钙结合蛋白CR在坐骨神经压榨模型脊髓内的表达

目的：研究大鼠坐骨神经压榨模型的钙结合蛋白Calretinin（CR）在脊髓的时空变化规律,为探讨其在神经再生中的作用提供实验依据。方法：36只SD大鼠随机分为假手术对照组和坐骨神经压榨组,实验组压榨后分别存活1d到21d,免疫组化结合图像分析技术观察CR在脊髓分布和含量的变化。结果：在对照组,CR样阳性神经元主要分布于腰髓背角Ⅰ,Ⅱ层,Ⅲ～Ⅵ层只观察到一些散在分布的CR样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见一些多极的中间型阳性神经元。坐骨神经压榨1d后,分布于腰髓背角H层内的CR样阳性神经元比对照组有轻微增加。3d后,CR样阳性神经元与对照组相比没有明显改变。7d后,CR样阳性神经元有轻微的减少;14d后,CR的表达显著下降;至21d,CR的表达有所恢复,但仍低于7d纽。脊髓后角CR免疫阳性产物灰度值测定结果显示：术后14d后角CR表达最低,与对侧和对照组相比有统计学意义（P〈0．05）。结论：坐骨神经压榨后CR表达变化呈现一定的时空模式,为进一步揭示CR在神经系统疾病中的作用提供实验依据。     

钙结合蛋白CR在坐骨神经压榨模型脊髓内的表达

目的:研究大鼠坐骨神经压榨模型的钙结合蛋白Calretinin(CR)在脊髓的时空变化规律,为探讨其在神经再生中的作用提供实验依据。方法:36只SD大鼠随机分为假手术对照组和坐骨神经压榨组,实验组压榨后分别存活1d到21d,免疫组化结合图像分析技术观察CR在脊髓分布和含量的变化。结果:在对照组,CR样阳性神经元主要分布于腰髓背角Ⅰ,Ⅱ层,Ⅲ～Ⅵ层只观察到一些散在分布的CR样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见一些多极的中间型阳性神经元。坐骨神经压榨1d后,分布于腰髓背角Ⅱ层内的CR样阳性神经元比对照组有轻微增加。3d后,CR样阳性神经元与对照组相比没有明显改变。7d后,CR样阳性神经元有轻微的减少;14d后,CR的表达显著下降;至21d,CR的表达有所恢复,但仍低于7d组。脊髓后角CR免疫阳性产物灰度值测定结果显示:术后14d后角CR表达最低,与对侧和对照组相比有统计学意义(P<0.05)。结论:坐骨神经压榨后CR表达变化呈现一定的时空模式,为进一步揭示CR在神经系统疾病中的作用提供实验依据。     

大鼠坐骨神经结扎后脊髓钙结合蛋白PV的表达变化

目的:研究大鼠坐骨神经结扎模型钙结合蛋白Parvalbumin(PV)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,采用免疫组化结合图像分析技术观察PV在脊髓的表达变化。结果:在对照组,PV免疫阳性神经元主要分布于腰髓背角Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的PV样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点PV样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角PV免疫阳性产物灰度值测定结果显示:术后14d后角PV表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后PV表达变化呈现一定的时空模式,为进一步揭示PV在神经系统疾病中的作用提供实验依据。     

Analysis of Gene Expression Following Sciatic Nerve Crush and Spinal Cord Hemisection in the Mouse by Microarray Expression Profiling

1. The responses of periphery (PNS) and central nervous systems (CNS) towards nerve injury are different: while injured mammalian periphery nerons can successfully undergo regeneration, axons in the central nervous system are usually not able to regenerate.2. In the present study, the genes which were differentially expressed in the PNS and CNS following nerve injury were identified and compared by microarray profiling techniques.3. Sciatic nerve crush and hemisection of the spinal cord of adult mice were used as the models for nerve injury in PNS and CNS respectively.4. It was found that of all the genes examined, 14% (80/588) showed changes in expression following either PNS or CNS injury, and only 3% (18/588) showed changes in both types of injuries.5. Among all the differentially expressed genes, only 8% (6/80) exhibited similar changes in gene expression (either up- or down-regulation) following injury in both PNS and CNS nerve injuries.6. Our results indicated that microarray expression profiling is an efficient and useful method to identify genes that are involved in the regeneration process following nerve injuries, and several genes which are differentially expressed in the PNS and/or CNS following nerve injuries were identified in the present study.     

Turnover of Glial Filaments in Mouse Spinal Cord

Twenty-day-old mice received a single tail vein injection of [guanido-14C]arginine. The cytoskeleton was extracted from the spinal cords at varying lengths of time thereafter. Glial fibrillary acidic protein (GFAP) formed a distinct, broad band that was widely separated from other protein bands in one-dimensional polyacrylamide gels. The purity of the GFAP band was verified by Western blot analysis of one- and two-dimensional electrophoretic patterns. In addition, enzyme-linked immunosorbent assay and quantitative Western blot analysis indicated that 95% of the total spinal cord GFAP was extracted in the cytoskeletal preparation. The specific activity of GFAP was obtained by eluting the protein from the cytoskeletal GFAP band in preparative one-dimensional gels. Specific activity reached a peak 2 h after injection with [14C]arginine. Forty percent of the incorporated radioactivity was still present in cytoskeletal GFAP at 9 weeks, indicating that a significant proportion of glial filaments turns over relatively slowly in vivo.     

大鼠坐骨神经结扎后脊髓钙结合蛋白Calbindin D-28k的表达变化

目的:研究大鼠坐骨神经结扎模型的钙结合蛋白(Calbindin D-28k,CB)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,免疫组化结合图像分析技术观察CB在脊髓的表达变化。结果:在对照组,CB阳性神经元主要分布于腰髓背角Ⅰ、Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的CB样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点CB样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角CB免疫阳性产物灰度值测定结果显示:术后14d后角CB表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后CB表达变化呈现一定的时空模式,为进一步揭示CB在神经系统疾病中的作用提供实验依据。     

Efficient Down-Regulation of Glia Maturation Factor Expression in Mouse Brain and Spinal Cord

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.     

Stage Dependent Effects of Progesterone on Motoneurons and Glial Cells of Wobbler Mouse Spinal Cord Degeneration

In the Wobbler mouse, a mutation in the Vps54 gene is accompanied by motoneuron degeneration and astrogliosis in the cervical spinal cord. Previous work has shown that these abnormalities are greatly attenuated by progesterone treatment of clinically afflicted Wobblers. However, whether progesterone is effective at all disease stages has not yet been tested. The present work used genotyped (wr/wr) Wobbler mice at three periods of the disease: early progressive (1–2 months), established (5–8 months) or late stages (12 months) and age-matched wildtype controls (NFR/NFR), half of which were implanted with a progesterone pellet (20 mg) for 18 days. In untreated Wobblers, degenerating vacuolated motoneurons were initially abundant, experienced a slight reduction at the established stage and dramatically diminished during the late period. In motoneurons, the cholinergic marker choline acetyltransferase (ChAT) was reduced at all stages of the Wobbler disease, whereas hyperexpression of the growth-associated protein (GAP43) mRNA preferentially occurred at the early progressive and established stages. Progesterone therapy significantly reduced motoneuron vacuolation, enhanced ChAT immunoreactive perikarya and reduced the hyperexpression of GAP43 during the early progressive and established stages. At all stage periods, untreated Wobblers showed high density of glial fibrillary acidic protein (GFAP)+ astrocytes and decreased number of glutamine synthase (GS) immunostained cells. Progesterone treatment down-regulated GFAP+ astrocytes and up-regulated GS+ cell number. These data reinforced the usefulness of progesterone to improve motoneuron and glial cell abnormalities of Wobbler mice and further showed that therapeutic benefit seems more effective at the early progressive and established periods, rather than on advance stages of spinal cord neurodegeneration.     

Altered Ultrasonic Vocalization and Impaired Learning and Memory in Angelman Syndrome Mouse Model with a Large Maternal Deletion from Ube3a to Gabrb3

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11–q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11–q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m−/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m−/p+), but not paternal (m+/p−), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.     

Protective Effects of Curcumin Against Paclitaxel-Induced Spinal Cord and Sciatic Nerve Injuries in Rats

Neurochemical Research - Paclitaxel (PTX) is an antineoplastic agent commonly used in the treatment of solid tumors and is known to cause dose-limiting peripheral neurotoxicity. This study was...     

Chromatographic Discrimination of Soluble Neuropathy Target Esterase Isoenzymes and Related Phenyl Valerate Esterases from Chicken Brain, Spinal Cord, and Sciatic Nerve

Abstract: Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 µ M paraoxon but sensitive to 250 µ M mipafox. Gel filtration chromatography with Sephacryl S-300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S-NTE1 and S-NTE2). The titration curve corresponding to inhibition by mipafox was studied over the 1–250 µ M range, in the presence of 40 µ M paraoxon. The data revealed that S-NTE1 and S-NTE2 have different sensitivities to mipafox with I₅₀ (30 min) values of 1.7 and 19 µ M , respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components ( V _o peak, or S-NTE1; and 100-K peak, or S-NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high-molecular-mass (>700-kDa) peak or S-NTE1 was obtained. It showed an I₅₀ of 5.2 µ M in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the V _o peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the V _o peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S-NTE2 should be a different entity than S-NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown.     

Angelman Syndrome Protein Ube3a Regulates Synaptic Growth and Endocytosis by Inhibiting BMP Signaling in Drosophila

Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the in vivo function of UBE3A in the nervous system, we generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders.     

Involvement of TRPM2 in Peripheral Nerve Injury-Induced Infiltration of Peripheral Immune Cells into the Spinal Cord in Mouse Neuropathic Pain Model

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP⁺) BM cells (TRPM2^BM+/Rec+, TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2^BM+/Rec+ mice was attenuated in TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice. The numbers of GFP⁺ BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP⁺ BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2^BM+/Rec+ mice. However, the numbers of GFP^–/Iba1⁺ resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.     

Recovery of CNS Pathway Innervating the Sciatic Nerve Following Transplantation of Human Neural Stem Cells in Rat Spinal Cord Injury

Stem cell research has been attained a greater attention in most fields of medicine due to its potential for many incurable diseases through replacing or helping the regeneration of damaged cells or tissues. Here, we demonstrated the functional recovery and structural connection of the central nervous system pathway innervating the sciatic nerve after total transection of the spinal cord followed by the transplantation of human neural stem cells (hNSC) in the injured rat spinal cord site. The limb function of hNSC-treated group recovered dramatically compared with that in the sham group by Basso–Beattie–Bresnahan (BBB) scores. Transplanted hNSC differentiated into astrocytes and neurons in the injured site. In addition, immunohistochemistry for growth-associated protein 43 showed axonal regeneration in the injured spinal cord site. The pseudorabies viral-Ba (PRV-Ba) tracing method revealed that transplanted hNSC and their differentiated neurons showed positive labeling after sciatic nerve injection. In addition, the PRV-Ba labeling was also observed in several nuclei in the brain innervating the sciatic nerve. This result implies that the rat CNS motor pathway could be reconstructed by hNSC transplantation, and it may contribute to the functional recovery of the limb.     

Assessment of Oxidative Parameters in Rat Spinal Cord After Chronic Constriction of the Sciatic Nerve

Although reactive oxygen species (ROS) are involved in neuropathic pain, the direct relationship between these species and chronic constriction of sciatic nerve (CCI) has not been studied in spinal cord. Thus, this study induced CCI in rats and these animals were sacrificed 3 and 10?days after the surgical procedure to determine the superoxide dismutase (SOD) and catalase activities, as well as ascorbic acid, hydrogen peroxide (H(2)O(2)) and lipid hydroperoxide levels in lumbosacral spinal cord. Von Frey Hair and hot plate tests were performed to assess the degree of mechanical and thermal hyperalgesia at days 0, 3 and 10. The results showed that CCI significantly induced mechanical and thermal hyperalgesia at days 3 and 10. Parallel there was increase in spinal cord lipid hydroperoxide at days 3 and 10 in rats submitted to CCI. In Sham rats a significant increase in this parameter occurred at day 10. H(2)O(2) decreased at day 10 only in CCI group. SOD activity was decreased in Sham and CCI groups at day 3, while catalase activity was increased in CCI rats at days 3 and 10. Ascorbic acid levels were reduced only in CCI rats at day 3. Although the role of such changes is unclear, many were not specific to neuropathic pain and the differences could be related to different degrees of central sensitization in Sham and CCI rats.     

Time Course of Immediate Early Gene Protein Expression in the Spinal Cord following Conditioning Stimulation of the Sciatic Nerve in Rats

Long-term potentiation induced by conditioning electrical stimulation of afferent fibers is a widely studied form of synaptic plasticity in the brain and the spinal cord. In the spinal cord dorsal horn, long-term potentiation is induced by a series of high-frequency trains applied to primary afferent fibers. Conditioning stimulation (CS) of sciatic nerve primary afferent fibers also induces expression of immediate early gene proteins in the lumbar spinal cord. However, the time course of immediate early gene expression and the rostral-caudal distribution of expression in the spinal cord have not been systematically studied. Here, we examined the effects of sciatic nerve conditioning stimulation (10 stimulus trains, 0.5 ms stimuli, 7.2 mA, 100 Hz, train duration 2 s, 8 s intervals between trains) on cellular expression of immediate early genes, Arc, c-Fos and Zif268, in anesthetized rats. Immunohistochemical analysis was performed on sagittal sections obtained from Th13- L5 segments of the spinal cord at 1, 2, 3, 6 and 12 h post-CS. Strikingly, all immediate early genes exhibited a monophasic increase in expression with peak increases detected in dorsal horn neurons at 2 hours post-CS. Regional analysis showed peak increases at the location between the L3 and L4 spinal segments. Both Arc, c-Fos and Zif268 remained significantly elevated at 2 hours, followed by a sharp decrease in immediate early gene expression between 2 and 3 hours post-CS. Colocalization analysis performed at 2 hours post-CS showed that all c-Fos and Zif268 neurons were positive for Arc, while 30% and 43% of Arc positive neurons were positive for c-Fos and Zif268, respectively. The present study identifies the spinal cord level and time course of immediate early gene (IEGP) expression of relevance for analysis of IEGPs function in neuronal plasticity and nociception.