclear factor erythroid-2 related factor-2 (Nrf2) promoted heme oxygenase-1 (HO-1) expression and decreased TXNIP (a protein that relate oxidative stress to activation of inflammasome) and ROS levels, which negatively regulated the expression of OGD-induced activation of NLRP3 inflammasomes. In addition, HO-1 weakened NLRP3 inflammation body activation, which suggests that Nrf2-regulated HO-1 could block the interaction between TXNIP and NLRP3 in OGD/R-treated cortical neurons by inhibiting ROS production. Our study has discovered the importance of Nrf2/HO-1 signaling cascade for inhibiting inflammasome of NLRP3, which negatively regulated necrosis. Therefore, NLRP3 is considered a potential target for a novel neuroprotective approach, which can expand the therapeutic windows of stroke drugs.

An interpretable machine learning approach for predicting 30-day readmission after stroke.

BACKGROUND: Stroke is the second leading cause of death worldwide and has a significantly high recurrence rate. We aimed to identify risk factors for stroke recurrence and develop an interpretable machine learning model to predict 30-day readmissions after stroke. METHODS: Stroke patients deposited in electronic health records (EHRs) in Xuzhou Medical University Hospital between February 1, 2021, and November 30, 2021, were included in the study, and deceased patients were excluded. We extracted 74 features from EHRs, and the top 20 features (chi-2 value) were used to build machine learning models. 80% of the patients were used for pre-training. Subsequently, a 20% holdout dataset was used for verification. The Shapley Additive exPlanations (SHAP) method was used to explore the interpretability of the model. RESULTS: The cohort included 6,558 patients, of whom the mean (SD) age was 65 (11) years, 3,926 were males (59.86 %), and 132 (2.01 %) were readmitted within 30 days. The area under the receiver operating characteristic curve (AUROC) for the optimized model was 0.80 (95 % CI 0.68-0.80). We used the SHAP method to identify the top 10 risk factors (i.e., severe carotid artery stenosis, weak, homocysteine, glycosylated hemoglobin, sex, lymphocyte percentage, neutrophilic granulocyte percentage, urine glucose, fresh cerebral infarction, and red blood cell count). The AUROC of a model with the 10 features was 0.80 (95 % CI 0.69-0.80) and was not significantly different from that of the model with 20 risk factors. CONCLUSIONS: Our methods not only showed good performance in predicting 30-day readmissions after stroke but also revealed risk factors that provided valuable insights for treatments.

Overexpression of vascular endothelial growth factor enhances the neuroprotective effects of bone marrow mesenchymal stem cell transplantation in ischemic stroke.

Although bone marrow mesenchymal stem cells (BMSCs) might have therapeutic potency in ischemic stroke, the benefits are limited. The current study investigated the effects of BMSCs engineered to overexpress vascular endothelial growth factor (VEGF) on behavioral defects in a rat model of transient cerebral ischemia, which was induced by middle cerebral artery occlusion. VEGF-BMSCs or control grafts were injected into the left striatum of the infarcted hemisphere 24 hours after stroke. We found that compared with the stroke-only group and the vehicle- and BMSCs-control groups, the VEGF-BMSCs treated animals displayed the largest benefits, as evidenced by attenuated behavioral defects and smaller infarct volume 7 days after stroke. Additionally, VEGF-BMSCs greatly inhibited destruction of the blood-brain barrier, increased the regeneration of blood vessels in the region of ischemic penumbra, and reducedneuronal degeneration surrounding the infarct core. Further mechanistic studies showed that among all transplant groups, VEGF-BMSCs transplantation induced the highest level of brain-derived neurotrophic factor. These results suggest that BMSCs transplantation with vascular endothelial growth factor has the potential to treat ischemic stroke with better results than are currently available.

Effect of S-Nitrosylation of RIP3 Induced by Cerebral Ischemia on its Downstream Signaling Pathway.

OBJECTIVES: Our preliminary experiments indicate that receptor-interacting protein 3 (RIP3) is S-nitrosylated and contributes to its autophosphorylation (activation) after 3 h of rat brain ischemia/reperfusion mediated by activation of the N-methyl-D-aspartate receptor (NMDAR)-dependent neuronal NO synthase (nNOS) and is involved in the process of neuronal injury. Here, we will to demonstrate whether S-nitrosylation of RIP3 facilitates the activation of the downstream signaling pathway and finally exacerbates ischemic neuron death. MATERIALS AND METHODS: Adult male Sprague-Dawley rat transient brain ischemia/reperfusion and cortical neurons oxygen and glucose deprivation (OGD)/reoxygenation models were performed. The hippocampal CA1 regions or cultured cells were homogenized and the cytosolic fraction were collected as tissue samples. Coimmunoprecipitation and western blot analysis were carried out for detecting phosphorylation of RIP1 and mixed lineage kinase-like domains (MLKL) and the Cleaved-Caspase8 (Cl-Caspase8). The activities of Glycogen phosphorylase (PYGL), Glutamate-ammonia ligase (GLUL) and Glutamate dehydrogenase (GLUD1) were detected with ultraviolet absorption method. RESULTS: This study showed that active RIP3 could phosphorylate RIP1 and MLKL through its kinase activity, promote the conversion of Caspase8 to active Cl-Caspase8, enhance the activities of PYGL, GLUL and GLUD1, and finally aggravate neuronal injury in cerebral ischemia/reperfusion. The inhibition of RIP3 S-nitrosylation inhibited the phosphorylation of RIP1 and MLKL, inhibited the activities of Caspase8, PYGL, GLUL, and GLUD1, and alleviated neuronal damage in cerebral ischemia/reperfusion. CONCLUSIONS: S-nitrosylation of RIP3 increased RIP1 and MLKL phosphorylation levels, Cl-Caspase8 content and PYGL, GLUL and GLUD1 activities and aggravated neuronal damage during cerebral ischemia/reperfusion and regulating the S-nitrosylation of RIP3 and its downstream signaling pathway might be a therapeutic target for stroke.

Hydrostatic pressure stimulates the osteogenesis and angiogenesis of MSCs/HUVECs co-culture on porous PLGA scaffolds.

In native bone tissue regeneration, blood vessels, providing oxygen and nutrition for tissues, can promote the regeneration of bone and accelerate the repair of a defected area. In this study, Poly(D, L-lactic-co-glycolic acid) (PLGA) inverse opal scaffolds with high pore interconnectivity were fabricated and further modified with vascular endothelial growth factor (VEGF). The rat bone marrow derived mesenchymal stem cells (rMSCs) and human umbilical vein endothelial cells (HUVECs) were co-cultured onto the scaffolds to enhance vascularization for bone tissue regeneration. Cell attachment, viability, proliferation, and morphology were detected by cell counting kit-8 (CCK-8) assay, live and dead staining and scanning electron microscopy (SEM). Hydrostatic pressure with 0-279 KPa and 1 Hz one hour per day for 7 days was applied to tissue engineered bone constructs to investigate whether the loading stimulation can promote osteogenesis and angiogenesis mutually evaluated in parallel by multiple in vitro assays and in an in vivo chicken chorioallantoic membrane (CAM) model. The results indicated that the immobilization of VEGF can improve biocompatibility of PLGA scaffolds and promote cell attachment and proliferation. The cell-scaffold constructs showed higher CD31 expression because of the angiogenic differentiation of rMSCs in hydrostatic loading culture condition in vitro. The in vivo CAM model experiment demonstrated that hydrostatic loading stimulated angiogenic differentiation of rMSCs can accelerate tubulogenesis. Furthermore, the new capillaries formed in cell-scaffold constructs were conducive to calcium deposition in vivo.

Photobiomodulation Promotes Hippocampal CA1 NSC Differentiation Toward Neurons and Facilitates Cognitive Function Recovery Involving NLRP3 Inflammasome Mitigation Following Global Cerebral Ischemia.

Our recent study revealed that photobiomodulation (PBM) inhibits delayed neuronal death by preserving mitochondrial dynamics and function following global cerebral ischemia (GCI). In the current study, we clarified whether PBM exerts effective roles in endogenous neurogenesis and long-lasting neurological recovery after GCI. Adult male rats were treated with 808 nm PBM at 20 mW/cm2 irradiance for 2 min on cerebral cortex surface (irradiance ∼7.0 mW/cm2, fluence ∼0.8 J/cm2 on the hippocampus) beginning 3 days after GCI for five consecutive days. Cognitive function was evaluated using the Morris water maze. Neural stem cell (NSC) proliferation, immature neurons, and mature neurons were examined using bromodeoxyuridine (BrdU)-, doublecortin (DCX)-, and NeuN-staining, respectively. Protein expression, such as NLRP3, cleaved IL1ß, GFAP, and Iba1 was detected using immunofluorescence staining, and ultrastructure of astrocyte and microglia was observed by transmission electron microscopy. The results revealed that PBM exerted a markedly neuroprotective role and improved spatial learning and memory ability at 58 days of ischemia/reperfusion (I/R) but not at 7 days of reperfusion. Mechanistic studies revealed that PBM suppressed reactive astrocytes and maintained astrocyte regeneration at 7 days of reperfusion, as well as elevated neurogenesis at 58 days of reperfusion, as evidenced by a significant decrease in the fluorescence intensity of GFAP (astrocyte marker) but unchanged the number of BrdU-GFAP colabeled cells at the early timepoint, and a robust elevation in the number of DCX-NeuN colabeled cells at the later timepoint in the PBM-treated group compared to the GCI group. Notably, PBM treatment protected the ultrastructure of astrocyte and microglia cells at 58 days but not 7 days of reperfusion in the hippocampal CA1 region. Furthermore, PBM treatment significantly attenuated the GCI-induced immunofluorescence intensity of NLRP3 (an inflammasome component), cleaved IL1ß (reflecting inflammasome activation) and Iba1, as well as the colocalization of NLRP3/GFAP or cleaved IL-1ß/GFAP, especially in animals subjected to I/R at 58 days. Taken together, PBM treatment performed postischemia exerted a long-lasting protective effect on astrocytes and promoted endogenous neurogenesis in the hippocampal CA1 region, which might contribute to neurological recovery after GCI.

Curcumin ameliorates ischemic stroke injury in rats by protecting the integrity of the blood-brain barrier.

The blood-brain barrier (BBB) is critical for proper cerebral homeostasis and its dysfunction during ischemic stroke can result in significant neurological injury. The major goal of the present study was to identify whether curcumin pretreatment possessed protective effects on BBB integrity during the 24 h of acute ischemic brain injury. To investigate the protective effects of curcumin, male Sprague-Dawley rats were divided into multiple groups, including sham, middle cerebral artery occlusion/reperfusion (MCAO/R) vehicle and curcumin pretreated MCAO/R groups. The effects of curcumin were measured by analyzing neurological deficits, infarct size, BBB permeability and expression levels of permeability-related proteins in the brain. It was found that curcumin pretreatment significantly improved neurological scores, decreased infarct size, and protected synaptic remodeling of hippocampal neurons and upregulated the protein expression level of tight junction proteins, ZO-1, occludin and claudin-5 in ischemic rat brains. Furthermore, curcumin pretreatment before stroke was shown to downregulate the phosphorylation of NF-κB and MMP-9, which are central mediators of inflammation. The results from the present study indicated that curcumin pretreatment ameliorated ischemic stroke injury by protecting BBB integrity and synaptic remodeling, as well as inhibiting inflammatory responses.

Theta-burst transcranial magnetic stimulation promotes stroke recovery by vascular protection and neovascularization.

Rationale: The integrity and function of the blood-brain barrier (BBB) is compromised after stroke. The current study was performed to examine potential beneficial effects and underlying mechanisms of repetitive transcranial magnetic stimulation (rTMS) on angiogenesis and vascular protection, function, and repair following stroke, which are largely unknown. Methods: Using a rat photothrombotic (PT) stroke model, continuous theta-burst rTMS was administered once daily to the infarcted hemisphere for 5 min, beginning 3 h after PT stroke. This treatment was applied for 6 days. BBB integrity, blood flow, vascular associated proteins, angiogenesis, integrity of neuronal morphology and structure, and behavioral outcome were measured and analyzed at 6 and/or 22 days after PT stroke. Results: We report that rTMS significantly mitigated BBB permeabilization and preserved important BBB components ZO-1, claudin-5, occludin, and caveolin-1 from PT-induced degradation. Damage to vascular structure, morphology, and perfusion was ameliorated by rTMS, resulting in improved local tissue oxygenation. This was accompanied with robust protection of critical vascular components and upregulation of regulatory factors. A complex cytokine response was induced by PT, particularly at the late phase. Application of rTMS modulated this response, ameliorating levels of cytokines related to peripheral immune cell infiltration. Further investigation revealed that rTMS promoted and sustained post-ischemic angiogenesis long-term and reduced apoptosis of newborn and existing vascular endothelial cells. Application of rTMS also inhibited PT-induced excessive astrocyte-vasculature interactions and stimulated an A1 to A2 shift in vessel-associated astrocytes. Mechanistic studies revealed that rTMS dramatically increased levels of PDGFRß associated with A2 astrocytes and their adjacent vasculature. As well, A2 astrocytes displayed marked amplification of the angiogenesis-related factors VEGF and TGFß. PT induced a rise in vessel-associated expression of HIF-1α that was starkly intensified by rTMS treatment. Finally, rTMS preserved neuronal morphology, synaptic structure integrity and behavioral outcome. Conclusions: These results indicate that rTMS can exert powerful protective and restorative effects on the peri-infarct microvasculature after PT stroke by, in part, promoting HIF-1α signaling and shifting vessel-associated astrocytic polarization to the A2 phenotype. This study provides further support for the potent protective effects of rTMS in the context of ischemic stroke, and these findings implicate vascular repair and protection as an important underlying phenomenon.

gB co-immunization with GP96 enhances pulmonary-resident CD8 T cells and exerts a long-term defence against MCMV pneumonitis.

Human cytomegalovirus (HCMV) infection in the respiratory tract leads to pneumonitis in immunocompromised hosts without available vaccine. Considering cytomegalovirus (CMV) mainly invades through the respiratory tract, CMV-specific pulmonary mucosal vaccine development that provides a long-lasting protection against CMV challenge gains our attention. In this study, N-terminal domain of GP96 (GP96-NT) was used as a mucosal adjuvant to enhance the induction of pulmonary-resident CD8 T cells elicited by MCMV glycoprotein B (gB) vaccine. Mice were intranasally co-immunized with 50 µg pgB and equal amount of pGP96-NT vaccine 4 times at 2-week intervals, and then i.n. challenged with MCMV at 16 weeks after the last immunization. Compared with pgB immunization alone, co-immunization with pgB/pGP96-NT enhanced a long-lasting protection against MCMV pneumonitis by significantly improved pneumonitis pathology, enhanced bodyweight, reduced viral burdens and increased survival rate. Moreover, the increased CD8 T cells were observed in lung but not spleen from pgB/pGP96-NT co-immunized mice. The increments of pulmonary CD8 T cells might be mainly due to non-circulating pulmonary-resident CD8 T-cell subset expansion but not circulating CD8 T-cell populations that home to inflammation site upon MCMV challenge. Finally, the deterioration of MCMV pneumonitis by depletion of pulmonary site-specific CD8 T cells in mice that were pgB/pGP96-NT co-immunization might be a clue to interpret the non-circulating pulmonary-resident CD8 T subset expansion. These data might uncover a promising long-lasting prophylactic vaccine strategy against MCMV-induced pneumonitis.

Identification of Key mRNAs and lncRNAs in Neonatal Sepsis by Gene Expression Profiling.

Neonatal sepsis is one of the most prevalent causes of death of the neonates. However, the mechanisms underlying neonatal sepsis remained unclear. The present study identified a total of 1128 upregulated mRNAs and 1008 downregulated mRNAs, 28 upregulated lncRNAs, and 61 downregulated lncRNAs in neonatal sepsis. Then, we constructed PPI networks to identify key regulators in neonatal sepsis, including ITGAM, ITGAX, TLR4, ITGB2, SRC, ELANE, RPLP0, RPS28, RPL26, and RPL27. lncRNA coexpression analysis showed HS.294603, LOC391811, C12ORF47, LOC729021, HS.546375, HNRPA1L-2, LOC158345, and HS.495041 played important roles in the progression of neonatal sepsis. Bioinformatics analysis showed DEGs were involved in the regulation cellular extravasation, acute inflammatory response, macrophage activation of NF-kappa B signaling pathway, TNF signaling pathway, HIF-1 signaling pathway, Toll-like receptor signaling pathway, and ribosome, RNA transport, and spliceosome. lncRNAs were involved in regulating ribosome, T cell receptor signaling pathway, RNA degradation, insulin resistance, ribosome biogenesis in eukaryotes, and hematopoietic cell lineage. We thought this study provided useful information for identifying novel therapeutic markers for neonatal sepsis.

Beneficial Effects of Theta-Burst Transcranial Magnetic Stimulation on Stroke Injury via Improving Neuronal Microenvironment and Mitochondrial Integrity.

Recent work suggests that repetitive transcranial magnetic stimulation (rTMS) may beneficially alter the pathological status of several neurological disorders, although the mechanism remains unclear. The current study was designed to investigate the effects of rTMS on behavioral deficits and potential underlying mechanisms in a rat photothrombotic (PT) stroke model. From day 0 (3 h) to day 5 after the establishment of PT stroke, 5-min daily continuous theta-burst rTMS (3 pulses of 50 Hz repeated every 200 ms, intensity at 200 G) was applied on the infarct hemisphere. We report that rTMS significantly attenuated behavioral deficits and infarct volume after PT stroke. Further investigation demonstrated that rTMS remarkably reduced synaptic loss and neuronal degeneration in the peri-infarct cortical region. Mechanistic studies displayed that beneficial effects of rTMS were associated with robust suppression of reactive micro/astrogliosis and the overproduction of pro-inflammatory cytokines, as well as oxidative stress and oxidative neuronal damage especially at the late stage following PT stroke. Intriguingly, rTMS could effectively induce a shift in microglial M1/M2 phenotype activation and an A1 to A2 switch in astrocytic phenotypes. In addition, the release of anti-inflammatory cytokines and mitochondrial MnSOD in peri-infarct regions were elevated following rTMS treatment. Finally, rTMS treatment efficaciously preserved mitochondrial membrane integrity and suppressed the intrinsic mitochondrial caspase-9/3 apoptotic pathway within the peri-infarct cortex. Our novel findings indicate that rTMS treatment exerted robust neuroprotection when applied at least 3 h after ischemic stroke. The underlying mechanisms are partially associated with improvement of the local neuronal microenvironment by altering inflammatory and oxidative status and preserving mitochondrial integrity in the peri-infarct zone. These findings provide strong support for the promising therapeutic effect of rTMS against ischemic neuronal injury and functional deficits following stroke.

Photobiomodulation therapy for repeated closed head injury in rats.

Repeated traumatic brain injury, leads to cumulative neuronal injury and neurological impairments. There are currently no effective treatments to prevent these consequences. Growing interest is building in the use of transcranial photobiomodulation (PBM) therapy to treat traumatic brain injury. Here, we examined PBM in a repeated closed head injury (rCHI) rat model. Rats were administered a total of three closed head injuries, with each injury separated by 5 days. PBM treatment was initiated 2 hours after the first injury and administered daily for a total of 15 days. We found that PBM-treated rCHI rats had a significant reduction in motor ability, anxiety and cognitive deficits compared to CHI group. PBM group showed an increase of synaptic proteins and surviving neurons, along with a reduction in reactive gliosis and neuronal injury. These findings highlight the complexity of gliosis and neuronal injury following rCHI and suggest that PBM may be a viable treatment option to mitigate these effects and their detrimental consequences.

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds.

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.

The treatment of spinal cord injury in rats using bone marrow-derived neural-like cells induced by cerebrospinal fluid.

This study aimed to evaluate the effect of bone mesenchymal stem cells (BMSCs) and BMSC neural-like cells (BMSC-Ns) on the spinal cord injury (SCI) in the rat model of SCI. BMSC-Ns were prepared from the third passage of BMSCs by induction of healthy cerebrospinal fluid (CSF) of an adult human. The SCI rat model was established through a surgical procedure, and after 7 days the rats were randomly divided into 3 (A, B and C) groups. Groups A (BMSC-Ns) and B (BMSCs) were treated with 1 × 106/20 µl cells, while group C (saline) was treated with saline, all via intracerebroventricular injection. After transplantation, the BBB score of group A was significantly higher than that of group B, which in turn was significantly higher than that of group C (P < .05). The levels of Bdnf, Ngf, Ntf3 were statistically significantly higher in group A than those in groups B and C (P < .05). The levels of 5-HT, NA, Ach, DA, GABA in group A were significantly higher than those in groups B and C, whereas the level of Glu was significantly lower in group A than that in groups B and C (P < .05). The histopathological data showed remarkably less necrosis of the spinal cord in group A, compared to that in groups B and C. Transplanting BMSC-Ns or BMSCs into the lateral ventricles improved the neurological function of rats with SCI. Moreover, BMSC-Ns were significantly more effective than BMSCs, which provides a possible approach for the treatment of SCI.

After Treatment with Methylene Blue is Effective against Delayed Encephalopathy after Acute Carbon Monoxide Poisoning.

Delayed encephalopathy after acute carbon monoxide (CO) poisoning (DEACMP) is the most severe and clinically intractable complication that occurs following acute CO poisoning. Unfortunately, the mechanism of DEACMP is still vague. Growing evidence indicates that delayed cerebral damage after CO poisoning is related to oxidative stress, abnormal neuro-inflammation, apoptosis and immune-mediated injury. Our recent report indicated that methylene blue (MB) may be a promising therapeutic agent in the prevention of neuronal cell death and cognitive deficits after transient global cerebral ischaemia (GCI). In this study, we aimed to investigate the potential of MB therapy to ameliorate the signs and symptoms of DEACMP. Rats were exposed to 1000 ppm CO for 40 min. in the first step; CO was then increased to 3000 ppm, which was maintained for another 20 min. The rats were implanted with 7-day release Alzet osmotic mini-pumps subcutaneously under the back skin, which provided MB at a dose of 0.5 mg/kg/day 1 hr after CO exposure. The results showed that MB significantly suppressed oxidative damage and expression of pro-inflammatory factors, including tumour necrosis factor-α and interleukin (IL)-1ß. MB treatment also suitably modulated mitochondrial fission and fusion, which is helpful in the preservation of mitochondrial function. Furthermore, MB dramatically attenuated apoptosis and neuronal death. Lastly, behavioural studies revealed that MB treatment preserved spatial learning and memory in the Barnes maze test. Our findings indicated that MB may have protective effects against DEACMP.

Inhibition of PDE2 reverses beta amyloid induced memory impairment through regulation of PKA/PKG-dependent neuro-inflammatory and apoptotic pathways.

Beta amyloid peptides (Aß) are known risk factors involved in cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Phosphodiesterase 2 (PDE2) inhibitors increase the intracellular cAMP and/or cGMP activities, which may ameliorate cognitive deficits associated with AD. However, it remains unclear whether PDE2 mediated neuroapoptotic and neuroinflammatory events, as well as cognitive performance in AD are related to cAMP/cGMP-dependent pathways. The present study investigated how the selective PDE2 inhibitor BAY60-7550 (BAY) affected Aß-induced learning and memory impairment in two classic rodent models. IL-22 and IL-17, Bax and Bcl-2, PKA/PKG and the brain derived neurotropic factor (BDNF) levels in hippocampus and cortex were detected with immunoblotting assay. The results showed that BAY reversed Aß-induced cognitive impairment as shown in the water maze test and step-down test. Moreover, BAY treatment reversed the Aß-induced changes in IL-22 and IL-17 and the ratio of Bax/Bcl-2. Changes in cAMP/cGMP levels, PKA/PKG and BDNF expression were also prevented by BAY. These effects of BAY on memory performance and related neurochemical changes were partially blocked by the PKG inhibitor KT 5823. These findings indicated that the protective effects of BAY against Aß-induced memory deficits might involve the regulation of neuroinflammation and neuronal apoptotic events.

Involvement of IL-17 in Secondary Brain Injury After a Traumatic Brain Injury in Rats.

The pro-inflammatory activity of interleukin 17, which is produced by the IL-23/IL-17 axis, has been associated with the pathogenesis of traumatic brain injury (TBI). The study investigated the potential role of IL-17 in secondary brain injury of TBI in a rat model. Our data showed that the levels of IL-17 increased from 6 h to 7 days and peaked at 3 days, in both the CNS and serum, which were consistent with the severity of secondary brain injury. The IL-23 inhibitor suberoylanilide hydroxamic acid (SAHA) treatment markedly decreased the expressions of IL-17 and apoptosis-associated proteins cleaved caspase-3 and increased the protein ratio of Bcl-2 (B cell lymphoma/leukemia-2)/Bax (Bcl-2-associated X protein). Meanwhile, neuronal apoptosis was reduced, and neural function was improved after SAHA treatment. This study suggests that IL-17 is involved in secondary brain injury after TBI. Administering an IL-23 inhibitor and thereby blocking the IL-23/IL-17 axis may be beneficial in the treatment of TBI.