大鼠脊髓压迫损伤后COX-2基因和蛋白的表达

目的观察大鼠脊髓压迫损伤后COX-2mRNA和蛋白在脊髓组织内表达的时间特征,以及COX-2的空间分布特点。方法以压迫装置致脊髓损伤后,在伤后不同时间点(30min、3h、6h、24h、72h、1w)分别应用RT-PCR、Western blotting、免疫组化技术检测COX-2mRNA和蛋白表达和分布。结果RT-PCR和Western blotting显示,COX-2mRNA和蛋白伤后30min表达开始增加,于伤后6h达到峰值,伤后1w表达接近基础水平。免疫组化提示,COX-2蛋白在假手术组表达仅见于脊髓血管内皮细胞,伤后COX-2蛋白表达见于损伤区附近脊髓灰质内的神经元和血管内皮细胞,损伤中心部位COX-2免疫阳性细胞少见。结论脊髓压迫损伤早期可快速诱导COX-2基因的表达上调和蛋白的合成,伤后COX-2蛋白分布发生变化,主要位于脊髓神经元和血管内皮细胞。     

Arterial Blood Supply to the Spinal Cord in Animal Models of Spinal Cord Injury. A Review

Animal models are used to examine the results of experimental spinal cord injury. Alterations in spinal cord blood supply caused by complex spinal cord injuries contribute significantly to the diversity and severity of the spinal cord damage, particularly ischemic changes. However, the literature has not completely clarified our knowledge of anatomy of the complex three‐dimensional arterial system of the spinal cord in experimental animals, which can impede the translation of experimental results to human clinical applications. As the literary sources dealing with the spinal cord arterial blood supply in experimental animals are limited and scattered, the authors performed a review of the anatomy of the arterial blood supply to the spinal cord in several experimental animals, including pigs, dogs, cats, rabbits, guinea pigs, rats, and mice and created a coherent format discussing the interspecies differences. This provides researchers with a valuable tool for the selection of the most suitable animal model for their experiments in the study of spinal cord ischemia and provides clinicians with a basis for the appropriate translation of research work to their clinical applications. Anat Rec, 300:2091–2106, 2017. © 2017 Wiley Periodicals, Inc.     

New Minimally Invasive Model of Spinal Cord Ischemia in Rats

We developed a new minimally invasive model of spinal cord ischemia in rats: intravascular occlusion of the abdominal aorta and its branches. This model can be used on small laboratory animals and allows qualitatively and quantitatively evaluating the morphofunctional state of the nervous system during spinal cord ischemia by clinical manifestations and histological changes. Selective intravascular occlusion determines minimal invasiveness and adequacy of the proposed model to in vivo pathological processes. This model of spinal cord ischemia can be used in experimental pharmacology for evaluation of neuroprotective activity of various drugs and bioactive substances.     

Combination Therapy by Tissue-Specific Suicide Gene and Bevacizumab in Intramedullary Spinal Cord Tumor

PurposeMalignant gliomas are aggressive spinal cord tumors. In this study, we hypothesized that combination therapy using an anti-angiogenic agent, bevacizumab, and hypoxia-inducible glioblastoma-specific suicide gene could reduce tumor growth.Materials and MethodsIn the present study, we evaluated the effect of combination therapy using bevacizumab and pEpo-NI2-SV-TK in reducing the proliferation of C6 cells and tumor growth in the spinal cord. Spinal cord tumor was generated by the injection of C6 cells into the T5 level of the spinal cord. Complexes of branched polyethylenimine (bPEI)/pEpo-NI2-SV-TK were injected into the spinal cord tumor. Bevacizumab was then administered by an intraperitoneal injection at a dose of 7 mg/kg. The anti-cancer effects of combination therapy were analyzed by histological analyses and magnetic resonance imaging (MRI). The Basso, Beattie and Bresnahan scale scores for all of the treatment groups were recorded every other day for 15 days to assess the rat hind-limb strength.ResultsThe complexes of bPEI/pEpo-NI2-SV-TK inhibited the viability of C6 cells in the hypoxia condition at 5 days after treatment with ganciclovir. Bevacizumab was decreased in the cell viability of human umbilical vein endothelial cells. Combination therapy reduced the tumor size by histological analyses and MRI. The combination therapy group showed improved hind-limb function compared to the other groups that were administered pEpo-NI2-SV-TK alone or bevacizumab alone.ConclusionThis study suggests that combination therapy using bevacizumab with the pEpo-NI2-SV-TK therapeutic gene could be useful for increasing its therapeutic benefits for intramedullary spinal cord tumors.     

人胎儿脊髓星形胶质细胞形态及分布

目的　观察星形胶质细胞在胎儿脊髓不同发育阶段形态和分布的变化。方法　在引产 19例胎儿脊髓 ,多克隆抗体GFAP ,应用SP免疫组织化学染色和图像分析。结果　星形胶质细胞在中央管、毛细血管周围密度大 ,染色深 ,环绕血管呈辐射状排列。 16W时已有少突起GFAP阳性细胞出现 ,胞体小 ,分布于脊髓白质的周边部 ,细长突起指向中央管方向 ;突起较短的细胞分布于脊髓前后正中沟两侧和中央管周围 ;灰质内阳性细胞主分布于背侧部的两侧 ,突起多而短 ,细胞核大。 2 4W时 ,多突起细胞增多 ,GFAP阳性细胞染色强度、细胞密度接近出生时水平。结论　人胎儿脊髓星形胶质细胞 16W时最多 ,2 4W时逐渐减少至出生时水平     

Therapeutic Effect of Curcumin and Methylprednisolone in the Rat Spinal Cord Injury

In addition to imperiling an individual's daily life, spinal cord injury (SCI), a catastrophic medical damage, can permanently impair an individual's body function. Methylprednisolone (MP), a medically accepted therapeutic drug for SCI, is highly controversial for the lack of consensus on its true therapeutic effect. In recent years, curcumin has served as a potential and novel therapeutic drug in SCI. Our study was intended to investigate the precise effect of MP and curcumin in SCI. We examined the function of MP and curcumin in a SCI model rat, both in vivo and in vitro, and found that there was a momentous improvement in Basso‐Beattie‐Bresnahan scores in the MP‐treated group when compared with Cur‐treated group within 14 days. Results obtained from the histological, immunohistochemistry and ultrastructural examinations evidenced the curative effect of MP was better than curcumin before Day 14. Nonetheless, there was a significant variation in the treatment effect between the MP‐treated and Cur‐treated groups after 14 days. The curcumin's effectiveness was more obvious than MP after 14 days following SCI. As such, we surmise that curcumin has a better therapeutic potential than MP with a prolong treatment time in the wake of SCI. Anat Rec, 301:686–696, 2018. © 2017 Wiley Periodicals, Inc.     

Curcumin-Loaded N,O-Carboxymethyl Chitosan Nanoparticles for Cancer Drug Delivery

Abstract

Chitosan (CS) and its carboxymethyl derivatives are smart biopolymers that are non-toxic, biocompatible and biodegradable, and, hence, suitable for various biomedical applications, such as drug delivery, gene therapy and tissue engineering. Curcumin is a major chemotherapeutic agent with antioxidant, anti-inflammatory, anti-proliferative, anticancer and antimicrobial effects. However, the potential of curcumin as a chemotherapeutic agent is limited by its hydrophobicity and poor bioavailability. In this work, we developed a nanoformulation of curcumin in a carboxymethyl chitosan (CMC) derivative, N,O-carboxymethyl chitosan (N,O-CMC). The curcumin-loaded N,O-CMC (curcumin-N,O-CMC) nanoparticles were characterized using DLS, AFM, SEM, FT-IR and XRD. DLS studies revealed nanoparticles with a mean diameter of 150 ± 30 nm. AFM and SEM confirmed that the particles have a spherical morphology within the size range of 150 ± 30 nm. Curcumin was entrapped with in N,O-CMC nanopartcles with an efficiency of 80%. The in vitro drug-release profile was studied at different pH (7.4 and 4.5) at 37°C for different incubation periods with and without lysozyme. Cytotoxicity studies using MTT assay indicated that curcumin-N,O-CMC nanoparticles showed specific toxicity towards cancer cells and non-toxicity to normal cells. Cellular uptake of curcumin-N,O-CMC nanoparticles was analyzed by fluorescence microscopy and was reconfirmed by flow cytometry. Overall, these results indicate that like previously reported curcumin loaded O-CMC nanoparticles, N,O-CMC will also be an efficient nanocarrier for delivering curcumin to cancer cells.     

The Effects of Age on the Morphometry of the Cervical Spinal Cord and Spinal Column in Adult Rats: An MRI‐Based Study

Rat models are commonly used to investigate the pathophysiological pathways and treatment outcomes after spinal cord injury (SCI). The high incidence of fall‐induced SCI in older adults has created a need for aging models of SCI in rats to investigate potential age‐related differences in SCI severity and outcomes. The aims of this study were to determine the influences of age and vertebral level on the geometries of the cervical spinal cord and spinal column in a rat model. Three young (3 months) and three aged (12 months) Fischer 344 rats were imaged in a high field (7 T) small‐animal magnetic resonance imaging system. All spinal cord geometry variables (including depth, width, and axial cross‐sectional area) and one spinal canal variable (depth) were significantly larger in the aged specimens by an average of 8.1%. There were main effects of vertebral level on all spinal cord variables and four spinal canal variables with values generally larger at C4 as compared to C6 (average increases ranged from 5.7% to 12.9% in spinal cord measures and 5.4% to 6.8% in spinal canal measures). High inter‐rater reliability between two measurers was observed with a mean intraclass correlation of 0.921 and percent difference of 0.9% across all variables measured. This study clearly demonstrates that cervical spinal cord geometry changes between the ages of 3 and 12 months in Fischer 344 rats. This information can aid in the planning and interpretation of studies that use a rat model to investigate the influence of age on cervical SCI. Anat Rec, 297:1885–1895, 2014. © 2014 Wiley Periodicals, Inc.     

The Location of the Major Ascending and Descending Spinal Cord Tracts in all Spinal Cord Segments in the Mouse: Actual and Extrapolated

Information on the location of the major spinal cord tracts in the mouse is sparse. We have collected published data on the position of these tracts in the mouse and have used data from other mammals to identify the most likely position of tracts for which there is no mouse data. We have plotted the position of six descending tracts (corticospinal, rubrospinal, medial and lateral vestibulospinal, rostral and caudal reticulospinal) and eight ascending tracts (gracile; cuneate; postsynaptic dorsal columns; dorsolateral, lateral, and anterior spinothalamic; dorsal and ventral spinocerebellar) on diagrams of transverse sections of all mouse spinal cord segments from the first cervical to the third coccygeal segment. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Nitrergic Neurons in the Spinal Cord of the Bottlenose Dolphin (Tursiops truncatus)

Nitric oxide (NO) is a freely diffusible gaseous neurotransmitter generated by a selected population of neurons and acts as a paracrine molecule in the nervous system. NO is synthesized from l ‐arginine by means of the neuronal nitric oxide synthase (nNOS), an enzyme requiring nicotine adenine dinucleotide phosphate (NADPH) as cofactor. In this study, we used histochemical and immunohistochemical techniques to investigate the distribution of NADPH‐diaphorase (NADPH‐d) and nNOS in the spinal cord of the bottlenose dolphin (Tursiops truncatus). Cells with a fusiform‐shaped somata were numerous in the laminae I and II. The intermediolateral horn showed darkly‐stained cells with a multipolar morphology. Neurons with a multipolar or fusiform morphology were observed in the ventral horn. Multipolar and fusiform neurons were the most common cell types in lamina X. Nitrergic fibers were numerous especially in the dorsal and intermediolateral horns. The presence of nitrergic cells and fibers in different laminae of the spinal cord suggests that NO may be involved in spinal sensory and visceral circuitries, and potentially contribute to the regulation of the complex retia mirabilia. Anat Rec, 296:1603–1614, 2013. © 2013 Wiley Periodicals, Inc.     

Initiation of Locomotor Activity in Spinal Cats by Epidural Stimulation of the Spinal Cord

Acute and chronic experiments on lower spinal (T10–T12) cats were performed to investigate the effects of epidural stimulation of the dorsal surface of the spinal cord on the initiation of locomotor activity. A zone located at the border between segments L4 and L5 was identified, stimulation of which induces locomotor activity. The parameters of epidural stimulation of the spinal cord effective in activating the stepping movement generator were identified. Epidural stimulation leading to the initiation of movement activity was shown to depend on intracentral and peripheral mechanisms activating the segmental, intersegmental and propriospinal reflex systems of the spinal cord. A leading role was demonstrated for the propriospinal system of the dorsolateral funiculi in activating the generators of stepping movements in epidural stimulation of the spinal cord.     

Stem Cells: Current Approach and Future Prospects in Spinal Cord Injury Repair

Spinal cord injury (SCI) invariably results in the loss of neurons and axonal degeneration at the lesion site, leading to permanent paralysis and loss of sensation. There has been no successful treatment for severe spinal cord injuries to recover back to normal function yet. Studies have shown that the transplantation of stem cells may provide an effective treatment for SCI because of the self‐renewing and multipotential nature of these cells. Stem cells have the capability to repair injured nervous tissue through replacement of damaged cells, neuroprotection, or the creation of an environment conducive to regeneration by endogenous cells. Up to today several types of stem cells have been transplanted into the injured spinal cord. However, the question of which cell type is most beneficial for SCI treatment is still unresolved. There are still several limitations to the current data sets which require further investigation. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Combined Muscle Motor and Somatosensory Evoked Potentials for Intramedullary Spinal Cord Tumour Surgery

Purpose

To evaluate whether intraoperative neurophysiologic monitoring (IONM) with combined muscle motor evoked potentials (mMEPs) and somatosensory evoked potentials is useful for more aggressive and safe resection in intramedullary spinal cord tumour (IMSCT) surgery.

Materials and Methods

We reviewed data from consecutive patients who underwent surgery for IMSCT between 1998 and April 2012. The patients were divided into two groups based on whether or not IONM was applied. In the monitored group, the procedures were performed under IONM using 75% muscle amplitude decline weaning criteria. The control group was comprised of patients who underwent IMSCT surgery without IONM. The primary outcome was the rate of gross total excision of the tumour on magnetic resonance imaging at one week after surgery. The secondary outcome was the neurologic outcome based on the McCormick Grade scale.

Results

The two groups had similar demographics. The total gross removal tended to increase when intraoperative neurophysiologic monitoring was used, but this tendency did not reach statistical significance (76% versus 58%; univariate analysis, p=0.049; multivariate regression model, p=0.119). The serial McCormick scale score was similar between the two groups (based on repeated measure ANOVA).

Conclusion

Our study evaluated combined IONM of trans-cranial electrical (Tce)-mMEPs and SEPs for IMSCT. During IMSCT surgery, combined Tce-mMEPs and SEPs using 75% muscle amplitude weaning criteria did not result in significant improvement in the rate of gross total excision of the tumour or neurologic outcome.     

NT-4在成年猴脊髓的表达

目的探讨NT-4在成年猴脊髓的表达分布情况,为成年猴脊髓中该生长因子的表达提供可靠的免疫组织化学证据。方法云南成年健康雄性猕猴5只,平均体重6 kg左右,氯氨酮(0.25 ml/kg)肌肉注射麻醉后,Zamboni液经心脏主动脉插管灌注固定动物,取T8脊髓节段入4%多聚甲醛后固定(4℃)6小时。制作20μm厚的连续冰冻切片,间隔取片后,分别以抗NT-4抗体行免疫组化ABC染色。观察各生长因子在猴脊髓的表达分布情况。结果NT-4的免疫阳性反应产物在成年猴脊髓腹角神经元、脊髓背角神经元和脊髓中央管(Ⅹ板层)均有分布。结论成年猴脊髓存在多肽生长因子NT-4的表达,提示多肽生长因子在成年猴脊髓的生理过程中发挥作用。     

Expression of the c-Fos Gene in Spinal Cord and Brain Cells in Rats Subjected to Stress in Conditions of Exposure to Various Types of Halothane Anesthesia

The influences of different treatments on the expression of the c-fos gene in the spinal cord and brain (hypothalamus) was studied in rats using various types of anesthesia. Synthesis of c-Fos-like proteins occurred only in the spinal cord in conditions of constant 1.5% halothane anesthesia. Use of induction anesthesia with 1.5% halothane allowed detection of c-Fos-like protein expression in cells of the rat spinal cord (lumbar segments) and brain, both when animals were placed in a hammock and when mechanical pain stimulation or electromagnetic irradiation of the skin with UHF currents were applied. The pattern of brain structures reacting to mechanical pain stimulation with expression of c-Fos-like protein was identified. This type of stimulation was shown to induce increases in the quantity of c-Fos-positive cells in the lateral hypothalamic area (LHA), the ventromedial (VMH) and dorsomedial (DMH) hypothalamic nuclei, and in the ventral hypothalamic area (AHA) by 116%, 167%, 101%, and 157% respectively as compared with controls. Skin irradiation with UHF currents decreased the intensity of mechanical pain stimulation-induced synthesis of c-Fos-like protein in most structures (LHA, VMH, DMN, and AHA by 32.8%, 29%, 15%, and 33% respectively). Only induction halothane anesthesia allowed identification of hypothalamic structures reacting to mechanical pain stimulation and the modifying effects of irradiating the skin with UHF currents on the intensity of these reactions.     

壳聚糖纳米颗粒包裹对鼠白细胞介素2基因表达和调节小鼠免疫效应的影响

本实验制备壳聚糖纳米颗粒 (CNP) ,包裹小鼠白细胞介素 2基因 (mIL 2 )真核表达质粒 (VRMIL 2 ) ,肌注接种 2 1d昆明小鼠 ,观察mIL 2基因体内表达及其对免疫应答和免疫保护的影响。实验结果发现 :CNP包裹VRMIL 2注射小鼠血液中IgG、IgM和IgA不同程度地增多 ,均显著高于CNP包裹空白质粒组 (P <0 .0 5 ) ;其血清中IL 2、IL 4和IL 6的含量明显升高 ,与对照组差异显著 (P <0 .0 5 ) ;外周血液的白细胞和淋巴细胞数量也较对照组显著增加。免疫后 35d以大肠杆菌口服攻毒实验小鼠 ,检测发现 :CNP包裹VRMIL 2组小鼠的上述免疫指标除中性粒细胞外均显著多于对照小鼠 ,VRMIL 2接种小鼠均健康存活 ,而对照小鼠均发病 ;尽管CNP包裹VRMIL 2接种小鼠的体液和细胞免疫指标与未包裹VRMIL 2免疫鼠差异不显著 (P >0 .0 5 ) ,但剂量仅为后者的 1/ 5。这些结果表明 :壳聚糖纳米颗粒包裹VRMIL 2可显著提高外源IL 2基因体内表达水平 ,明显增强体液和细胞免疫水平的效应 ,增强对大肠杆菌的抗病力 ,提示壳聚糖纳米颗粒包裹IL 2基因可明显增强动物的体液和细胞免疫 ,可作为有效的抗感染免疫调节剂。     

巢蛋白在胚胎及新生大鼠脊髓的表达

目的　检测不同发育时期巢蛋白 (nestin)在脊髓神经干细胞的表达水平 ,探讨脊髓神经干细胞的发育规律 ,为分离、富集和增殖均质的神经干细胞以及后期的研究工作如神经干细胞定向分化及移植治疗等研究提供实验依据。方法　将Wistar大鼠按胎龄及日龄分组 ,剥离脊髓 ,制成单细胞悬液 ,经间接荧光法标记nestin抗原 ,用流式细胞仪检测和分析。结果　从E13到P7整个发育过程中都有nestin抗原的表达 ,呈升高趋势 ,其中P1期表达水平最高。出生后表达下降 ,趋近于 90d(同型对照 )表达水平。结论　巢蛋白在脊髓的表达出现于胚胎早期 ,表达水平随胎龄轻微波动 ,呈升高趋势 ,P1达到高峰 ,出生后表达下降 ,趋于成鼠的微量表达     

胚胎脊髓移植对大鼠脊髓全横断后功能修复及NOS表达的影响

目的　探讨胚胎脊髓移植对脊髓全横断大鼠脊髓功能修复及NOS表达的影响。方法　在脊髓全横断处移植胚胎脊髓 6 0d后 ,应用NADPH d组织化学方法检侧脊髓内一氧化氮合酶神经元的分布及形态变化 ,并做图像分析。用BBB评分法观察大鼠后肢运动功能的恢复。结果　胚胎大鼠脊髓移植后可以使全横断脊髓前角内NOS表达增加 ,与损伤对照组比较有显著性差异 (P <0 .0 1) ;大鼠后肢的自主运动功能恢复好于损伤对照组。结论　胚胎脊髓移植对成年大鼠损伤脊髓功能恢复有促进作用 ,NO可能参与修复作用。     

Spinal Cord Hemangioblastomas in von Hippel-Lindau Disease: Management of Asymptomatic and Symptomatic Tumors

Purpose

Standard treatment of asymptomatic spinal cord hemangioblastoma in von Hippel-Lindau (VHL) disease has yet to be established. The purpose of this study was to propose guidelines for the treatment of asymptomatic spinal cord hemangioblastomas in VHL disease.

Materials and Methods

VHL disease patients treated for spinal cord hemangioblastomas between 1999 and 2009 were included. All spinal cord hemangioblastomas were divided into three groups: Group 1, asymptomatic tumors at initial diagnosis followed with serial imaging studies; Group 2, asymptomatic tumors at initial diagnosis that were subsequently resected; and Group 3, symptomatic tumors at initial diagnosis, all of which were resected.

Results

We identified 24 spinal cord hemangioblastomas in 12 patients. Groups 1, 2 and 3 comprised 13, 4 and 7 tumors, respectively. Group 1 exhibited a smaller tumor volume (257.1 mm³) and syrinx size (0.8 vertebral columns) than those of Group 2 (1304.5 mm³, 3.3 vertebral columns) and Group 3 (1787.4 mm³, 6.1 vertebral columns). No difference in tumor volume or syrinx size was observed between Groups 2 and 3. Five tumors in Group 1 were resected during follow-up because symptoms had developed or the tumor had significantly grown. Finally, among 17 asymptomatic tumors at the initial diagnosis, nine tumors were resected. Only one tumor of these nine tumors resulted in neurological deficits, while five of seven symptomatic tumors caused neurological deficits.

Conclusion

Selective resection of asymptomatic tumors before they cause neurological deficits might bring about better outcomes.     

X线照射对受损脊髓轴索和髓鞘的作用

目的 :探讨 X线对受损脊髓髓鞘和轴索作用。方法 :实验组行脊髓胸腰段半横断 ,术后次日行 X线照射 ,剂量是 35 Gy。对照组行同样手术 ,但不照射。2 4周时行 SP(streptavidin peroxidase conjugataed method,链霉素抗生素蛋白 -过氧化酶连接法 )免疫组化染色和超微结构观察。结果 :实验组脊髓损伤平面上、下段 MBP(myelin basicprotein,髓磷脂基蛋白 )阳性髓鞘数量明显低于对照组 ;实验组损伤平面上段 NF(neuron filament,神经纤维丝 )阳性神经纤维数量与对照组无明显差别 ,实验组损伤平面下段 NF阳性神经纤维数量高于对照组。电镜下实验组脊髓出现明显脱髓鞘改变 ,损伤平面下段可见新生的细小神经纤维束。结论 :35 Gy的 X线照射可以使脊髓出现较多的脱髓鞘变化 ,有促进损伤平面下段 NF阳性纤维增多的作用