水通道蛋白-4在实验性脊髓损伤大鼠的表达

为了观察大鼠脊髓损伤后水通道蛋白4(AQP 4)的表达和对后肢功能的影响,本实验建立Allen脊髓损伤动物模型,于术后1、3、7、14和21d分别对大鼠进行了BBB评分检查后肢功能,处死动物后应用免疫组织化学技术检测脊髓组织内AQP 4的表达,用图像分析仪对脊髓损伤后神经组织的AQP 4的变化进行了定量分析。结果表明:脊髓损伤后第1d,在脊髓损伤组受损伤脊髓灰质和白质中均可见到AQP 4的表达明显增加;第3d时均达到高峰。在第7、14、和21d,AQP 4的表达与对照组均有显著性差异(P<0. 01)。本研究结果提示:脊髓损伤后大鼠脊髓组织中AQP 4的表达显著增加。     

Limited minocycline neuroprotection after balloon-compression spinal cord injury in the rat

Minocycline (MC), a second-generation tetracycline and anti-inflammatory agent reportedly provides neuroprotection following CNS injury. The objective of this study was to examine the neuroprotective effects of short and long-term MC treatment using balloon-compression spinal cord injury (SCI) in the rat. Rats subjected to SCI were treated with MC for 1 day (1DMC group; total dose 180 mg/kg) or 5 days (5DMC group; total dose 450 mg/kg) or placebo. The effects of MC treatment on locomotor recovery (BBB scale) and spinal cord white and gray matter sparing were evaluated for up to 28 days. Morphometric analysis showed that while MC treatment spared spinal cord white and gray matter rostral to the lesion epicenter in both, 1DMC and 5DMC groups, sparing of white and gray matter areas was not observed caudal to the traumatic lesion. In addition, MC treatment had no effect on final locomotor recovery. Limited improvement of spinal cord post-compression consequences raises questions about the neuroprotection efficiency of MC treatment following compression SCI in the rat.     

Longitudinal assessment of recovery after spinal cord injury with behavioral measures and diffusion,quantitative magnetization transfer and functional magnetic resonance imaging

Spinal cord injuries (SCIs) are a leading cause of disability and can severely impact the quality of life. However, to date, the processes of spontaneous repair of damaged spinal cord remain incompletely understood, partly due to a lack of appropriate longitudinal tracking methods. Noninvasive, multiparametric magnetic resonance imaging (MRI) provides potential biomarkers for the comprehensive evaluation of spontaneous repair after SCI. In this study in rats, a clinically relevant contusion injury was introduced at the lumbar level that impairs both hindlimb motor and sensory functions. Quantitative MRI measurements were acquired at baseline and serially post‐SCI for up to 2 wk. The progressions of injury and spontaneous recovery in both white and gray matter were tracked longitudinally using pool‐size ratio (PSR) measurements derived from quantitative magnetization transfer (qMT) methods, measurements of water diffusion parameters using diffusion tensor imaging (DTI) and intrasegment functional connectivity derived from resting state functional MRI. Changes in these quantitative imaging measurements were correlated with behavioral readouts. We found (a) a progressive decrease in PSR values within 2 wk post‐SCI, indicating a progressive demyelination at the center of the injury that was validated with histological staining, (b) PSR correlated closely with fractional anisotropy and transverse relaxation of free water, but did not show significant correlations with behavioral recovery, and (c) preliminary evidence that SCI induced a decrease in functional connectivity between dorsal horns below the injury site at 24 h. Findings from this study not only confirm the value of qMT and DTI methods for assessing the myelination state of injured spinal cord but indicate that they may also have further implications on whether therapies targeted towards remyelination may be appropriate. Additionally, a better understanding of changes after SCI provides valuable information to guide and assess interventions.     

Excitotoxic injury to thoracolumbar gray matter alters sympathetic activation and thermal pain sensitivity

Studies of humans, monkeys and rodents have implicated combined gray and white matter damage as important for development of chronic pain following spinal cord injury (SCI). Below-level chronic pain and hyperalgesia following injury to the spinal white matter, including the spinothalamic tract (STT), can be enhanced by excitotoxic influences within the gray matter at the site of SCI. Also, excitotoxic injury of thoracic gray matter without interruption of the STT results in below-level heat hyperalgesia. The present study evaluates the possibility that thoracolumbar gray matter injury increases sensitivity to nociceptive heat stimulation by altering spinal sympathetic outflow. Thermal preferences of rats for heat (45 °C) versus cold (15 °C) were evaluated before and after thoracolumbar injections of quisqualic acid (QUIS). A pre-injury preference for heat changed to a post-injury preference for cold. Systemic activation of the sympathetic nervous system by restraint stress decreased the heat preference pre-injury and increased the cold preference post-injury. The heat aversive effect of stress was magnified and prolonged post-injury, compared to pre-injury. Also, peripheral sympathetic activation by nociceptive stimulation was evaluated pre- and post-injury by measuring thermal transfer through a hindpaw during stimulation with 44.5 °C. Skin temperature recordings revealed enhanced sympathetic activation by nociceptive heat stimulation following spinal QUIS injury. However, increased sympathetic activation with peripheral vasoconstriction should enhance cold aversion, in contrast to the observed increase in heat aversion. Thus, peripheral sympathetic vasoconstriction can be ruled out as a mechanism for heat hyperalgesia following excitotoxic gray matter injury.     

Quantitative MRI in a non‐surgical model of cervical spinal cord injury

Quantitative T₂ (qT2), diffusion tensor imaging (DTI), and histology were used to investigate a cervical model of spinal cord injury (SCI) in the rat. While quantitative MRI can significantly increase the specificity in the presence of pathology, it must be validated for each type of injury or disease. In the case of traumatic SCI most models are difficult to image, either due to the location of the injury, or as a result of damage to surrounding tissues resulting from invasive surgical procedures. In this study a non‐surgical cervical model of SCI, produced using a combination of focused ultrasound and microbubbles, was used to produce pathology similar to that seen in models of contusive and compressive injuries. qT2 and DTI were performed at 24 h and 1 and 2 weeks following injury, and compared with H&E and luxol fast blue histology. In the injured spinal cord, in addition to intra/extracellular (I/E) water and myelin water in white matter, qT2 revealed a large component with very short T₂ of about 3 ms, which was highly correlated with the presence of hemorrhage in both gray and white matter at 24 h, and with the presence of hemosiderin in gray matter at 2 weeks following injury. The T₂ of the I/E water peak was also elevated at 24 h in both gray and white matter, which was correlated with the presence of vacuolation/edema on histology. Cystic cavities were only seen at the 1 or 2 week timepoints, and were correlated with the presence of a water peak with T₂ > 250 ms. No significant changes in diffusivity parameters were observed. Pathologies were often co‐occurring, with opposite effects on the average T₂ in a given voxel, reducing the visibility of injured tissue on standard T₂‐weighted MR images. Copyright © 2015 John Wiley & Sons, Ltd.     

Visualisation of the extent of damage in a rat spinal cord injury model using MR microsopy of the water diffusion tensor

Magnetic Resonance Diffusion Tensor Imaging (DTI) of the control and traumatic injured spinal cord of a rat in vitro is reported. Experiments were performed on excised spinal cords from 10 Wistar rats, using a home-built 6.4 T MR microscope. MRI and histopathological results were compared. Presented results show that DTI of the spinal cord, perfused with formalin 10 minutes after the injury, can detect changes in water diffusion in white matter (WM) and in gray matter (GM), in areas extending well beyond the region of direct impact. Histology of neurons of the GM shows changes that can be attributed to ischemia. This is in agreement with the observed decrease of diffusion in the injured regions, which may be attributed to the cytotoxic edema due to ischemia. However, the diffusion changes in highly anisotropic WM seem to be caused by a direct action of mechanical force of impact, which significantly distorts the nerve fibers.     

Diffusion tensor imaging in the human spinal cord: development, limitations, and clinical applications

Diffusion tensor imaging (DTI) is currently the only non-invasive in vivo assessment of white matter tract integrity. Capitalizing on the diffusion properties of water within an axon, DTI enables the visualization of tissue structure at a microscopic scale. Furthermore, measurements of anisotropy and diffusivity enable the detection of subtle details of the effects of injury that cannot be detected using conventional magnetic resonance techniques. Recently, DTI has been applied to the spinal cord, and results have demonstrated it to be a valuable tool for assessing the extent of white matter damage in numerous spinal cord-related conditions including multiple sclerosis, spinal cord injury, amyotrophic lateral sclerosis, myelitis, and spinal cord tumors. The purpose of this review is to discuss the technical limitations of the imaging method within the spinal cord, review possible solutions, and highlight the current uses and the potential clinical application of this technique.     

小鼠颈脊髓半侧挫伤模型的建立及其组织学特点

目的　建立基于位移控制的C57/6J小鼠C5脊髓半侧挫伤模型,观察其脊髓组织学改变。 方法　C57BL/6小鼠在麻醉状态下行C5左侧椎板切除术,打击头（直径0.75 mm）对准C5左侧,由电磁伺服材料试验机驱动挫伤脊髓,设定打击位移0.9 mm,打击速度50 mm/s。损伤后1周脊髓标本取材,EC染色,作组织学定量分析。 结果　打击参数结果稳定性与重复性良好。打击位移、打击速度和打击力分别为（0.880±0.035）mm、（48.146±4.367）mm/s、（0.407±0.129）N,损伤中心的脊髓组织学表现为：伤侧脊髓有明显的出血及正常组织结构破坏,脊髓背侧束、脊髓后角和部分前角有破坏;健侧脊髓结构基本保持完整。计算损伤中心平面的残存灰质比例、残存白质比例及损伤面积比例分别为（19±7）%、（88±9）%及（28±4）%。 结论　本研究成功建立小鼠颈脊髓半侧挫伤模型,此模型具有重复性较好的力学参数,表现出典型的单侧颈脊髓损伤的组织学特征,可为脊髓损伤分子机制和治疗研究奠定基础。     

Withanoside IV improves hindlimb function by facilitating axonal growth and increase in peripheral nervous system myelin level after spinal cord injury

Although methylprednisolone is the clinically standard medication and almost the only therapy for spinal cord injury (SCI), its effect on functional recovery remains questionable. Transplantation strategies using sources such as neural stem cells and embryonic spinal cord still have some hurdles to overcome before practical applications become available. We therefore aimed to develop a practical medication for SCI. Per oral treatment with withanoside IV, which was previously shown to regenerate neuronal networks in the brain, improved locomotor functions in mice with SCI. In the spinal cord after SCI, axons were crushed in the white matter and gray matter, and central nervous system (CNS) myelin level decreased. In mice treated with withanoside IV (10micromol/kg body weight/day, for 21 days), axonal density and peripheral nervous system (PNS) myelin level increased. The loss of CNS myelin and increase in reactive gliosis were not affected by withanoside IV. These results suggest that oral administration of withanoside IV may ameliorate locomotor functions by facilitating both axonal regrowth and increase in PNS myelin level.     

Diffusion tensor imaging detects axonal injury and demyelination in the spinal cord and cranial nerves of a murine model of globoid cell leukodystrophy

Globoid cell leukodystrophy is an inherited neurodegenerative disorder caused by a deficiency of the lysosomal enzyme galactosylceramidase. In both human patients and the authentic murine Twitcher model, pathological findings include demyelination as well as axonal damage in both the central and peripheral nervous system. Diffusion tensor imaging (DTI) has emerged as a powerful noninvasive technique that is sensitive to these white matter disease processes. Increases in radial diffusivity (λ?) and decreases in axial diffusivity (λ∥) correlate with histopathological evidence of demyelination and axonal damage, respectively. Compared to age‐matched, normal littermates, DTI of optic nerve and trigeminal nerve in end‐stage Twitcher mice displayed a statistically significant increase in λ? and decrease in λ∥, consistent with previously characterized demyelination and axonal damage in these regions. In the Twitcher spinal cord, a statistically significant decrease in λ∥ was identified in both the dorsal and ventrolateral white matter, relative to normal controls. These results were consistent with immunofluorescence evidence of axonal damage in these areas as detected by staining for nonphosphorylated neurofilaments (SMI32). Increase in λ? in Twitcher spinal cord white matter relative to normal controls reached statistical significance in the dorsal columns and approached statistical significance in the ventrolateral region. Correlative reduced levels of myelin basic protein were detected by immunofluorescent staining in both these white matter regions in the Twitcher spinal cord. Fractional anisotropy, a nonspecific but sensitive indicator of white matter disease, was significantly reduced in the optic nerve, trigeminal nerve, and throughout the spinal cord white matter of Twitcher mice, relative to normal controls. This first reported application of spinal cord DTI in the setting of GLD holds potential as a noninvasive, quantitative assay of therapeutic efficacy in future treatment studies. Copyright © 2009 John Wiley & Sons, Ltd.     

The Neuroprotective Effects of Reg‐2 Following Spinal Cord Transection Injury

This study was designed to elucidate the potential neuroprotective effects of Reg‐2 (regeneration gene protein 2) in a rodent model of spinal cord transection injury at the ninth thoracic level. Reg‐2 at 100 and 500 μg, recombinant rat ciliary neurotrophic factor, or vehicle were delivered intrathecally using Alzet miniosmotic pumps. We found that Reg‐2 treatment significantly reduced neuronal death in the spinal cord. There was also an attenuation of inflammation at the injury site and an increase in white matter sparing and retained myelination. Retrograde tracing revealed that Reg‐2 protected axons of long descending pathways at 6 weeks post‐SCI, and the number of FluoroGold‐labeled neurons in spinal and supraspinal regions was also significantly increased. Immunofluorescent staining confirmed that the spared white matter contained neurofilament‐positive axons. Moreover, behavioral improvements were revealed by Basso Beattie Bresnahan locomotor rating scores and grid‐walk analysis. These results suggest that Reg‐2 might promote functional recovery by increasing axonal growth, inhibiting neuronal apoptosis, and attenuating spinal cord secondary injury after SCI. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

紫外线照射充氧自血回输对家兔脊髓损伤后血液循环的影响

家兔用改良Allen 氏系统造成不完全性脊髓损伤,随机分为对照组、损伤组和治疗组。分别测定血液和脊髓组织TXB 2、 6-keto-PGF 1 a 和ET-1 含量;以及脊髓组织灰质、白质和总血流量,研究紫外线照射和充氧自血回输疗法（UBIO）治疗脊髓损伤的作用机制。实验发现：损伤组血液和脊髓组织TXB 2 含量升高、6-keto-PGF 1 a 含量下降、ET-1 含量升高。脊髓组织灰质和白质血流量以及脊髓组织总血流量均下降。治疗组用UBIO 治疗后,血液和脊髓组织TXB 2 含量下降、6-keto-PGF 1 a 含量升高、ET-1 含量下降。脊髓组织灰质和白质血流量以及脊髓组织总血流量均上升。实验说明：UBIO 可以改善全身和脊髓组织的血液循环。     

Effect of space flight on composition of soluble proteins of the spinal cord and spinal ganglia of rats

The content of individual protein fractions extracted from the spinal cord, at the level of the lumbar enlargement, and the spinal ganglia of rats consecutively with distilled water, 0.85% NaCl solution, and 0.1 N NaOH solution was investigated. A significant decrease in the content of water-soluble proteins was found in the white and gray matter of the spinal cord 12 h after space flight. The content of salt-soluble and alkali-soluble proteins in the structures of the spinal cord and spinal ganglia per milligram wet weight of tissue was not significantly altered. A significant increase in the content of water-soluble proteins compared with the control was found in the gray matter of the spinal cord 25 days after space flight. The content of water-soluble proteins in the white matter of the spinal cord was increased to the control level. A significant increase also was observed in the content of alkali-soluble proteins in the spinal ganglia.Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 168–171, August, 1977.     

Computational Model of Interstitial Transport in the Spinal Cord using Diffusion Tensor Imaging

Local drug delivery methods, including convection-enhanced delivery (CED), are being used to increase distribution in selected regions of nervous tissue. There is a need for 3D models that predict spatial drug distribution within these tissues. A methodology was developed to process magnetic resonance microscopy (MRM) and diffusion tensor imaging (DTI) scans, segment gray and white matter regions, assign tissue transport properties, and model the interstitial transport of macromolecules. Fiber tract orientation was derived from DTI data and used to assign directional dependence of hydraulic conductivity, K, and tracer diffusivity, D _t , transport tensors. Porous media solutions for interstitial fluid pressure, velocity, and albumin distribution were solved using a finite volume method. To test this DTI-based methodology, a rat spinal cord transport model was developed to simulate CED into the dorsal white matter column. Predicted distribution results correspond well with small volume (∼1 μl) trends found experimentally, although albumin loss was greater at larger infusion volumes (>2 μl). Simulations were similar to those using fixed transport properties due to the bulk alignment of white matter fibers along the cord axis. These findings help to validate the DTI-based methodology which can be applied to modeling regions where fiber tract organization is more complex, e.g., the brain.     

The spinal cord development in guinea pig: a morphometric study on an image analysis system

Using an image analysis system, the Authors carried out a morphometric study on guinea pig spinal cord in order to determine volumetric changes of white and gray matter during development. White and gray matter volumes were determined by measuring the area occupied by these matters in 10 micrograms sections of spinal cord in 1 day and 90 days old subjects. Several topographic correspondences in the localisation of the lowest and highest volumetric values were observed in the two groups of subjects. Such correspondences were more marked for white than gray matter. Moreover, during growth white matter volume showed an increase double that observed in gray matter.     

Dynamic induction of the myelin-associated growth inhibitor Nogo-A in perilesional plasticity regions after human spinal cord injury

The myelin-associated inhibitor Nogo-A (Reticulon 4, RTN4) restricts axonal outgrowth, plasticity, and neural circuitry formation in experimental models of spinal cord injury (SCI) and is targeted in clinical interventions starting treatment within 4 weeks post-SCI. Specifically, Nogo-A expressed by oligodendroglia restricts compensatory neurite sprouting. To interrogate the hypothesis of an inducible, lesion reactive Nogo-A expression over time, we analyzed the spatiotemporal Nogo-A expression at the spinal lesion core (region of tissue necrosis and axonal damage/pruning) and perilesional rim (region of plasticity formation). Spinal cord specimens of SCI subjects (n = 22) were compared to neuropathologically unaltered controls (n = 9). Nogo-A expression was investigated ranging from acute (0–3 days), early subacute (4–21 days), late subacute (22–90 days) to early chronic–chronic (91 days to 1.5 years after SCI) stages after SCI. Nogo-A expression in controls is confined to motoneurons in the anterior horn and to oligodendrocytes in gray and white matter. After SCI, the number of Nogo-A⁺ and TPPP/p25⁺ oligodendrocytes (i) inclined at the organizing perilesional rim specifically, (ii) increased further over time, and (iii) peaked at chronic stages after SCI. By contrast, at the lesion core, the number of Nogo-A⁺ and TPPP/p25⁺ oligodendrocytes did not increase. Increasing numbers of Nogo-A⁺ oligodendrocytes coincided with oligodendrogenesis corroborated by Nogo-A coexpression of Ki67⁺, TPPP/p25⁺ proliferating oligodendrocytes. Nogo-A oligodendrocyte expression emerges at perilesional (plasticity) regions over time and suggests an extended therapeutical window for anti-Nogo-A pathway targeting interventions beyond 4 weeks in patients after SCI.     

Distribution of Internal Strains Around Bony Prominences in Pigs

Deep tissue injury (DTI) is a type of pressure ulcer in which tissue breakdown initiates at the bone-muscle interface under intact skin. Excessive deformation in the soft tissue, particularly around bony prominences, is believed to be one of the causes leading to the development of DTI. The main goal of this study was to measure the magnitude and distribution of strains within muscles surrounding the ischial tuberosities, induced by levels of external loading that encompass the range of loading experienced by the soft tissue in seated individuals. The experiments were conducted in adult pigs with intact spinal cords (n = 5) and pigs with partial spinal cord injury (SCI) (n = 2), one of which also had a DTI. A secondary goal was to obtain a preliminary assessment regarding the capacity of intermittent electrical stimulation (IES), an intervention for preventing the formation of DTI, to counteract the muscle compression caused by external loading. In intact animals, muscles subjected to external loads equivalent to 25% of body weight experienced maximal principal strains, minimal principal strains, and shear strains of 0.68, -0.3, and 0.4, respectively. These magnitudes increased by 91.9, 17.6, and 87.5%, respectively, when external loading increased to 50% body weight. Minimal to no further increases in strain magnitudes were seen with the 75% body weight loading level. In one animal with SCI and no DTI, strain magnitudes were on average 9.7% higher than those in the intact animals at the corresponding loading levels. The presence of a DTI in another animal with SCI reduced strain magnitudes by 28% compared to intact animals. The regions in the muscle that underwent the largest deformations were those between the ischial tuberosity and the external surface, and up to 2 cm ventral to the ischial tuberosity (furthest measured). Muscle contractions produced by IES increased the thickness of the tissue between the ischial tuberosities and skin during the period of stimulation by 10-20% for loading levels up to 75% of body weight in both intact and spinal cord injured pigs. This study provides the first measurements of strain around the ischial tuberosities in an animal model that resembles humans.     

Quantification of microvasculature in the canine spinal cord

Capillary density and capillary orientation in canine spinal cords were estimated by calculating actual lengths, surfaces, and volumes of capillary segments in tissue sections. Transverse, sagittal, and frontal section planes were sampled from dorsal, ventral, and lateral funiculi and from dorsal and ventral gray horns of spinal segments C3, T6, and L3 from three dogs. Capillaries were defined as vessels less than 10 μm in diameter. Electron microscopy of 104 such vessels revealed no muscle coat but collagen fibrils between endothelium and astrocyte process in 68% of the white matter capillaries and 16% of those in gray matter. Capillary diameter was significantly different among regions in some cases, but consistent patterns of variation were not found. Capillary density was four to five times greater in gray matter than in white matter. Capillary density differed significantly among the same-size dogs, but within dogs, density was similar among segments and within gray matter and white matter regions. In 62% of the transverse sections, capillary orientation was significant but mean direction was variable. Significant capillary orientation was found in 89% of the sagittal and frontal sections, and the mean direction was always along the craniocaudal axis of the spinal cord. The craniocaudal orientation was significant in 96% of the white matter sections and 78% of the gray sections, and in 97% of the cervical and thoracic sections but only 73% of the lumbar sagittal and frontal sections. Because capillary orientation is neither isotropic nor regular, unbiased, lowvariance estimates of capillary density cannot be expected without resorting to excessive sampling. An efficient method of quantifying spinal capillaries for comparative purposes by counting number of profiles per unit area is recommended.     

Activation of endothelial nitric oxide synthase following spinal cord injury in mice

Endothelial nitric oxide synthase (eNOS) plays a neuroprotective role after cerebral ischemia through the production of NO, which enhances cerebral blood flow. However, precise details regarding activation of eNOS after spinal cord injury (SCI) largely remain to be elucidated. In the present study we investigated chronological alteration and cellular location of eNOS and phosphorylated (p)-eNOS at Ser(1177) following SCI in mice. Western blot analysis showed eNOS to be significantly phosphorylated at Ser(1177) from 1 to 2 days after mild SCI, with gradual decrease thereafter. Immunohistochemistry revealed the p-eNOS to be mainly expressed in the endothelial cells of microvessels within gray matter under these conditions. These findings suggest that mild SCI activates eNOS in the subacute stage, which increases spinal cord blood flow and may be involved in protective and repair responses.     

人工合成高分子支架材料治疗脊髓损伤

背景：治疗脊髓损伤的人工合成高分子支架材料是目前的研究热点之一。 目的：综述国内外人工合成高分子支架材料在治疗脊髓损伤方面的研究进展。 方法：应用计算机检索万方医学、中国知网、PubMed、EBSCO数据库中2000年1月至2012年1月关于人工合成高分子支架材料治疗脊髓损伤方面的文章,在标题和摘要中以“脊髓损伤,组织工程,人工合成高分子材料”或 “spinal cord injury,tissue engineering,synthetic polymer material”为检索词进行检索。 结果与结论：治疗脊髓损伤的人工合成高分子材料很多,主要有聚乳酸、聚羟基酸、聚β-羟丁酸、合成水凝胶、聚乙二醇及其他人工合成高分子材料。每种材料都有其优缺点,都无法达到完全的组织相容性和可降解性,这些支架材料不能完全模仿脊髓的三维多孔立体结构,移植后这些支架材料的位置相对于脊髓结构来讲是随机的,并未与脊髓的灰、白质组织学结构吻合,更没有和白质中主要纤维相对应,所以均未应用于临床。人工合成高分子支架材料在治疗脊髓损伤方面需要进一步研究。