The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Baseline Hemodynamic Impairment and Revascularization Outcome in Newly Diagnosed Adult Moyamoya Disease Determined by Pseudocontinuous Arterial Spin Labeling.

OBJECTIVE: The study aimed to investigate the hemodynamic features and independent predictors of neoangiogenesis after revascularization in moyamoya disease (MMD) by pseudocontinuous arterial spin labeling magnetic resonance imaging (pCASL MRI). METHODS: Thirty-nine MMD patients were categorized into infarction group, hemorrhagic group, and atypical group. All patients underwent combined bypass surgery and pCASL MRI with postlabeling delays (PLD) of 1525 ms and 2525 ms. Absolute CBFMCA (cerebral blood flow in middle cerebral artery territory), relative CBFMCA (CBFMCA 2525 ms/CBFMCA 1525 ms), and spatial coefficient of variation of MCA (CoVMCA) were analyzed. Relationships between CBFMCA and the following clinical parameters were assessed: Suzuki stage, modified Rankin scale (mRS), cerebrovascular accident lesion score, and deep medullary veins score. Potential predictors for favorable neoangiogenesis and hemodynamic changes were explored as well. RESULTS: Preoperative CBFMCA differed among MMD patients with variable clinical presentations, Matsushima stages, modified Rankin Scale scores, CVA scores, and deep medullary vein scores. After bypass surgery, mean CBFMCA increased significantly in the infarction group (P = 0.027) and decreased in the hemorrhagic group (P = 0.043), while spatial CoVMCA was observed to decline in all groups. Higher preoperative relative CBFMCA and spatial CoVMCA were independent predictors for robust neoangiogenesis after bypass. The cutoff value of 0.330 of spatial CoVMCA at long PLD yielded the best sensitivity at 82.1% and specificity at 81.8%. Furthermore, both preoperative relative CBFMCA and spatial CoVMCA showed mild positive correlations with ΔmRS in MMD patients. CONCLUSIONS: pCASL-MRI with multiple PLDs could reflect preoperative hemodynamic impairment and predict the neoangiogenesis after combined bypass surgery in moyamoya patients.

Caveolin-1 Promoted Collateral Vessel Formation in Patients With Moyamoya Disease.

Background: Caveolin-1 (Cav-1) plays pivotal roles in the endothelial function and angiogenesis postischemia. Moyamoya disease (MMD) is characterized by progressive artery stenosis with unknown etiology. We aim to determine whether serum Cav-1 levels of patients with MMD were associated with collateral vessel formation after bypass surgery. Methods: We studied serum Cav-1 levels of 130 patients with MMD (16 with RNF213 p.R4810K mutation and 114 without RNF213 p.R4810K mutation), 15 patients with acute stroke, and 33 healthy controls. Cerebral perfusion and collateral circulation were evaluated preoperation and at 6 months after operation using pseudocontinuous arterial spin labeling MRI (pCASL-MRI) and digital subtraction angiography (DSA), respectively. Endothelial expression of Cav-1 was verified in the superficial temporal artery (STA) wall of patients with MMD by immunofluorescence double staining. We also investigated whether overexpression of Cav-1 affects cell migration and tube formation using human microvascular endothelial cells (HMECs). Results: The serum Cav-1 level of patients with MMD intermediated between the stroke group and healthy controls and it was enhanced after the bypass surgery (681.87 ± 311.63 vs. 832.91 ± 464.41 pg/ml, p = 0.049). By 6 months after bypass surgery, patients with MMD with better collateral compensation manifested higher postoperative/preoperative Cav-1 ratio (rCav-1) than bad compensation patients. Consistently, cerebral blood flow (CBF) determined by pCASL-MRI (nCBFMCA ratio) was positively in line with rCav-1 ratio (r = 0.8615, p < 0.0001). Cav-1 was expressed in the endothelial cells of the STA vessels of patients with MMD. Overexpression of Cav-1 by plasmid transfection in HMECs promoted tube formation and cell migration. Conclusion: This study indicated that Cav-1 may be a potential driver to promote angiogenesis and collateral formation after bypass surgery in patients with MMD, providing a better understanding of MMD pathophysiology and potential non-surgical targets of MMD.

LncRNA-mRNA Co-expression Profiles Relative to Vascular Remodeling in Moyamoya Patients Without RNF213 Mutation.

OBJECTIVE: Moyamoya disease (MMD) is an idiopathic cerebrovascular disease with unknown etiology. Long noncoding RNA (lncRNA) and messenger RNA (mRNA) profiles in MMD remain unknown. In this current study, we aim to investigate lncRNA-mRNA co-expression pattern and their biological functions in superficial temporal artery (STA) of MMD. METHODS: STA of 3 MMD patients without RNF213 mutation and 3 age-matched controls were obtained for transcriptomic RNA sequencing. Bioinformatics analysis was performed to investigate their molecular functions and interactions. Then, differentially expressed genes relative to vascular remodeling were further validated by quantitative real-time polymerase chain reaction and immunofluorescence. WNT5A functions were tested by tube formation assay and wound scratching assay in human microvascular endothelial cells (HMECs). RESULTS: We detected 6235 different lncRNAs and 2065 different mRNAs from the RNA-sequencing between MMD patients and controls (P < 0.05; fold change >2.0). Gene ontology showed that altered mRNAs were enriched for endothelial cell morphogenesis and positive regulation of angiogenesis, which were closely related with vascular remodeling. We then searched 76 altered genes related with vascular remodeling and applied Kyoto Encyclopedia of Genes and Genomes analysis. Integrated analysis of lncRNA-TF-mRNA co-expression networks and gene verifications indicated that molecular including WNT5A, TEK, and GATA2 may contribute to the vascular malformation of MMD. Overexpression of WNT5A in HMECs promoted tube formation and cell migration. CONCLUSIONS: In MMD patients, genes related to vascular remodeling including WNT5A and their regulators were aberrantly disrupted. These results will help elucidate the complicated pathogenic mechanism of MMD and develop potential therapeutic targets facilitating MMD angiogenesis in the future.

Effect of Hormone Levels and Aging on Cognitive Function of Patients with Pituitary Adenomas Prior to Medical Treatment.

BACKGROUND: Cognitive impairments have been reported in patients with pituitary adenomas (PAs). The aim of this research was to demonstrate the effects of hormones and age on cognitive decline in patients with PAs. METHODS: A total of 64 patients with PA and 69 healthy control subjects (HCs) were recruited for this study. Both PAs and HCs were divided into a younger group (<50 years of age) and an older group (≥50 years of age). Neurocognitive domains were assessed using the Wechsler Adult Intelligence Scale-Chinese Revision (WAIS-RC) and Wechsler Memory Scale-Chinese Revision (WMS-RC) tests. Furthermore, we also investigated the relationship between cognitive domains and tumor volume, and the hormone levels and age of patients with PA. RESULTS: Several of the cognitive impairments found on the WAIS-RC and WMS-RC tests were more frequently observed in untreated patients with PA. Importantly, no significant correlations were found between cognitive domains and tumor volume after controlling age, sex, and educational levels. Furthermore, several significant correlations were found between cognitive domains and hormone levels, such as free thyroxine and adrenocorticotropic hormone, after controlling age, sex, and educational levels. Finally, the age of the patients was found to correlate with a decrease in memory after controlling sex and educational levels. CONCLUSIONS: Our findings demonstrate a significant decline in the cognitive performance of patients with PA prior to medical treatment, especially in older patients, which suggests that hormones and age have the ability to interact and aggravate cognitive decline in patients with PA.

Clinical and angiographic outcomes after combined direct and indirect bypass in adult patients with moyamoya disease: A retrospective study of 76 procedures.

The present retrospective study was performed to evaluate the clinical outcome, as well as post-operative collateral formation and revascularization patterns in combined bypass. Surgical revascularization has been the mainstay of treatment for moyamoya patients. A total of 76 hemispheres from 64 moyamoya patients undergoing combined superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis and encephalo-duro-myo-synangiosis (EDMS) were retrospectively reviewed. Computed tomography perfusion and DSA were routinely performed. Modified Rankin scale (mRS) scoring and Kaplan-Meier analysis were performed, and post-operative collateral grading on digital subtraction angiography (DSA), reconstruction patterns and vessel diameter were measured to evaluate the revascularization area and vascular compensatory effect. During the follow-up period, the chief complaint was partially alleviated in 57/64 patients (89.1%). In all patients, mRS was significantly decreased after the operation. Good revascularization was established in most patients at 6 months after the surgery: 92.1% of sides were scored as 2 and 77.6% were scored as 3 by post-operative collateral grading on DSA. Dominant collateral revascularization originating from EDMS was more frequent than that from STA-MCA anastomosis (31 vs. 19 hemispheres) at 6-month follow-up. Morphologically, the calibers of the STA main trunk (2.60±0.65 mm) increased profoundly at the first 10 post-operative days (3.32±1.05 mm) and shrank back to pre-operative status at 6 months (2.20±1.01 mm) and 12 months (2.36±0.73 mm) according to DSA examination. Middle meningeal artery (MMA) and deep temporal artery (DTA) demonstrated continuous augmentation during the follow-up time (from 1.30±0.46 to 1.87±0.69 mm for MMA and 1.11±0.25 to 2.11±1.16 mm for DTA). These results strongly suggested that combined STA-MCA bypass and EDMS provided efficient revascularization and reduced acute cerebral events. The direct STA-MCA bypass provided early augmentation of cerebral perfusion, whereas the indirect EDMS provided a more durable long-term revascularization, indicating a complementary association between the two revascularization methods.

Prognostic value of plasma galectin-3 levels after aneurysmal subarachnoid hemorrhage.

BACKGROUND: Inflammatory responses are correlated with secondary brain injury after aneurysmal subarachnoid hemorrhage (aSAH). Galectin-3 (Gal-3) is a novel biomarker reflecting inflammation status, and its elevated circulating levels are associated with poor prognosis of some inflammatory diseases. The aim of this study was to evaluate the relationship between Gal-3 plasma levels and prognosis in a group of aSAH patients. MATERIALS AND METHODS: We assessed plasma Gal-3 levels in 120 patients and 120 healthy individuals. 6-month clinical outcomes included mortality and unfavorable outcome (Glasgow Outcome Scale score of 1-3). Associations of plasma Gal-3 levels with clinical outcomes were investigated using multivariate analysis. RESULTS: Patients showed significantly higher Gal-3 levels as compared to controls. Circulating Gal-3 was significantly and independently associated with 6-month clinical outcomes in the logistic regression analysis. Moreover, we observed a significant correlation between circulating Gal-3 and World Federation of Neurological Surgeons scores and modified Fisher scores. Furthermore, Gal-3 possessed high area under receiver operating characteristic curve for prognostic assessment. CONCLUSION: Our findings indicate the associations between Gal-3 levels and the severity and poor prognosis following aSAH. This suggests the possible role of Gal-3 in the prognostic prediction after aSAH.

Early expression of serum neutrophil gelatinase-associated lipocalin (NGAL) is associated with neurological severity immediately after traumatic brain injury.

PURPOSE: Intracranial bleeding and inflammatory reactions are common consequences of traumatic brain injury (TBI). Neutrophil gelatinase-associated lipocalin (NGAL), an iron-handling and acute phase protein, may participate in the pathogenesis of TBI. Therefore, we hypothesize that NGAL may be of high diagnostic and therapeutic relevance in the prognosis of TBI. METHODS: 74 subjects were recruited in this study. 30 TBI patients receiving emergent operation were designated as severe TBI group (sTBI), 24 TBI patients receiving conservative treatment as mild TBI group (mTBI), while 20 age-matched healthy volunteers as healthy controls (CNT). We detected the expression and localization of NGAL in brain tissue by Q-PCR, western blotting, and immunofluorescence. Serum NGAL was evaluated by ELISA. Clinical manifestations and outcomes were measured by Glasgow Score (GCS), Trauma score (TS), Revised Trauma score (RTS), APACHEII, Sequential Organ Failure Assessment (SOFA), and Abbreviated Injury Scale (AIS) 85 at admission. Glasgow outcome score (GOS) and Karnofsky Performance Score (KPS) were documented at discharge. RESULTS: NGAL mRNA and protein levels in brain tissue from sTBI group were profoundly higher than control tissue. Double labeled NGAL with GFAP, NeuN and Iba-1 by immunofluroscence demonstrated that increased NGAL was mainly located in neurons. Compared to CNT and mTBI groups, serum NGAL were significantly increased in sTBI group (sTBI: 532.6±71.77ng/ml vs. mTBI: 230.5±29.59ng/ml, p<0.01; sTBI: 532.6±71.77ng/ml vs. CNT 178.0±19.83ng/ml, p<0.01). Linear regression analysis indicated that there was a negative correlation between the NGAL levels and GCS (r=-0.427, p=0.033), TS (r=-0.429, p=0.032), RTS (r=-0.413, p=0.040) in sTBI group. However, NGAL levels did not correlated with GOS and KPS scores. The NAGL cut-off value of 244.13ng/ml yielded good sensitivity at 84% and specificity at 78.9%. CONCLUSION: NGAL may be a novel biomarker reflecting TBI severity, which increased obviously and negatively correlated with GCS, TS, and RTS scores; additionally, this characteristic of NGAL may be helpful in guiding clinical TBI therapeutic strategies.

Inhibition of the Rac1-WAVE2-Arp2/3 signaling pathway promotes radiosensitivity via downregulation of cofilin-1 in U251 human glioma cells.

The Ras-related C3 botulinum toxin substrate 1 (Rac1)-WASP-family verprolin-homologous protein-2 (WAVE2)-actin-related protein 2/3 (Arp2/3) signaling pathway has been identified to be involved in cell migration and invasion in various types of cancer cell. Cofilin­1 (CFL­1), which is regulated by the Rac1­WAVE2­Arp2/3 signaling pathway, may promote radioresistance in glioma. Therefore, the present study aimed to investigate the potential role of the Rac1­WAVE2­Arp2/3 signaling pathway in radioresistance in U251 human glioma cells and elucidate its affect on CFL­1 expression. Western blot analysis was performed to evaluate the protein expression of CFL­1. In the present study, Rac1 was inhibited by NSC 23766, WAVE2 was inhibited by transfection with short hairpin (sh)RNA­WAVE2 using Lipofectamine™ 2000 and Arp2/3 was inhibited by CK­666. Cell viability was measured using the 3­(4,5­dimethylthiazol­2­yl)-2,5­diphenyltetrazolium bromide assay, the cell migration ability was examined by a wound­healing assay, and the cell invasion ability was assessed using a Transwell culture chamber system. The results showed that inhibition of the Rac1­WAVE2­Arp2/3 signaling pathway using NSC 23766, shRNA­WAVE2 or CK­666 reduced the cell viability, migration and invasion abilities in U251 human glioma cells, concordant with a reduced expression of CFL­1. Furthermore, the expression of CFL­1 was significantly increased in radioresistant U251 glioma cells when compared with normal U251 human glioma cells. These findings indicate that inhibition of the Rac1­WAVE2­Arp2/3 signaling pathway may promote radiosensitivity, which may partially result from the downregulation of CFL­1 in U251 human glioma cells.

Deferoxamine Attenuated the Upregulation of Lipocalin-2 Induced by Traumatic Brain Injury in Rats.

Intracranial hemorrhage is one of the most common consequences of traumatic brain injury (TBI). The release of iron from the breakdown of hemoglobin during intracerebral hematoma resolution results in an increase in perihematomal non-heme iron. Lipocalin 2 (LCN-2) is a siderophore-binding protein that mediates transferrin-independent iron transport. This study examined the effects of TBI (lateral fluid percussion) on LCN-2 expression in Sprague-Dawley rats. LCN-2 protein levels were markedly increased in the ipsilateral cortex and hippocampus after TBI, with the highest level at day 1. Most LCN-2-positive cells appeared to be astrocytes. Treatment with an iron chelator, deferoxamine (100 mg/kg, intramuscularly), attenuated the TBI-induced upregulation of LCN-2. In summary, TBI resulted in upregulation of LCN-2 and deferoxamine reduced TBI-induced LCN-2 increase, suggesting LCN-2 may have a role in iron-trafficking after TBI.

Downregulation of phosphoglycerate kinase 1 by shRNA sensitizes U251 xenografts to radiotherapy.

Phosphoglycerate kinase 1 (PGK1) has been demonstrated to be involved in radioresistance. The present study was designed to investigate the effect of PGK1 on the radioresistance in vivo. U251 glioma cells were transfected with the short hairpin RNA (shRNA)-PGK1 and pcDNA3.1-PGK1 using Lipofectamine 2000. The radiosensitivity of U251 xenografts was observed by tumor growth curve following radiotherapy. Quantitative PCR, western blot analysis and immunohistochemistry were performed to evaluate PGK1 expression in the xenografts from the different tumor models. The expression of PGK1 was maximally inhibited in response to shRNA4 at 24 h after the transfection in vitro. Tumor growth of the U251 xenografts was significantly inhibited following treatment with shRNA-PGK1 and radiotherapy. The expression of PGK1 in vivo at the mRNA and protein levels was downregulated by the treatment of shRNA1 when compared to levels following treatment with shNC and PBS after radiotherapy. The results showed that suppression of PGK1 enhanced the radiosensitivity of U251 xenografts and suggest that PGK1 may serve as a useful target in the treatment of radioresistant glioma.

Deferoxamine attenuates acute hydrocephalus after traumatic brain injury in rats.

Acute post-traumatic ventricular dilation and hydrocephalus are relatively frequent consequences of traumatic brain injury (TBI). Several recent studies have indicated that high iron levels in brain may relate to hydrocephalus development after intracranial hemorrhage. However, the role of iron in the development of post-traumatic hydrocephalus is still unclear. This study was to determine whether or not iron has a role in hydrocephalus development after TBI. TBI was induced by lateral fluid-percussion in male Sprague-Dawley rats. Some rats had intraventricular injection of iron. Acute hydrocephalus was measured by magnetic resonance T2-weighted imaging and brain hemorrhage was determined by T2* gradient-echo sequence imaging and brain hemoglobin levels. The effect of deferoxamine on TBI-induced hydrocephalus was examined. TBI resulted in acute hydrocephalus at 24 h (lateral ventricle volume: 24.1 ± 3.0 vs. 9.9 ± 0.2 mm(3) in sham group). Intraventricular injection of iron also caused hydrocephalus (25.7 ± 3.4 vs. 9.0 ± 0.6 mm(3) in saline group). Deferoxamine treatment attenuated TBI-induced hydrocephalus and heme oxygenase-1 upregulation. In conclusion, iron may contribute to acute hydrocephalus after TBI.

Elevated cerebral cortical CD24 levels in patients and mice with traumatic brain injury: a potential negative role in nuclear factor κb/inflammatory factor pathway.

Increasing evidence indicates that sterile inflammatory response contributes to secondary brain injury following traumatic brain injury (TBI). However, the specific mechanisms remain largely unknown, as is whether CD24, known as an important regulator in the non-infectious inflammatory response, plays a role in secondary brain injury after TBI. Here, the expression of CD24 was detected in samples from patients with TBI by quantitative real-time polymerase chain reaction (PCR), western blotting, immunohistochemistry and immunofluorescence. RNA interference was used to investigate the effects of CD24 on inflammatory response in a mouse model of TBI. Nuclear factor kappa B (NF-κB) DNA-binding activity was measured by electrophoretic mobility shift assay, and the levels of downstream pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and Interleukin 1ß (IL-1ß) were detected by real-time PCR. The results indicated that both the mRNA and protein levels of CD24 were markedly elevated after TBI in humans and mice, showing a time-dependent expression. The expression of CD24 could be observed in neurons, astrocytes and microglia in both humans and mice. Meanwhile, downregulation of CD24 significantly induced an increase of NF-κB DNA-binding activity and mRNA levels of TNF-α and IL-1ß. These findings indicated that CD24 expression could negatively regulate the NF-κB/inflammatory factor pathway after experimental TBI in mice, thus providing a novel target for therapeutic intervention of TBI.

Phosphoglycerate kinase 1 promotes radioresistance in U251 human glioma cells.

Phosphoglycerate kinase 1 (PGK1) has been found to be increased in radioresistant astrocytomas. The present study was designed to investigate the potential role of PGK1 in the radioresistance in U251 human cells. Quantitative PCR and western blot analysis were performed to evaluate PGK1 expression for mRNA levels and protein levels, respectively. The short hairpin RNA (shRNA)-PGK1 and the high expression plasmids were transfected to radioresistant U251 cells (RR-U251 cells) or normal U251 cells using lipofectamine™ 2000. The cell viability was determined by MTT assay. The wound-healing assay (WHA) was used to evaluate cell migration ability. Cell invasion abilities were examined using a Transwell culture chamber system. Our results showed that the expression of PGK1 was significantly increased in RR-U251 cells compared to normal U251 cells. Following irradiation, the cell viability as well as the migration and invasion ability were significantly higher in RR-U251 cells compared with normal U251 cells. Downregulating PGK1 using shRNA induced a significantly downregulated cell viability and decreased migration and invasion ability, and overexpression of PGK1 contributed to upregulated cell viability and increased migration and invasion ability, both in RR-U251 cells and normal U251 cells. These findings suggest that PGK1 could promote radioresistance in U251 human cells.

Drug-induced hypothermia in stroke models: does it always protect?

Ischemic stroke is a common neurological disorder lacking a cure. Recent studies show that therapeutic hypothermia is a promising neuroprotective strategy against ischemic brain injury. Several methods to induce therapeutic hypothermia have been established; however, most of them are not clinically feasible for stroke patients. Therefore, pharmacological cooling is drawing increasing attention as a neuroprotective alternative worthy of further clinical development. We begin this review with a brief introduction to the commonly used methods for inducing hypothermia; we then focus on the hypothermic effects of eight classes of hypothermia-inducing drugs: the cannabinoids, opioid receptor activators, transient receptor potential vanilloid, neurotensins, thyroxine derivatives, dopamine receptor activators, hypothermia-inducing gases, adenosine, and adenine nucleotides. Their neuroprotective effects as well as the complications associated with their use are both considered. This article provides guidance for future clinical trials and animal studies on pharmacological cooling in the setting of acute stroke.

Suppression of JAK2/STAT3 signaling reduces end-to-end arterial anastomosis induced cell proliferation in common carotid arteries of rats.

BACKGROUND: JAK2/STAT3 pathway was reported to play an essential role in the neointima formation after vascular intima injury. However, little is known regarding this pathway to the whole layer injury after end-to-end arterial anastomosis (AA). Here, we investigated the role of JAK2/STAT3 pathway in common carotid arterial (CCA) anastomosis-induced cell proliferation, phenotypic change of vascular smooth muscle cells (VSMCs) and re-endothelialization. METHODS: CCAs of adult male Wistar rats were resected at 3, 7, 14, and 30 days after end-to-end CCA anastomosis. Activation of JAK2/STAT3 pathway was detected by Western blotting and Immunofluorescence, and expression of proliferating cell nuclear antigen (PCNA) was detected by Q-PCR and Western blotting. Under the treatment with AG490 (a JAK2 inhibitor), protein levels of JAK2, STAT3 and PCNA, morphological changes of artery, phenotypic change of VSMCs, and re-endothelialization were measured by Western blotting, H&E, Q-PCR, and Evans blue staining respectively. RESULTS: The protein levels of p-JAK2, p-STAT3, and PCNA were up-regulated, peaked on the 7(th) day in the vessel wall after AA. AG490 down-regulated the levels of p-JAK2, p-STAT3, and PCNA on the 7(th)-day-group, resulting in reduced vessel wall proliferation on the 7(th) and 14(th) day after AA. Besides, AG490 switched the phenotypic change of VSMCs after AA representing inhibited mRNA levels of synthetic phase markers (osteopoitin and SMemb) and up-regulated contractile phase markers (ASMA, SM2 and SM22α). Furthermore, AG490 did not affect the re-endothelialization process on all indicated time points after AA (the 3(rd), 7(th), 14(th), and 30(th) day). CONCLUSION: Our study indicated that JAK2/STAT3 signaling pathway played an important role on cell proliferation of the injured vessel wall, and probably a promising target for the exploration of drugs increasing the patency or reducing the vascular narrowness after AA.

Recombinant high-mobility group box 1 protein (HMGB-1) promotes myeloid differentiation primary response protein 88 (Myd88) upregulation in mouse primary cortical neurons.

Myeloid differentiation primary response protein 88 (Myd88) is a vital factor for inflammation and immunity, and high-mobility group box 1 protein (HMGB-1) can be released from neurons after injury and may contribute to the initial stages of inflammatory response. Therefore, the current study was intended to investigate the expression of Myd88 in cultured neurons following recombinant HMGB-1 (rHMGB-1) addition and to clarify the potential role of Myd88 after neuron injury in vitro. The cultured neurons were randomly divided into six groups: control group and rHMGB-1 groups at hours 1, 6, 12, 24, and 48. The cultured neurons in rHMGB-1 groups were subjected to rHMGB-1 addition. The expression of Myd88 was assessed by quantitative real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence, and nuclear factor kappa B (NF-κB) DNA-binding activity was detected by electrophoretic mobility shift assay, and the levels of tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß) were measured by quantitative real-time PCR. The elevated mRNA and protein levels of Myd88, peaking at 24 h, were detected after rHMGB-1 addition. NF-κB, TNF-α, and IL-1ß also ascended significantly after rHMGB-1 addition. Interestingly, Myd88 increasingly expressed in a parallel time course to the upregulation of NF-κB, TNF-α, and IL-1ß. These findings indicated a possible role of Myd88 in the inflammatory response after neuron injury, and might provide an attractive therapeutic approach of targeting the Myd88 cascade to achieve better outcomes for patients with central nervous system injury.

The potential role of JAK2/STAT3 pathway on the anti-apoptotic effect of recombinant human erythropoietin (rhEPO) after experimental traumatic brain injury of rats.

Previous studies indicate that administration of recombinant human erythropoietin (rhEPO) protects cortical neurons following traumatic brain injury (TBI). The mechanisms of rhEPO's neuroprotection are complex and interacting, including anti-apoptosis. Here we aim to demonstrate the role of janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway on the anti-apoptotic effect of rhEPO in Feeney free falling TBI model. Activation of JAK2/STAT3 in pericontusional cortex was analyzed among rats in Sham, TBI, TBI+rhEPO, TBI+rhEPO+AG490 groups (rhEPO: 5000 U/kg day; JAK2 inhibitor AG490: 5 mg/kg day, intraperitoneal) through Western blotting, electrophoretic mobility shift assay. Bcl-2 and Bcl-xl expression (Q-PCR, Western blotting) and cell apoptosis (TUNEL) in pericontusional cortex were also detected in each group. As a result, we found that TBI could activate JAK2 and STAT3, and increase cell apoptosis in pericontusional cortex. RhEPO enhanced the expression of p-JAK2 and p-STAT3, up-regulated the mRNA and protein levels of Bcl-2 and Bcl-xl, followed by increased cell survival. Moreover, AG490 attenuated rhEPO's neuroprotection by down-regulating rhEPO-induced activation of JAK2/STAT3, and inhibiting Bcl-2 and Bcl-xl. These results suggest the essential role of JAK2/STAT3 pathway on the anti-apoptotic benefit of post-TBI rhEPO treatment.

Beneficial effects of ethyl pyruvate through inhibiting high-mobility group box 1 expression and TLR4/NF-κB pathway after traumatic brain injury in the rat.

Ethyl pyruvate (EP) has demonstrated neuroprotective effects against acute brain injury through its anti-inflammatory action. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from dying cells. This study was designed to investigate the protective effects of EP against secondary brain injury in rats after Traumatic Brain Injury (TBI). Adult male rats were randomly divided into three groups: (1) Sham + vehicle group, (2) TBI + vehicle group, and (3) TBI + EP group (n = 30 per group). Right parietal cortical contusion was made by using a weight-dropping TBI method. In TBI + EP group, EP was administered intraperitoneally at a dosage of 75 mg/kg at 5 min, 1 and 6 h after TBI. Brain samples were harvested at 24 h after TBI. We found that EP treatment markedly inhibited the expressions of HMGB1 and TLR4, NF-κB DNA binding activity and inflammatory mediators, such as IL-1ß, TNF-α and IL-6. Also, EP treatment significantly ameliorated beam walking performance, brain edema, and cortical apoptotic cell death. These results suggest that the protective effects of EP may be mediated by the reduction of HMGB1/TLR4/NF-κB-mediated inflammatory response in the injured rat brain.