Surgical intervention combined with weight-bearing walking training promotes recovery in patients with chronic spinal cord injury:a randomized controlled study

For patients with chronic spinal cord injury,the co nventional treatment is rehabilitation and treatment of spinal cord injury complications such as urinary tract infection,pressure sores,osteoporosis,and deep vein thrombosis.Surgery is rarely perfo rmed on spinal co rd injury in the chronic phase,and few treatments have been proven effective in chronic spinal cord injury patients.Development of effective therapies fo r chronic spinal co rd injury patients is needed.We conducted a randomized controlled clinical trial in patients with chronic complete thoracic spinal co rd injury to compare intensive rehabilitation(weight-bearing walking training)alone with surgical intervention plus intensive rehabilitation.This clinical trial was registered at ClinicalTrials.gov(NCT02663310).The goal of surgical intervention was spinal cord detethering,restoration of cerebrospinal fluid flow,and elimination of residual spinal cord compression.We found that surgical intervention plus weight-bearing walking training was associated with a higher incidence of American Spinal Injury Association Impairment Scale improvement,reduced spasticity,and more rapid bowel and bladder functional recovery than weight-bearing walking training alone.Overall,the surgical procedures and intensive rehabilitation were safe.American Spinal Injury Association Impairment Scale improvement was more common in T7-T11 injuries than in T2-T6 injuries.Surgery combined with rehabilitation appears to have a role in treatment of chronic spinal cord injury patients.

Are newborn rat-derived neural stem cells more sensitive to lead neurotoxicity?

Lead ion （Pb2＋） has been proven to be a neurotoxin due to its neurotoxicity on mammalian nervous system, especially for the developing brains of juveniles. However, many reported studies involved the negative effects of Pb2＋ on adult neural cells of humans or other mammals, only few of which have examined the effects of Pb2＋ on neural stem cells. The purpose of this study was to reveal the biological effects of Pb2＋from lead acetate [Pb （0H30OO）2] on viability, proliferation and differentiation of neural stem cells derived from the hippocampus of newborn rats aged 7 days and adult rats aged 90 days, respectively. This study was carried out in three parts. In the first part, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay （MTT viability assay） was used to detect the effects of Pb2＋ on the cell viability of passage 2 hippocampal neural stem cells after 48-hour exposure to 0-200 pM Pb2＋. In the second part, 10 pM bromodeoxyuridine was added into the culture medium of passage 2 hippocampal neural stem cells after 48-hour exposure to 0- 200 pM Pb2＋, followed by immunocytochemical staining with anti-bromodeoxyuridine to demonstrate the effects of Pb2＋ on cell proliferation. In the last part, passage 2 hippocampal neural stem cells were allowed to grow in the differentiation medium with 0-200 pM Pb2＋. Immunocytochemical staining with anti-microtubule-associated protein 2 （a neuron marker）, anti-glial fibrillary acidic protein （an astrocyte marker）, and anti-RIP （an oligodendrocyte marker） was performed to detect the differentiation commitment of affected neural stem cells after 6 days. The data showed that Pb2~ inhibited not only the viability and proliferation of rat hippocampal neural stem cells, but also their neuronal and oligodendrocyte differentiation in vitro. Moreover, increased activity of astrocyte differentiation of hippocampal neural stem cells from both newborn and adult rats was observed after exposure to high concentration of lead ion in vitro. These findings suggest that hippocampal neural stem cells of newborn rats were more sensitive than those from adult rats to Pb2＋cytotoxicity.

A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Surgical intervention combined with weight-bearing walking training improves neurological recoveries in 320 patients with clinically complete spinal cord injury:a prospective self-controlled study

Although a large number of trials in the SCI field have been conducted,few proven gains have been realized for patients.In the present study,we determined the efficacy of a novel combination treatment involving surgical intervention and long-term weight-bearing walking training in spinal cord injury(SCI)subjects clinically diagnosed as complete or American Spinal Injury Association Impairment Scale(AIS)Class A(AIS-A).A total of 320 clinically complete SCI subjects(271 male and 49 female),aged 16–60 years,received early(≤7 days,n=201)or delayed(8–30 days,n=119)surgical interventions to reduce intraspinal or intramedullary pressure.Fifteen days post-surgery,all subjects received a weight-bearing walking training with the“Kunming Locomotion Training Program(KLTP)”for a duration of 6 months.The neurological deficit and recovery were assessed using the AIS scale and a 10-point Kunming Locomotor Scale(KLS).We found that surgical intervention significantly improved AIS scores measured at 15 days post-surgery as compared to the pre-surgery baseline scores.Significant improvement of AIS scores was detected at 3 and 6 months and the KLS further showed significant improvements between all pair-wise comparisons of time points of 15 days,3 or 6 months indicating continued improvement in walking scores during the 6-month period.In conclusion,combining surgical intervention within 1 month post-injury and weight-bearing locomotor training promoted continued and statistically significant neurological recoveries in subjects with clinically complete SCI,which generally shows little clinical recovery within the first year after injury and most are permanently disabled.This study was approved by the Science and Research Committee of Kunming General Hospital of PLA and Kunming Tongren Hospital,China and registered at ClinicalTrials.gov(Identifier:NCT04034108)on July 26,2019.

ISP and PAP4 peptides promote motor functional recovery after peripheral nerve injury

Both intracellular sigma peptide(ISP) and phosphatase and tensin homolog agonist protein(PAP4) promote nerve regeneration and motor functional recovery after spinal cord injury. However, the role of these two small peptides in peripheral nerve injury remains unclear. A rat model of brachial plexus injury was established by crush of the C6 ventral root. The rats were then treated with subcutaneous injection of PAP4(497 μg/d, twice per day) or ISP(11 μg/d, once per day) near the injury site for 21 successive days. After ISP and PAP treatment, the survival of motoneurons was increased, the number of regenerated axons and neuromuscular junctions was increased, muscle atrophy was reduced, the electrical response of the motor units was enhanced and the motor function of the injured upper limbs was greatly improved in rats with brachial plexus injury. These findings suggest that ISP and PAP4 promote the recovery of motor function after peripheral nerve injury in rats. The animal care and experimental procedures were approved by the Laboratory Animal Ethics Committee of Jinan University of China(approval No. 20111008001) in 2011.

Calcitonin gene-related peptide in anterior and posterior horns of spinal cord after brachial plexus injury

BACKGROUND： The changes of calcitonin gene-related peptide （CGRP） expression are closely associated with peripheral nerve injury, whereas it should be further investigated whether the damage of central nerve can lead to the changes of CGRP expression, and whether it is associated with the neural regeneration and repair. OBJECTIVE： To observe the changing law of CGRP expression in the anterior and posterior horns of spinal cord following brachial plexus injury. DESIGN： A randomized controlled trial. SETTINGS： Department of Anatomy, Yunyang Medical College; Department of Anatomy, Basic Medical College, Sun Yat-sen University. MATERIALS： Sixty-five adult male SD rats of clean degree, weighing 180 - 220 g, provided by the experimental animal center of the Basic Medical College, Sun Yat-sen University, were randomly divided into control group （n =5） and experimental group （n =60）, and the latter was subdivided into three damage groups： avulsion of anterior root group （n =20）, disjunction of posterior root group （n =20） and transection of spinal cord group （n =20）. Diaminobenzidine （DAB） chromogen, rabbit anti-CGRP polyclonal antibody were the products of Sigma Company; Leica image analytical apparatus was produced by QUIN Company （Germany）; Histotome by Sigma Company. METHODS： The experiments were carried out in the Department of Anatomy, Basic Medical College, Sun Yat-sen University from September 2004 to March 2005. Three kinds of models of brachial plexus injury were established： In the avulsion of anterior root group, right C7 anterior root was avulsed, and the distal nerve residual root was transected. In the disjunction of posterior root group, right C7 anterior root was avulsed and right C5 - T1 posterior horns were cut to block the sensory afferent pathway. In the transection of spinal cord group, right C7 anterior root was avulsed and C5-6 segments of right spinal cord were semi-transected to block the cortical descending pathway. In the control group, C5 - T1 vertebral plates were prayed open, and then the skin was sutured. The C7 segments of spinal cord were removed on the 1^st, 3^rd, 7^th and 14^th days postoperatively respectively, and the CGRP expressions in the anterior and posterior horns of spinal cord were determined and analyzed using immunohistochemical method and image analysis. MAIN OUTCOME MEASURES： ① Number of CGRP immuno-positive motor neurons in the anterior horn of spinal cord; ② Total area ofCGRP immuno-positive fibers in the posterior horn of spinal cord. RESULTS： All the 65 rats were involved in the analysis of results. ① Number of CGRP immuno-positive motor neurons in the anterior horn of spinal cord： CGRP immuno-positive motor neurons could be observed in the anterior horns of C7 spinal cord in the control group and damage groups, the neurons had big cell body with stained cytoplasm, appeared as brown granules, and mainly distributed in the ventral lateral anterior horn of spinal cord. On the Ist day postoperatively, the number of CGRP positive neurons was obviously higher in the in the avulsion of anterior root group than in the control group （P 〈 0.01）, whereas obviously lower in the disjunction of posterior root group than in the control group （P 〈 0.01）, and there was no obvious difference between the transection of spinal cord group and the control group （P 〉 0.05）. On the 7^th day, the numbers of CGRP positive neurons in the damage groups were obviously higher than that in the control group （P 〈 0.01）, also obviously different from those on the 1^st day in the same group respectively （P 〈 0.01）. On the 14^th day, the number of CGRP positive neurons in the disjunction of posterior root group was decreased, but there was no obvious difference as compared with that in the control group, whereas those in the avulsion of anterior root group and transection of spinal cord group were still obviously higher than that in the control group （P 〈 0.01）. The number of CGRP positive neurons was the most in the avulsion of anterior root group, followed by the transection of spinal cord group, and the least in the disjunction of posterior root group, and there were significant differences among them （P 〈 0.01）. ② Total area of CGRP immuno-positive fibers in the posterior horn of spinal cord： Dense CGRP immuno-positive nerve fibers distributed in the layers Ⅰ and Ⅱ of the C7 posterior horn of spinal cord in the control group. On the 1^st day postoperatively, the total areas of CGRP positive fibers in the avulsion of anterior root group and transection of spinal cord group were obviously larger than that in the control group （P 〈 0.01）, whereas there was no obvious difference between the disjunction of posterior root group and control group. On the 7^th day, the CGRP expression in the posterior horn of spinal cord decreased to the lowest level in the disjunction of posterior root group, whereas there were no obvious differences in the avulsion of anterior root group and transection of spinal cord group as compared with that in the control group （P 〉 0.05）. On the 14^th day, the area continued to decrease in the avulsion of anterior root group and transection of spinal cord group, and it was obviously lower in the transection of spinal cord group than in the control group （P 〈 0.01）, and it was slightly increased in the disjunction of posterior root group as compared with that on 7^th day, but still obviously lower than that in the control group （P 〈 0.01）. CONCLUSION： The expression and role of CGRP are in discrepancy in the anterior and posterior horns of spinal cord after brachial plexus injury. The CGRP in anterior horn of spinal cord are derived from the cell body of motor neurons, and may be involved in the repairing mechanism of nerve injury regeneration; Whereas those in the posterior horn are mainly derived from posterior root ganglion, and may be associated with the conduction of noxious stimulations.

Spinal root avulsion:an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Spinal root avulsion is an excellent model for studying the re- sponse of motoneurons to severe injury to their axons （Koliat- sos et al., 1994）. In this model （‘Avulsion Model＇）, spinal roots are torn off from spinal cord without removing the vertebra at different levels of spinal segments, usually at cervical and lum- bar segments. Step-by-step procedures are described in detail elsewhere （Chu and Wu, 2009）. The Avulsion Model resembles very well brachial plexus injuries in human beings. Around 70% of severe brachial plexus injuries in human involved avulsion of one or more roots （Narakas, 1985） and the main causes of traumatic brachial plexus injuries were motor vehicle accidents, sport injuries and difficult deliveries （Terzis et al., 2001）. The Avulsion Model involves injury to both central nervous system （CNS） and peripheral nervous system （PNS） while nerve axoto- my, transection and crush injuries only involve PNIS.

Self-assembling peptide nanofibrous hydrogel as a promising strategy in nerve repair after traumatic injury in the nervous system

Following injury in central nervous system（CNS）,there are pathological changes in the injured region,which include neuronal death,axonal damage and demyelination,inflammatory response and activation of glial cells.The proliferation of a large number of astrocytes results in the formation of glial scar.

P75 and phosphorylated c-Jun are differentially regulated in spinal motoneurons following axotomy in rats

The neurotrophin receptor （p75） activates the c-Jun N-terminal kinase （JNK） pathway. Activation of JNK and its substrate c-Jun can cause apoptosis. Here we evaluate the role of p75 in spinal motoneurons by comparing immunoreactivity for p75 and phosphorylated c-Jun （p-c-Jun）, the production of JNK activation in axotomized motoneurons in postnatal day （PN）I, PN7, PN14 and adult rats. Intensive p-c-Jun was induced in axotomized motoneurons in PN1 and PN7. In PN14, p-c-Jun expression was sharply reduced after the same injury. The decreased expression of p-c-Jun at this age coincided with a developmental switch of re-expression of p75 in axotomized cells. In adult animals, no p-c-Jun but intensive p75 was detected in axotomized motoneurons. These results indicate differential expression or turnover of phosphorylation of c-Jun and p75 in immature versus mature spinal motoneurons in response to axonal injury. The non-co-occurrence of p75 and p-c-Jun in injured motoneurons indicated that p75 may not activate JNK pathway, suggesting that the p75 may not be involved in cell death in axotomized motoneurons.

Spastin interacts with collapsin response mediator protein 3 to regulate neurite growth and branching

Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury.Spastin plays an important role in the regulation of microtubule severing.Both spastin and collapsin response mediator proteins can regulate neurite growth and branching;however,whether spastin interacts with collapsin response mediator protein 3(CRMP3)during this process remains unclear,as is the mechanism by which CRMP3 participates in the repair of spinal cord injury.In this study,we used a proteomics approach to identify key proteins associated with spinal cord injury repair.We then employed liquid chromatography-mass spectrometry to identify proteins that were able to interact with glutathione S-transferase-spastin.Then,co-immunoprecipitation and staining approaches were used to evaluate potential interactions between spastin and CRMP3.Finally,we co-transfected primary hippocampal neurons with CRMP3 and spastin to evaluate their role in neurite outgrowth.Mass spectrometry identified the role of CRMP3 in the spinal cord injury repair process.Liquid chromatography-mass spectrometry pulldown assays identified three CRMP3 peptides that were able to interact with spastin.CRMP3 and spastin were co-expressed in the spinal cord and were able to interact with one another in vitro and in vivo.Lastly,CRMP3 overexpression was able to enhance the ability of spastin to promote neurite growth and branching.Therefore,our results confirm that spastin and CRMP3 play roles in spinal cord injury repair by regulating neurite growth and branching.These proteins may therefore be novel targets for spinal cord injury repair.The Institutional Animal Care and Use Committee of Jinan University,China approved this study(approval No.IACUS-20181008-03)on October 8,2018.

Time-lapse changes of in vivo injured neuronal substructures in the central nervous system after low energy two-photon nanosurgery

There is currently very little research regarding the dynamics of the subcellular degenerative events that occur in the central nervous system in response to injury. To date, multi-photon excitation has been primarily used for imaging applications; however, it has been recently used to selectively disrupt neural structures in living animals. However, understanding the complicated processes and the essential underlying molecular pathways involved in these dynamic events is necessary for studying the underlying process that promotes neuronal regeneration. In this study, we introduced a novel method allowing in vivo use of low energy（less than 30 m W） two-photon nanosurgery to selectively disrupt individual dendrites, axons, and dendritic spines in the murine brain and spinal cord to accurately monitor the time-lapse changes in the injured neuronal structures. Individual axons, dendrites, and dendritic spines in the brain and spinal cord were successfully ablated and in vivo imaging revealed the time-lapse alterations in these structures in response to the two-photon nanosurgery induced lesion. The energy（less than 30 m W） used in this study was very low and caused no observable additional damage in the neuronal sub-structures that occur frequently, especially in dendritic spines, with current commonly used methods using high energy levels. In addition, our approach includes the option of monitoring the time-varying dynamics to control the degree of lesion. The method presented here may be used to provide new insight into the growth of axons and dendrites in response to acute injury.

Substance P mRNA expression in the rat spinal cord following selective brachial plexus injury

BACKGROUND： The neuropeptide, substance P, has various bioactivities and is widely distributed in the central nervous system. Substance P participates in neural transmission in the spinal cord and plays an important role in regeneration and repair of nerve injury. OBJECTIVE： To investigate substance P mRNA expression in the anterior horn of the spinal cord following brachial plexus injury. DESIGN, TIME AND SETTING： A molecular cell biology randomized controlled study was performed at the Department of Anatomy, Zhongshan Medical College, Sun Yat-sen University and the DaAn Gene Laboratory in May 2005. MATERIALS： A total of 29 adult male Sprague Dawley rats were randomly assigned to a control group （n = 5） and an injury group （n = 24）. METHODS： The injury group was divided into three subgroups. In subgroup A, the right seventh cervical vertebra （C7） anterior root was avulsed, and the residual nerve root at the distal end was removed. In subgroup B, the right C7 anterior root was avulsed, and the right C5 first thoracic vertebrae （T1） posterior root was incised. Thus afferent pathways of the posterior root that connected with the anterior horn motor neurons were blocked. In subgroup C, the right C7 anterior root was avulsed, and a right C56 hemisection was performed. Thus the descending fiber pathways of the cortex that connected with anterior horn motor neurons were blocked. In the control group, the C5-T1 vertebral plate was opened, and then the skin was sutured. MAIN OUTCOME MEASURE： Substance P mRNA expression in the anterior horn of the spinal cord was quantified using fluorescent quantitative reverse transcription-polymerase chain reaction. RESULTS： Substance P mRNA expression was low in the anterior horn of the rat spinal cord in the control group. Substance P mRNA expression in the anterior horn of the spinal cord was upregulated and was significantly higher in the injury group compared with the control group （P 〈 0.01）. Substance P mRNA expression was highest in subgroup B. CONCLUSION： Brachial plexus anterior root avulsion is responsible for increased substance P expression in the anterior horn of the rat spinal cord. Pathway disjunction in efferent fibers of the posterior root or cortex does not have an effect on substance P expression in the anterior horn of the spinal cord.

Three-dimensional histology:new visual approaches to morphological changes during neural regeneration

Three-dimensional（3D） histology utilizes tissue clearing techniques to turn intact tissues transparent,allowing rapid interrogation of tissue architecture in three dimensions.In this article,we summarized the available tissue clearing methods and classified them according to their physicochemical principles of operation,which provided a framework for one to choose the best techniques for various research settings.Recent attempts in addressing various questions regarding the degenerating and regenerating nervous system have been promising with the use of 3D histological techniques.

Functional self-assembling peptide nanofiber hydrogel for peripheral nerve regeneration

Peripheral nerves are fragile and easily damaged,usually resulting in nervous tissue loss,motor and sensory function loss.Advances in neuroscience and engineering have been significantly contributing to bridge the damage nerve and create permissive environment for axonal regrowth across lesions.We have successfully designed two self-assembling peptides by modifying RADA 16-I with two functional motifs IKVAV and RGD.Nanofiber hydrogel formed when combing the two neutral solutions together,defined as RADA 16-Mix that overcomes the main drawback of RADA16-I associated with low pH.In the present study,we transplanted the RADA 16-Mix hydrogel into the transected rat sciatic nerve gap and effect on axonal regeneration was examined and compared with the traditional RADA16-I hydrogel.The regenerated nerves were found to grow along the walls of the large cavities formed in the graft of RADA16-I hydrogel,while the nerves grew into the RADA 16-Mix hydrogel toward distal position.RADA 16-Mix hydrogel induced more axons regeneration and Schwann cells immigration than RADA16-I hydrogel,resulting in better functional recovery as determined by the gait-stance duration percentage and the formation of new neuromuscular junction structures.Therefore,our results indicated that the functional SAP RADA16-Mix nanofibrous hydrogel provided a better environment for peripheral nerve regeneration than RADA16-I hydrogel and could be potentially used in peripheral nerve injury repair.

Contrasting neuropathology and functional recovery after spinal cord injury in developing and adult rats

Conflicting findings exist regarding the link between functional recovery and the regrowth of spinal tracts across the lesion leading to the restoration of functional contacts. In the present study, we investigated whether functional locomotor recovery was attributable to anatomical regeneration at postnatal day 1 （PN1）, PN7, PN14 and in adult rats two months after transection injury at the tenth thoracic segment of the spinal cord. The Basso, Beattie, and Bresnahan scores showed that transection led to a failure of hindlimb locomotor function in PN14 and adult rats. However, PN1 and PN7 rats showed a significant level of stepping function after complete spinal cord transection. Unexpectedly, unlike the transected PN14 and adult rats in which the spinal cord underwent limited secondary degeneration and showed a scar at the lesion site, the rats transected at PN1 and PN7 showed massive secondary degeneration both anterograde and retrograde, leaving a 〉5-mm gap between the two stumps. Furthermore, retrograde tracing with fluorogold （FG） also showed that FG did not cross the transection site in PN1 and PN7rats as in PN14 and adult rats, and re-transection of the cord caused no apparent loss in locomotor performance in the rats transected at PN1. Thus, these three lines of evidence strongly indicated that the functional recovery after transection in neonatal rats is independent of regrowth of spinal tracts across the lesion site. Our results support the notion that the recovery of locomotor function in developing rats may be due to intrinsic adaptations in the spinal circuitry below the lesion that control hindlimb locomotor activity rather than the regrowth of spinal tracts across the lesion. The difference in secondary degeneration between neonatal and adult rats remains to be explored.

Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array

Parallelized fluorescence imaging has been a long-standing pursuit that can address the unmet need for a comprehensive three-dimensional(3D)visualization of dynamical biological processes with minimal photodamage.However,the available approaches are limited to incomplete parallelization in only two dimensions or sparse sampling in three dimensions.We hereby develop a novel fluorescence imaging approach,called coded light-sheet array microscopy(CLAM),which allows complete parallelized 3D imaging without mechanical scanning.Harnessing the concept of an“infinity mirror”,CLAM generates a light-sheet array with controllable sheet density and degree of coherence.Thus,CLAM circumvents the common complications of multiple coherent light-sheet generation in terms of dedicated wavefront engineering and mechanical dithering/scanning.Moreover,the encoding of multiplexed optical sections in CLAM allows the synchronous capture of all sectioned images within the imaged volume.We demonstrate the utility of CLAM in different imaging scenarios,including a light-scattering medium,an optically cleared tissue,and microparticles in fluidic flow.CLAM can maximize the signal-to-noise ratio and the spatial duty cycle,and also provides a further reduction in photobleaching compared to the major scanning-based 3D imaging systems.The flexible implementation of CLAM regarding both hardware and software ensures compatibility with any light-sheet imaging modality and could thus be instrumental in a multitude of areas in biological research.