Environmental enrichment combined with fasudil promotes motor function recovery and axonal regeneration after stroke

Fasudil,a Rho-associated protein kinase(ROCK)inhibitor,has a protective effect on the central nervous system.In addition,environmental enrichment is a promising technique for inducing the recovery of motor impairments in ischemic stroke models.The present study aimed to explore whether environmental enrichment combined with fasudil can facilitate motor function recovery and induce cortical axonal regeneration after stroke.First,a mouse model of ischemic cerebral stroke was established by photochemical embolization of the left sensorimotor cortex.Fasudil solution(10 mg/kg per day)was injected intraperitoneally for 21 days after the photothrombotic stroke.An environmental enrichment intervention was performed on days 7-21 after the photothrombotic stroke.The results revealed that environmental enrichment combined with fasudil improved motor function,increased growth-associated protein 43 expression in the infarcted cerebral cortex,promoted axonal regeneration on the contralateral side,and downregulated ROCK,p-LIM domain kinase(LIMK)1,and p-cofilin expression.The combined intervention was superior to monotherapy.These findings suggest that environmental enrichment combined with fasudil treatment promotes motor recovery after stroke,at least partly by stimulating axonal regeneration.The underlying mechanism might involve ROCK/LIMK1/cofilin pathway regulation.This study was approved by the Institutional Animal Care and Use Committee of Fudan University,China(approval No.20160858A232)on February 24,2016.

Biotinylated dextran amine anterograde tracing of the canine corticospinal tract

In this study, biotinylated dextran amine （BDA） was microinjected into the left cortical motor area of the canine brain. Fluorescence microscopy results showed that a large amount of BDA-labeled pyramidal cells were visible in the left cortical motor area after injection. In the left medulla oblongata, the BDA-labeled corticospinal tract was evenly distributed, with green fluorescence that had a clear boundary with the surrounding tissue. The BDA-positive corticospinal tract entered into the right lateral funiculus of the spinal cord and descended into the posterior part of the right lateral funiculus, close to the posterior horn, from cervical to sacral segments. There was a small amount of green fluorescence in the sacral segment. The distribution of BDA labeling in the canine central nervous system was consistent with the course of the corticospinal tract. Fluorescence labeling for BDA gradually diminished with time after injection. Our findings indicate that the BDA anterograde tracing technique can be used to visualize the localization and trajectory of the corticospinal tract in the canine central nervous system.

A Simple Method Designed for the Separation of Biotinylated Ligate from Free Biotin

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Chondroitinase ABC treatment of injured spinal cord in rats Evaluation of long-term outcomes

Chondroitin sulfate proteoglycans （CSPGs） which are produced by mature oligodendrocytes and reactive astrocytes can be upregulated after spinal cord injury and contribute to regenerative failure. Chondroitinase ABC （ChABC） digests glycosaminoglycan chains on CSPGs and can thereby overcome CSPG-mediated inhibition. However, many current studies have used an incomplete spinal cord injury model, and examined results after 8-12 weeks of ChABC treatment. In this study, a complete rat spinal cord transection injury model was used to study the long-term effects of ChABC treatment by subarachnoid catheter. Pathology of spinal cord regeneration was compared with control 24 weeks following ChABC treatment using immunohistochemistry and axon tracing techniques. At 24 weeks after injury, neurofilament 200 expression was significantly greater in the ChABC treatment group compared with the transection group. In the ChABC treatment group, axonal growth was demonstrated by a large number of biotinylated dextran amine positive axons caudal to, or past, the epicenter of injury. Biotinylated dextran amine-labeled fibers were found in the proximal end of the spinal cord in the transection alone group. These results confirm that ChABC can promote axon growth, neural regeneration, and repair after spinal cord injury in rats long after the initial injury.

Biotinylated dextran amine is an ideal anterograde tracer for the corticospinal tract in a goat model of ischemic corticospinal tract injury

Existing visualized tracer studies of the corticospinal tract have been focused on rodents, which have markedly different spinal cord structures compared with humans. In this study, the segmental artery feeding the spinal cord was embolized with digital subtraction angiography to establish a goat model of ischemic spinal cord injury. Biotinylated dextran amine was injected into the motor function areas of the cortex in goats with ischemic spinal cord injury. The corticospinal tract originates from the cerebral cortex motor function area, and travels towards the lateral funiculus at the contralateral spinal dorsal horn after decussation at the pyramid. The number of corticospinal tract positive fibers was found to be gradually reduced. These findings indicate that digital subtraction angiography can be applied to a goat model of ischemic spinal cord injury. Biotinylated dextran amine visualizes the course of the goat corticospinal tract in the spinal cord, which is similar to the human spinal cord. Biotinylated dextran amine is an ideal tracer for the corticospinal tract.

Biotinylated dextran amine as a neural tracer in the rat corticospinal tract

BACKGROUND： The corticospinal tract is the core structure of cerebral control of extremity movement and plasticity, which are prerequisites for movement rehabilitation after brain injury. The measurement and assessment of plasticity changes within the corticospinal tract has become one of the key goals in this field. OBJECTIVE： To explore the effects of biotinylated dextran amine （BDA） as a neural tracer in the rat corticospinal tract and the possibilities of assessing plasticity within the corticospinal tract. DESIGN： An observational experiment. SETTING： Department of Acupuncture of Chinese Medical College, Chongqing Medical University, Department of Neurology, the Second Affiliated Hospital, Chongqing Medical University. MATERIALS： Eighteen male adult Sprague Dawley （SD） rats of clean grade, weighing 200-250 g, were provided by the experimental animal center of Chongqing Medical University. The animal procedures in this study were in accordance with the animal ethics standards. BDA was provided by Vector Laboratories Company （USA, catalogue Sp- 1140; serial number R0721 ）. METHODS. This experiment was performed in the Laboratory of Chongqing Medical University between September and December 2006. Adult SD rats were used in the experiment and 15% BDA was injected slowly with a mini-syringe through two round （3 mm diameter） holes into the left sensory and motor cortex. The center of one hole was located 3 mm anterior from the anterior fontanel and 1.5 mm left of the midline; the second hole was located 1.5 mm posterior from the anterior fontanel and 4 mm left of the midline. Three injections were made at each hole at three different levels： 1.4, 1.2, and 1 mm ventral from the surface of the flat skull. After 14 days, the brains and spinal cords were removed and frozen. Sections were cut on a cryostat and BDA transportation absorbed by axons was observed under a fluorescence microscope. MAIN OUTCOME MEASURES： Axonal absorption and transportation of BDA was observed under fluorescence microscope. RESULTS： Eighteen SD rats were enrolled in this experiment; 12 rats were included in the final analysis and six were eliminated, resulting in a dropout rate of 33% （6/18）. BDA injected into the left cortex was absorbed in the axons, and fluorescence was observed throughout the pyramidal neurons and axons of the left cerebral cortex. At 14 days after rejection, BDA was detected in the midbrain and cervical enlargement along the CST, and axonal structures and Ranvier nodes were clearly observed with 200x magnification. CONCLUSION： BDA injected into the cerebral cortex effectively traces the corticospinal tract and is biologically stable over long distance transportation. In addition, the method of BDA tracing is fairly simple to perform.

Biotinylated dextran amine tracing of nerve tracts determines regeneration of corticospinal tracts after neural stem cell transplantation

We investigated regeneration of the corticospinal tract and rearrangement of corticospinal nerves after spinal cord injury by biotinylated dextran amine （BDA） nerve tract tracing after neural stem cell transplantation. Neural stem cell transplantation increased motor function scores of rats at 3 weeks after spinal cord transection injury at the thoracic 10 segment. A proportion of BDA-labeled corticospinal tract regenerated through the spinal cord injury site at 12 weeks after transplantation. Electron microscopy revealed that the regenerated BDA-labeled nerve terminals formed new synaptic connections with neurons at the distal end of the injured site. These findings indicate that BDA nerve tract tracing effectively provides anatomic and morphological evidence of recovery after spinal cord injury.

Differential expression of autocrine motility factor receptor (AMFR) mRNA in normal and cancer cells detected by in situ hybridization

The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.

High Level Expression of HLA-A ~*0203-BSP Fusion Protein in Escherichia coli and Construction of Soluble HLA-A ~*0203 Monomer and Tetramer Loaded with Epstein-Barr Virus Peptide

Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with Epstein- Barr virus EBNA3596-604 peptide （SVRDRLARL, SVR）. Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A＊0203 fused with a BirA substrate peptide （HLA-A＊0203-BSP） was constructed and the expression conditions of the fusion protein in Escherichia coli （E. coli） were optimized. The fusion protein was highly expressed in inclusion bodies within E. coil It was then refolded in the presence of 132-microglobulin and SVR peptide to form a soluble HLA-A＊0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4：1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8^＋ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8^＋ T cells from HLA-A＊0203 subjects.

Visualizing the Transport of Porcine Reproductive and Respiratory Syndrome Virus in Live Cells by Quantum Dots-Based Single Virus Tracking

Quantum dots(QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time.The porcine reproductive and respiratory syndrome virus(PRRSV)is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide.A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies.In this study,we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells.Our results indicated that the labeling method had negligible effect on viral infectivity.We also observed that prior to the entry,PRRSV vibrated on the plasma membrane,and entered the cells via endosome mediated cell entry pathway.Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell.Our results also showed that once inside the cell,PRRSV moved along the microtubule,microfilament and vimentin cytoskeletal elements.During the transport process,virus particles also made contacts with non-muscle myosin heavy chainⅡ-A(NMHCⅡ-A),visualized as small spheres in cytoplasm.This study can facilitate the application of QDs in virus infection imaging,especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism.

Characterization of amyloid-β precursor protein intracellular domain-associated transcriptional complexes in SH-SY5Y neurocytes

Objective Alzheimer＇s disease （AD） is one of the major disorders worldwide. Recent research suggests that the amyloid-β precursor protein intracellular domain （AICD） is a potential contributor to AD development and progression. The small AICD is rapidly degraded after processing from the full-length protein. The present study aimed to apply a highly efficient biotinylation approach in vitro to study AICD-associated complexes in neurocytes. Methods By coexpressing Escherichia coli biotin ligase with biotinyl-tagged AICD in the SH-SY5Y neuronal cell line, the effects of AICD overexpression on cell proliferation and apoptosis were analyzed. Besides, AICD-associated nuclear transcriptional complexes were purified and then examined by mass spectrometry. Results Our data showed that AICD overexpression not only affected cell proliferation but also led to apoptosis in differentiated SH-SY5Y cells. Moreover, biotinylation allowed single-step purification of biotinylated AICD-associated complexes from total nuclear extract via high-affinity biotin-streptavidin binding. Following this by mass spectrometry, we identified physically associated proteins, some reported previously and other novel binding partners, CUX1 and SPT5. Conclusion Based on these results, a map of the AICD-associated nuclear interactome was depicted. Specifically, AICD can activate CUX1 transcriptional activity, which may be associated with AICD-dependent neuronal cell death. This work helps to understand the AICD-associated biological events in AD progression and provides novel insights into the development of AD.

DETECTION OF GENES FOR HEAT-STABLE ENTEROTOXIN IN ESCHERICHIA COLI BY BIOTINYLATED ST-DNA PROBES

Reference strains of enterotoxigenic Escherichia coli (ETEC), non-enterotoxigenic Escherichia coli (non-ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), and other enteropathogenic bacteria were used to prove the reliability of BIO-ST-DNA probe hybridization. In addition, 417 strains of E. coli isolated from children with diarrheal diseases in Shanxi Children's Hospital were examined for BIO-ST-DNA probe hybridization. In the test, BIO-ST-DNA hybridization was compared with suckling mouse assay in identifying ST-ETEC. The results obtained by both methods showed no significant difference. It was found that identification of ST-ETEC using hybridization is a simple, sensitive and more practical method.

Synthesis of N^(11)-anchoring biotinylated artemisinin derivatives and their preliminary biological assessment

Unique endoperoxide moiety of artemisinin and its derivatives has been considered the functionality exhibiting highly potent antimalarial and anticancer activities.To investigate the mechanisms of their biological actions,development of suitable molecular probes including biotinylated derivatives is of extreme significance.The synthesis and preliminary biological assessment of four new biotinylated artemisinin derivatives have been reported in this work.

Caimodulin Binding Proteins (CaMBPs) in lschemia and Control Mice Brains

Brain ischemia is one of the major causes of mortality and physical disability in recent years. In order to find out an effective method to cure the disease, many researches on brain ischemia mechanism have been done. Calcium is an especially interesting factor in cell injury induced by brain ischemia. Much evidence has been obtained from the studies on nerve and other tissues, implying that Ca2+ is a major initiator of ischemic cell injury. With ionsensitive microelectrodes, a marked reduction of Ca2+ in extracellular fluid was de-