Amniotic fluid brain-specific proteins are biomarkers for spinal cord injury in experimental myelomeningocele

Myelomeningocele (MMC), the most severe form of spina bifida (SB), causes neurological deficit. Injury to the spinal cord is thought to begin in utero. We investigated whether brain-specific proteins (BSPs) would enable us to monitor the development of MMC-related tissue damage during pregnancy in an animal model with naturally occurring SB (curly tail/loop tail mouse; n = 256). Amniotic fluid levels of neurofilament heavy chain (NfH), glial acidic fibrillary protein (GFAP) and S100B were measured by standard ELISA techniques. The amniotic fluid levels of all BSPs were similar in SB and control mice on embryonic day (E) 12.5 and 14.5, whereas a significant increase was observed for GFAP in SB mice on E16.5. Levels of all BSPs were significantly increased in SB mice on E18.5. The rapid increase in GFAP, paralleled by a moderate increase in NfH and S100B, suggests that spinal cord damage starts to accelerate around E16.5. The macroscopic size of the MMC was related to NfH level on E16.5 and E18.5, suggesting that axonal degeneration is most severe in large MMC. Amniotic fluid BSP measurements may provide important information for balancing the risks and benefits to mother and child of in utero surgery for MMC.     

Neuropeptides in spinal cord injury: Comparative experimental models

The possible role of endogenous opioids in the pathophysiology of spinal cord injury was evaluated utilizing a variety of experimental models and species. In the cat, we have shown that β-endorphin-like immunoreactivity was increased in plasma following traumatic spinal injury; such injury was associated with a decrease in spinal cord blood flow (SCBF) which was reversed by the opiate receptor antagonist naloxone. Naloxone treatment also significantly improved functional neurological recovery after severe injury. Thyrotropin-releasing hormone (TRH), possibly through its “anti-endorphin” actions, was even more effective than naloxone in improving functional recovery in the cat. In a rat model, utilizing a similar trauma method, TRH proved superior to naloxone in improving SCBF after injury. In addition, naloxone at high doses attenuated the hindlimb paralysis produced by temporary aortic occlusion in the rabbit. The high doses of naloxone required to improve neurological function after spinal injury suggest that naloxone's actions, if opiate receptor mediated, may be mediated by non-μ receptors. Dynorphin, an endogenous opioid with a high affinity for the κ receptor, produced hindlimb paralysis following intrathecal administration in rats. Taken together, these findings suggest that endogenous opioids, possibly acting at κ receptors in the spinal cord, may serve as pathophysiological factors in spinal cord injury.     

Cellular imaging of inflammation after experimental spinal cord injury

The ability to visualize the cellular inflammatory responses after experimental spinal cord injury (SCI) was investigated using a clinical 1.5-T magnetic resonance imaging scanner, a custom-built, high-strength gradient coil insert, a 3-D fast imaging employing steady-state acquisition (FIESTA) imaging sequence and a superparamagnetic iron oxide (SPIO) contrast agent. An "active labeling" approach was used, with SPIO administered intravenously at different time points following SCI. Our results show that this strategy can be used to visualize clusters of iron-labeled cells associated with the inflammatory response in SCI. Of particular importance for this application was the finding that in FIESTA images hemorrhage does not cause signal loss. In T2-weighted spin echo or T2*-weighted gradient-echo images, which are more commonly used to detect signal loss associated with SPIO, the signal loss associated with hemorrhage interferes with the detection of iron-induced signal loss. FIESTA, therefore, allowed us to discriminate between iron associated with blood products in hemorrhage that occurs in acute SCI and the iron associated with SPIO-labeled cells accumulating in the injured cord.     

Immunocytochemical study of the distribution of cytoskeletal proteins in neurons from mouse embryo spinal cord in culture

Monoclonal FITC and Rhodamine-labeled antibodies were used to investigate distribution of tubulin, actin, and neurofilament protein with a molecular weight of 160 kDa in neurons from mouse embryo spinal cord cultivated in a monolayer. It was found that migration of tubulin from the neuronal soma occurs during the course of nerve cell development. The extent to which neuronal processes became filled with tubulin varied according to the stage of cell differentiation. Neuronal actin concentration was negligible and was found at points along the processes close to focal contacts with lining cells. The main body of neurofilament protein is concentrated in the neuronal soma and proximal portions of neurites.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 618–623, 1988.     

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with³H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of³²P into purified neurofilament proteins when they were incubated with³²P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.     

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells

The goal of this research was to evaluate the roles of calpains and their interactions with the proteasome and the lysosome in degradation of individual sarcomeric and cytoskeletal proteins in cultured muscle cells. Rat L8-CID muscle cells, in which we expressed a transgene calpain inhibitor (CID), were used in the study. L8-CID cells were grown as myotubes after which the relative roles of calpain, proteasome and lysosome in total protein degradation were assessed during a period of serum withdrawal. Following this, the roles of proteases in degrading cytoskeletal proteins (desmin, dystrophin and filamin) and of sarcomeric proteins (alpha-actinin and tropomyosin) were assessed. Total protein degradation was assessed by release of radioactive tyrosine from pre-labeled myotubes in the presence and absence of protease inhibitors. Effects of protease inhibitors on concentrations of individual sarcomeric and cytoskeletal proteins were assessed by Western blotting. Inhibition of calpains, proteasome and lysosome caused 20, 62 and 40% reductions in total protein degradation (P<0.05), respectively. Therefore, these three systems account for the bulk of degradation in cultured muscle cells. Two cytoskeletal proteins were highly-sensitive to inhibition of their degradation. Specifically, desmin and dystrophin concentrations increased markedly when calpain, proteasome and lysosome activities were inhibited. Conversely, sarcomeric proteins (alpha-actinin and tropomyosin) and filamin were relatively insensitive to the addition of protease inhibitors to culture media. These data demonstrate that proteolytic systems work in tandem to degrade cytoskeletal and sarcomeric protein complexes and that the cytoskeleton is more sensitive to inhibition of degradation than the sarcomere. Mechanisms, which bring about changes in the activities of the proteases, which mediate muscle protein degradation are not known and represent the next frontier of understanding needed in muscle wasting diseases and in muscle growth biology.     

Clonidine in spinal cord injury.

A 16-year-old girl, one of dizygotic twins, presented in 1976 complaining of a 1-year history of a lack of coordination and an inability to run. The results of biochemical tests confirmed the diagnosis of classic abetalipoproteinemia. In addition to the recognized neurologic features of this disorder, she had a reduced evoked motor unit potential and markedly elevated serum levels of muscle enzymes, which suggested myositis. The serum vitamin E level was markedly decreased. Oral therapy with vitamin E, 800 mg daily, was begun, and in 1981 the dosage was increased to 3200 mg daily. Over the 7 years of follow-up she improved clinically, there was an increase in the evoked motor unit potential, the serum levels of some of the muscle enzymes decreased to normal, and the serum and tissue vitamin E levels increased significantly. It was concluded that treatment with high doses of vitamin E was responsible for the arrest of the usually progressive neuropathy and myopathy.     

Regenerative treatment in spinal cord injury

Spinal cord injury is a devastating, traumatic event, and experienced mainly among young people. Until the modern era, spinal cord injury was so rapidly fatal that no seriously injured persons would survive long enough for regeneration to occur. Treatment of spinal cord injury can be summarized as follows: prevent further cord injury, maintain blood flow, relieve spinal cord compression, and provide secure vertebral stabilization so as to allow mobilization and rehabilitation, none of which achieves functional recovery. Previous studies have focused on analyzing the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as the tissue damage and neural cell death associated with secondary injury. Now, there are hundreds of current experimental and clinical regenerative treatment studies. One of the most popular treatment method is cell transplantation in injured spinal cord. For this purpose bone marrow stromal cells, mononuclear stem cells, mesenchymal stem cells, embryonic stem cells, neural stem cells, and olfactory ensheathing cells can be used. As a result, cell transplantation has become a promising therapeutic option for spinal cord injury patients. In this paper we discuss the effectiveness of stem cell therapy in spinal cord injury.     

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.     

Metabolite profiles correlate closely with neurobehavioral function in experimental spinal cord injury in rats

Traumatic spinal cord injury (SCI) results in direct physical damage and the generation of local factors contributing to secondary pathogenesis. Untargeted metabolomic profiling was used to uncover metabolic changes and to identify relationships between metabolites and neurobehavioral functions in the spinal cord after injury in rats. In the early metabolic phase, neuronal signaling, stress, and inflammation-associated metabolites were strongly altered. A dynamic inflammatory response consisting of elevated levels of prostaglandin E2 and palmitoyl ethanolamide as well as pro- and anti-inflammatory polyunsaturated fatty acids was observed. N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) were significantly decreased possibly reflecting neuronal cell death. A second metabolic phase was also seen, consistent with membrane remodeling and antioxidant defense response. These metabolomic changes were consistent with the pathology and progression of SCI. Several metabolites, including NAA, NAAG, and the ω-3 fatty acids docosapentaenoate and docosahexaenoate correlated greatly with the established Basso, Beattie and Bresnahan locomotive score (BBB score). Our findings suggest the possibility of a biochemical basis for BBB score and illustrate that metabolites may correlate with neurobehavior. In particular the NAA level in the spinal cord might provide a meaningful biomarker that could help to determine the degree of injury severity and prognosticate neurologic recovery.     

Mechanisms of inflammation in spinal cord injury

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI-induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real-time PCR and immunoblots, respectively. Both the contusion injured animals and the time-matched sham controls exhibited elevated pro-enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post-SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post-SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real-time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up-regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

PTEN和mTOR信号转导通路在实验性脊髓损伤中的表达

目的:研究mTOR和PTEN蛋白在实验性脊髓损伤中的表达及其在脊髓损伤发生、发展中的作用.方法:用免疫组织化学方法和RT-PCR法,检测脊髓组织内mTOR和PTEN的表达.结果:与正常组织相比,免疫组化法和RT-PCR两种方法结果均显示,脊髓损伤后mTOR表达明显下降,而PTEN的表达明显增加;两者呈负相关(r=0.862,P＜0.01).结论:PI3 K/PTEN/AKT/mTOR信号转导通路中重要的调节位点和节点PTEN在实验性脊髓损伤中的表达明显增高,而mTOR的表达明显降低.提示该信号转导通路在介导实验性脊髓损伤的发生、发展的过程中起重要作用.     

Biocompatible hydrogels in spinal cord injury repair

Spinal cord injury results in a permanent neurological deficit due to tissue damage. Such a lesion is a barrier for "communication" between the brain and peripheral tissues, effectors as well as receptors. One of the primary goals of tissue engineering is to bridge the spinal cord injury and re-establish the damaged connections. Hydrogels are biocompatible implants used in spinal cord injury repair. They can create a permissive environment and bridge the lesion cavities by providing a scaffold for the regeneration of neurons and their axons, glia and other tissue elements. The advantage of using artificial materials is the possibility to modify their physical and chemical properties in order to develop the best implant suitable for spinal cord injury repair. As a result, several types of hydrogels have been tested in experimental studies so far. We review our work that has been done during the last 5 years with various types of hydrogels and their applications in experimental spinal cord injury repair.     

Brainstem-evoked muscle potentials: their prognostic value in experimental spinal cord injury in the rat

Recording myoelectric motor-evoked potentials is frequently used as an in vivo evaluation technique in experimental studies of spinal cord injury (SCI). The aim of the present study was to determine whether specific neuronal pathways conduct these potentials. Stainless steel screws were permanently implanted into the cranium of 18 rats for stimulation of brainstemevoked muscle potentials (B-MPs). Twelve rats were subjected to spinal cord lesions that restricted the continuity of the spinal cord to different discrete sections of the lateral and/or ventral white matter (WM) of the left hemicord. Sham rats ( n = 6) were subjected to laminectomy only. Left hind limb B-MPs and motor function (open field walking test) were recorded before surgery and weekly thereafter for six consecutive weeks. Motor function was severely affected by SCI in all rats but recovered significantly during the first 14 postoperative days. The degree of functional recovery depended not only on the amount of spared WM but also on the particular section of WM that had been spared. In contrast, B-MP amplitudes also were severely reduced by SCI, but did not recover during the survival period. Moreover, B-MP amplitudes correlated only weakly with the amount of spared WM and were not influenced by which section of WM had been spared. While functional recovery correlated significantly with the amount of spared WM, no correlation was found between B-MP amplitudes and functional recovery. B-MP conduction velocities were not affected by SCI. It is therefore believed that B-MPs have little prognostic value for experimental studies of SCI in the rat.     

Brainstem-evoked muscle potentials: their prognostic value in experimental spinal cord injury in the rat

Recording myoelectric motor-evoked potentials is frequently used as an in vivo evaluation technique in experimental studies of spinal cord injury (SCI). The aim of the present study was to determine whether specific neuronal pathways conduct these potentials. Stainless steel screws were permanently implanted into the cranium of 18 rats for stimulation of brainstem-evoked muscle potentials (B-MPs). Twelve rats were subjected to spinal cord lesions that restricted the continuity of the spinal cord to different discrete sections of the lateral and/or ventral white matter (WM) of the left hemicord. Sham rats (n = 6) were subjected to laminectomy only. Left hind limb B-MPs and motor function (open field walking test) were recorded before surgery and weekly thereafter for six consecutive weeks. Motor function was severely affected by SCI in all rats but recovered significantly during the first 14 postoperative days. The degree of functional recovery depended not only on the amount of spared WM but also on the particular section of WM that had been spared. In contrast, B-MP amplitudes also were severely reduced by SCI, but did not recover during the survival period. Moreover, B-MP amplitudes correlated only weakly with the amount of sparedWM and were not influenced by which section ofWM had been spared. While functional recovery correlated significantly with the amount of spared WM, no correlation was found between B-MP amplitudes and functional recovery. B-MP conduction velocities were not affected by SCI. It is therefore believed that B-MPs have little prognostic value for experimental studies of SCI in the rat.     

Pregnancy following spinal cord injury.

Each year about 2,000 women of childbearing age in the United States have a spinal cord injury. Only a few mostly anecdotal reports describe pregnancy after such an injury. In a retrospective study of 16 women with a spinal cord injury, half of whom have a complete injury and about half quadriplegia, 25 pregnancies occurred, with 21 carried to full term. The women delayed pregnancy an average of 6.5 years after their injury, with an average age at first pregnancy of 26.8 years. Cesarean section was necessary in 4 patients because of inadequate progress of labor. In 5 deliveries an episiotomy and local anesthesia were required, 7 required epidural anesthesia, including all cesarean sections, and 10 did not require anesthesia. Several complications have been identified in the antepartum, intrapartum, and postpartum periods including autonomic hyperreflexia, premature labor, pressure sores, urinary tract infections, abnormal presentation, and failure to progress. Ultrasonography and amniocentesis were used selectively. Women with spinal cord injuries can have healthy children, although there are significant risks and these women have special needs.