Increased level of exosomal miR-20b-5p derived from hypothermia-treated microglia promotes neurite outgrowth and synapse recovery after traumatic brain injury.

Mild hypothermia has been proven to inhibit microglia activation after TBI. Exosomal microRNA derived from microglia played a critical role in promoting neurite outgrowth and synapse recovery. Here, we aimed to investigate the role of microRNAs in microglial exosomes after hypothermia treatment on neuronal regeneration after TBI. For in vitro study, stretch-injured neurons were co-cultured with microglial exosomes. For in vivo study, C57BL/6 mice were under controlled cortical impact and injected with microglial exosomes. The results showed that MG-LPS-EXOHT increased the number of dendrite branches and total length of dendrites both in vitro and in vivo, elevated the expression levels of PSD-95 and GluR1 in stretch-injured neurons, and increased spine density in the pericontusion region. Moreover, MG-LPS-EXOHT improved motor function and motor coordination. A high-throughput sequencing showed that miR-20b-5p was upregulated in MG-LPS-EXOHT. Elevating miR-20b-5p promoted neurite outgrowth and synapse recovery of injured neurons both in vitro and in vivo. Following mechanistic study demonstrated that miR-20b-5p might promote neurite outgrowth and synapse recovery by directly targeting PTEN and activating PI3K-AKT pathway. In conclusion, mild hypothermia could modify the microRNA prolife of exosomes derived from LPS activated BV2 cells. Furthermore, high level of microglial exosomal miR-20b-5p induced by mild hypothermia could transfer into injured neurons and promote neurite outgrowth and synapse recovery after TBI via activating the PI3K-AKT pathway by suppressing PTEN expression.

Glial fibrillary acidic protein as a biomarker in severe traumatic brain injury patients: a prospective cohort study.

INTRODUCTION: Glial fibrillary acidic protein (GFAP) may serve as a serum marker of traumatic brain injury (TBI) that can be used to monitor biochemical changes in patients and gauge the response to treatment. However, the temporal profile of serum GFAP in the acute period of brain injury and the associated utility for outcome prediction has not been elucidated. METHODS: We conducted a prospective longitudinal cohort study of consecutive severe TBI patients in a local tertiary neurotrauma center in Shanghai, China, between March 2011 and September 2014. All patients were monitored and managed with a standardized protocol with inclusion of hypothermia and other intensive care treatments. Serum specimens were collected on admission and then daily for the first 5 days. GFAP levels were measured using enzyme-linked immunosorbent assay techniques. Patient outcome was assessed at 6 months post injury with the Glasgow Outcome Scale and further grouped into death versus survival and unfavorable versus favorable. RESULTS: A total of 67 patients were enrolled in the study. The mean time from injury to admission was 2.6 hours, and the median admission Glasgow Coma Scale score was 6. Compared with healthy subjects, patients with severe TBI had increased GFAP levels on admission and over the subsequent 5 days post injury. Serum GFAP levels showed a gradual reduction from admission to day 3, and then rebounded on day 4 when hypothermia was discontinued with slow rewarming. GFAP levels were significantly higher in patients who died or had an unfavorable outcome across all time points than in those who were alive or had a favorable outcome. Results of receiver operating characteristic curve analysis indicated that serum GFAP at each time point could predict neurological outcome at 6 months. The areas under the curve for GFAP on admission were 0.761 for death and 0.823 for unfavorable outcome, which were higher than those for clinical variables such as age, Glasgow Coma Scale score, and pupil reactions. CONCLUSIONS: Serum GFAP levels on admission and during the first 5 days of injury were increased in patients with severe TBI and were predictive of neurological outcome at 6 months.

Intranasal 15d-PGJ2 inhibits the growth of rat lactotroph pituitary neuroendocrine tumors by inducing PPARγ-dependent apoptotic and autophagic cell death.

PPARγ agonists have been reported to induce cell death in pituitary neuroendocrine tumor (PitNET) cell cultures. However, the therapeutic effects of PPARγ agonists in vivo remain unclear. In the present study, we found that intranasal 15d-PGJ2, an endogenous PPARγ agonist, resulted in growth suppression of Fischer 344 rat lactotroph PitNETs induced by subcutaneous implantation with a mini-osmotic pump containing estradiol. Intranasal 15d-PGJ2 reduced the volume and weight of the pituitary gland and the level of serum prolactin (PRL) in rat lactotroph PitNETs. 15d-PGJ2 treatment attenuated pathological changes and significantly decreased the ratio of PRL/pituitary-specific transcription factor 1 (Pit-1) and estrogen receptor α (ERα)/Pit-1 double-positive cells. Moreover, 15d-PGJ2 treatment induced apoptosis in the pituitary gland characterized by an increased ratio of TUNEL-positive cells, cleavage of caspase-3, and elevated activity of caspase-3. 15d-PGJ2 treatment decreased the levels of cytokines, including TNF-α, IL-1ß, and IL-6. Furthermore, 15d-PGJ2 treatment markedly increased the protein expression of PPARγ and blocked autophagic flux, as evidenced by the accumulation of LC3-II and SQSTM1/p62 and the decrease in LAMP-1 expression. Importantly, all these effects mediated by 15d-PGJ2 were abolished by cotreatment with the PPARγ antagonist GW9662. In conclusion, intranasal 15d-PGJ2 suppressed the growth of rat lactotroph PitNETs by inducing PPARγ-dependent apoptotic and autophagic cell death. Therefore, 15d-PGJ2 may be a potential new drug for lactotroph PitNETs.

Neural stem cell­derived exosomes transfer miR­124­3p into cells to inhibit glioma growth by targeting FLOT2.

Currently, exosomes (EXOs) are being explored as novel drug delivery carriers with greater advantages, including crossing the blood­brain­barrier and loading drugs. The present study utilized EXOs derived from neural stem cells (NSCs) for the delivery of molecular drugs to treat gliomas. miR­124­3p was selected according to previous studies by the authors, and the effects of the delivery of miR­124­3p to glioma cells by NSC­EXOs in vitro and in vivo were evaluated. It was found that NSC­EXOs successfully delivered miR­124­3p into glioma cells, and NSC­EXOs loaded with miR­124­3p significantly inhibited glioma cell proliferation, invasion and migration. Furthermore, the delivery of miR­124­3p by NSC­EXOs suppressed flotillin 2 (FLOT2) expression by specifically binding to the 3' untranslated region of the FLOT2 gene in gliomas; subsequently, AKT1 was found to be associated with the EXO­miR­124­3p/FLOT2 pathway. Moreover, the therapeutic effects of the delivery of miR­124­3p by NSC­EXOs were confirmed in a mouse tumor xenograft model of glioma. Thus, bio­carrier NSC­EXOs loaded with miR­124­3p suppressed glioma growth via the EXO­miR­124­3p/FLOT2/AKT1 pathway. On the whole, the present study provides insight into stem cell­free molecular­targeted therapy based on bio­carrier NSC­EXOs and provides a potential strategy for the treatment of glioma.

Decreased Level of Exosomal miR-5121 Released from Microglia Suppresses Neurite Outgrowth and Synapse Recovery of Neurons Following Traumatic Brain Injury.

Activated microglia can suppress neurite outgrowth and synapse recovery in the acute stage following traumatic brain injury (TBI). However, the underlying mechanism has not been clearly elucidated. Exosomes derived from microglia have been reported to play a critical role in microglia-neuron interaction in healthy and pathological brains. Here, we aimed to investigate the role of microglia-derived exosomes in regulating neurite outgrowth and synapse recovery following TBI. In our study, exosomes derived from microglia were co-cultured with stretch-injured neurons in vitro and intravenously injected into mice that underwent fluid percussion injury (FPI) by tail vein injection in vivo. The results showed that microglia-derived exosomes could be absorbed by neurons in vitro and in vivo. Moreover, exosomes derived from stretch-injured microglia decreased the protein levels of GAP43, PSD-95, GluR1, and Synaptophysin and dendritic complexity in stretch-injured neurons in vitro, and reduced GAP43+ NEUN cell percentage and apical dendritic spine density in the pericontusion region in vivo. Motor coordination was also impaired in mice treated with stretch-injured microglia-derived exosomes after FPI. A microRNA microarray showed that the level of miR-5121 was decreased most greatly in exosomes derived from stretch-injured microglia. Overexpression of miR-5121 in stretch-injured microglia-derived exosomes partly reversed the suppression of neurite outgrowth and synapse recovery of neurons both in vitro and in vivo. Moreover, motor coordination in miR-5121 overexpressed exosomes treated mice was significantly improved after FPI. Following mechanistic study demonstrated that miR-5121 might promote neurite outgrowth and synapse recovery by directly targeting RGMa. In conclusion, our finding revealed a novel exosome-mediated mechanism of microglia-neuron interaction that suppressed neurite outgrowth and synapse recovery of neurons following TBI.

Role and mechanism of neural stem cells of the subventricular zone in glioblastoma.

Glioblastoma multiforme (GBM), the most frequently occurring malignant brain tumor in adults, remains mostly untreatable. Because of the heterogeneity of invasive gliomas and drug resistance associated with the tumor microenvironment, the prognosis is poor, and the survival rate of patients is low. Communication between GBMs and non-glioma cells in the tumor microenvironment plays a vital role in tumor growth and recurrence. Emerging data have suggested that neural stem cells (NSCs) in the subventricular zone (SVZ) are the cells-of-origin of gliomas, and SVZ NSC involvement is associated with the progression and recurrence of GBM. This review highlights the interaction between SVZ NSCs and gliomas, summarizes current findings on the crosstalk between gliomas and other non-glioma cells, and describes the links between SVZ NSCs and gliomas. We also discuss the role and mechanism of SVZ NSCs in glioblastoma, as well as the interventions targeting the SVZ and their therapeutic implications in glioblastoma. Taken together, understanding the biological mechanism of glioma-NSC interactions can lead to new therapeutic strategies for GBM.

Depletion of Microglia Attenuates Dendritic Spine Loss and Neuronal Apoptosis in the Acute Stage of Moderate Traumatic Brain Injury in Mice.

Microglia are the primary immune cells in the central nervous system and undergo significant morphological and transcriptional changes after traumatic brain injury (TBI). However, their exact contribution to the pathogenesis of TBI is still debated and remains to be elucidated. In the present study, thy-1 GFP mice received a colony-stimulating factor 1 receptor inhibitor (PLX3397) for 21 consecutive days, then were subjected to moderate fluid percussion injury (FPI). Brain samples were collected at 1 day and 3 days after FPI for flow cytometry analysis, immunofluorescence, dendrite spine quantification, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and Western blot. We found that PLX3397 treatment significantly attenuated the percentages of resident microglia and infiltrated immune cells. Depletion of microglia promoted neurite outgrowth, preserved dendritic spines and reduced total brain cell and neuronal apoptosis after FPI, which was accompanied by decreased the protein levels of endoplasmic reticulum stress marker proteins, C/EBP-homologous protein and inositol-requiring kinase 1α. Taken together, these findings suggest that microglial depletion may exert beneficial effects in the acute stage of FPI.

Decreased liver stiffness by transient elastography indicates lower incidence of hepatocellular carcinoma in patients with chronic hepatitis B.

The prognositc value of dynamic liver stiffness (LS) variation on hepatocellular carcinoma (HCC) incidence in patients with chronic hepatitis B (CHB) remains to be explored. We aim to compare HCC incidence in patients with compensated CHB-related cirrhosis with increased and decreased LS after nucleos(t)ide analog (NA) regimens.A total of 168 patients with CHB-related compensated cirrhosis were divided into groups according to LS variation post to NA treatment. The laboratory results of 2 groups were reviewed and investigated. The probability of HCC development among each group was analyzed and compared.A total of 168 patients with CHB with compensated cirrhosis received NA treatment and Fibroscan. Child-Pugh score, alanine aminotransferase, total bilirubin level, status of hepatitis B e antigen, and serum hepatitis B virus DNA level were compared between groups. The cumulative probability of HCC development in patients with decreased LS was significantly lower than in patients with increased LS (P < .05). Multi-variant analysis indicated that decreased LS was significantly associated with lower probability of HCC development (hazard ratio, 0.65; 95% confidence interval range, 0.33-0.84, P < .05).Decreased LS after NA treatment indicates a lower HCC incidence in patients with CHB with compensated cirrhosis.

Mild hypothermia reduces endoplasmic reticulum stress-induced apoptosis and improves neuronal functions after severe traumatic brain injury.

BACKGROUND: Mild hypothermia is wildly used in clinical treatment of traumatic brain injury (TBI). However, the effect of mild hypothermia on endoplasmic reticulum (ER) stress-induced apoptosis after severe TBI is still unknown. METHODS: In the present study, we used BALB/c mice to investigate the efficacy of posttraumatic mild hypothermia in reducing ER stress. Severe TBI was induced by controlled cortical impact injury. Mild hypothermia treatment was performed immediately after surgery and maintained for 4 hr. The animals were euthanized at 1 and 7 days after severe TBI. The expression levels of ER stress marker proteins were evaluated using Western blot and immunofluorescence. Cell apoptosis rate was analyzed by TUNEL staining. Neuronal functions of the mice were assessed using rotarod test and Morris water maze. RESULTS: Our results revealed that mild hypothermia significantly attenuated ER stress marker proteins, including p-eIF2α/eIF2α, ATF4, CHOP and IRE-1α, and reduced apoptosis rate in the pericontusion region at 1 and 7 days after severe TBI. Interestingly, mild hypothermia also prevented the translocation of CHOP into nucleus. In addition, posttraumatic mild hypothermia significantly improved neuronal functions after severe TBI. CONCLUSIONS: Our findings illustrated that mild hypothermia could reduce ER stress-induced apoptosis and improve neuronal functions after severe traumatic brain injury.

Alteration in Long Non-Coding RNA Expression after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) causes a primary insult and initiates a secondary injury cascade. The mechanisms underlying the secondary injury are multifactorial and may include the aberrant expression of long non-coding RNA (lncRNA) post-TBI. Here, lncRNA microarray analysis was performed to profile the altered lncRNAs in the rat hippocampus after TBI. A total of 271 lncRNA probe sets and 1046 messenger RNA (mRNA) probe sets were differentially expressed after TBI. Gene ontology analysis showed that the main components of the most significantly changed categories were inflammation, DNA transcription, apoptosis, and necroptosis. Additionally, the pathway analysis and the pathway relation network revealed correlated pathways mainly involving inflammation, cell cycle, and apoptosis. A co-expression network of these aberrantly expressed lncRNAs and mRNAs was further constructed to predict the potential function of individual lncRNAs. Sub-co-expression networks were formed for the top three lncRNAs: NR_002704, ENSRNOT00000062543, and Zfas1. Thus, our study demonstrated differential expression of a series of lncRNAs in the rat hippocampus after TBI, which may be correlated with post-TBI physiological and pathological processes. The findings also may provide novel targets for further investigation of both the molecular mechanisms underlying TBI and potential therapeutic interventions.

Mild Hypothermia Promotes Pericontusion Neuronal Sprouting via Suppressing Suppressor of Cytokine Signaling 3 Expression after Moderate Traumatic Brain Injury.

Mild therapeutic hypothermia is a candidate for the treatment of traumatic brain injury (TBI). However, the role of mild hypothermia in neuronal sprouting after TBI remains obscure. We used a fluid percussion injury (FPI) model to assess the effect of mild hypothermia on pericontusion neuronal sprouting after TBI in rats. Male Sprague-Dawley rats underwent FPI or sham surgery, followed by mild hypothermia treatment (33°C) or normothermia treatment (37°C) for 3 h. All the rats were euthanized at 7 days after FPI. Neuronal sprouting that was confirmed by an increase in growth associated protein-43 (GAP-43) expression was evaluated using immunofluorescence and Western blot assays. The expression levels of several intrinsic and extrinsic sprouting-associated genes such as neurite outgrowth inhibitor A (NogoA), phosphatase and tensin homolog (PTEN), and suppressor of cytokine signaling 3 (SOCS3) were analyzed by quantitative real-time polymerase chain reaction (RT-PCR). Our results revealed that mild hypothermia significantly increased the expression level of GAP-43 and dramatically suppressed the expression level of interleukin-6 (IL-6) and SOCS3 at 7 days after FPI in the ipsilateral cortex compared with that of the normothermia TBI group. These data suggest that post-traumatic mild hypothermia promotes pericontusion neuronal sprouting after TBI. Moreover, the mechanism of hypothermia-induced neuronal sprouting might be partially associated with decreased levels of SOCS3.

The Role of Posttraumatic Hypothermia in Preventing Dendrite Degeneration and Spine Loss after Severe Traumatic Brain Injury.

Posttraumatic hypothermia prevents cell death and promotes functional outcomes after traumatic brain injury (TBI). However, little is known regarding the effect of hypothermia on dendrite degeneration and spine loss after severe TBI. In the present study, we used thy1-GFP transgenic mice to investigate the effect of hypothermia on the dendrites and spines in layer V/VI of the ipsilateral cortex after severe TBI. We found that hypothermia (33 °C) dramatically prevented dendrite degeneration and spine loss 1 and 7 days after CCI. The Morris water maze test revealed that hypothermia preserved the learning and memory functions of mice after CCI. Hypothermia significantly increased the expression of the synaptic proteins GluR1 and PSD-95 at 1 and 7 days after CCI in the ipsilateral cortex and hippocampus compared with that of the normothermia TBI group. Hypothermia also increased cortical and hippocampal BDNF levels. These results suggest that posttraumatic hypothermia is an effective method to prevent dendrite degeneration and spine loss and preserve learning and memory function after severe TBI. Increasing cortical and hippocampal BDNF levels might be the mechanism through which hypothermia prevents dendrite degeneration and spine loss and preserves learning and memory function.

Thalidomide Accelerates the Degradation of Extracellular Matrix in Rat Hepatic Cirrhosis via Down-Regulation of Transforming Growth Factor-ß1.

PURPOSE: The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and α-smooth muscle actin (α-SMA) protein in the liver, transforming growth factor ß1 (TGF-ß1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-ß1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. RESULTS: Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-ß1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. CONCLUSION: Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-ß1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.

Primary cerebellopontine angle ependymoma with spinal metastasis in an adult patient: A case report.

Subtentorial ependymoma is a common central nervous system tumor in young children, but is uncommon in adults. Ependymoma often arises from the cells lining the fourth ventricle. The present study reports a rare case of primary ependymoma that originated from the cerebellopontine angle, with local extension to the two internal auditory canals and remote spinal metastasis, in an adult male. A 50-year-old male presented with headache, tinnitus and bilateral hearing loss that had persisted for 4 months. Magnetic resonance imaging (MRI) revealed a mass in each of the cerebellopontine angles, which had spread to each internal auditory canal and wrapped the VII/VIII cranial nerve complex. A gross total resection was performed to remove the mass in the right side. Histological examination confirmed that the tumor was a World Health Organization grade II papillary ependymoma. Notably, the patient complained of urine retention post-surgery and massive occupational lesions in T3-T4 and L5-S2 were found on full spinal cord MRI. The patient then received combination therapy consisting of temozolomide, and whole-brain and spinal cord radiation. In the final follow-up examination, performed 13 months after treatment, slight shrinkage of the T3 lesion was observed, and no progression of the left cerebellopontine angle and S5-L2 lesions were identified on MRI. In summary, although this clinical entity is rare, the diagnosis of ependymoma and the possibility of spinal cord metastasis should be considered in subtentorial tumors.

ß-Catenin knockdown inhibits pituitary adenoma cell proliferation and invasion via interfering with AKT and gelatinases expression.

Pituitary adenomas are among the most prevalent forms of intrinsic brain tumors. Although most pituitary adenomas are benign, some of them may become invasive and cause significant mass effect and hormonal dysfunction. We have previously shown that ß-catenin is overexpressed in human pituitary adenomas and its level correlates to tumor grades. In the present study, we further investigated the role of ß-catenin in pituitary adenoma cell proliferation and invasion in vitro. Stable ß-catenin knockdown pituitary adenoma cell line was created by transfecting mouse growth hormone pituitary adenoma GT1.1 cells with ß-catenin shRNA plasmid. Cell proliferation and invasion were assessed using CCK-8 kit and transwell assay, respectively. Our data demonstrated that knockdown of ß-catenin with shRNA significantly inhibited the proliferation and invasion of GT1.1 cells. In ß-catenin shRNA transfected cells, the expression of AKT, STAT3, cyclin D1 and CDK4 were significantly suppressed, which accounted for the observed growth retardation following ß-catenin shRNA transfection. Moreover, ß-catenin shRNA transfection led to a drastic reduction in MMP-2/9 secretion into the conditioned media, which might be responsible for the reduced invasiveness of ß-catenin shRNA-transfected pituitary adenoma cells. These results indicate that ß-catenin may regulate the expression of AKT, STAT3, cyclin D1, CDK4 and MMP-2/9 to promote pituitary adenoma cell proliferation and invasion.