Assessment of hindlimb locomotor strength in spinal cord transected rats through animal-robot contact force

Robotic locomotor training devices have gained popularity in recent years, yet little has been reported regarding contact forces experienced by the subject performing automated locomotor training, particularly in animal models of neurological injury. The purpose of this study was to develop a means for acquiring contact forces between a robotic device and a rodent model of spinal cord injury through instrumentation of a robotic gait training device (the rat stepper) with miniature force/torque sensors. Sensors were placed at each interface between the robot arm and animal's hindlimb and underneath the stepping surface of both hindpaws (four sensors total). Twenty four female, Sprague-Dawley rats received mid-thoracic spinal cord transections as neonates and were included in the study. Of these 24 animals, training began for 18 animals at 21 days of age and continued for four weeks at five min/day, five days/week. The remaining six animals were untrained. Animal-robot contact forces were acquired for trained animals weekly and untrained animals every two weeks while stepping in the robotic device with both 60 and 90% of their body weight supported (BWS). Animals that received training significantly increased the number of weight supported steps over the four week training period. Analysis of raw contact forces revealed significant increases in forward swing and ground reaction forces during this time, and multiple aspects of animal-robot contact forces were significantly correlated with weight bearing stepping. However, when contact forces were normalized to animal body weight, these increasing trends were no longer present. Comparison of trained and untrained animals revealed significant differences in normalized ground reaction forces (both horizontal and vertical) and normalized forward swing force. Finally, both forward swing and ground reaction forces were significantly reduced at 90% BWS when compared to the 60% condition. These results suggest that measurement of animal-robot contact forces using the instrumented rat stepper can provide a sensitive and reliable measure of hindlimb locomotor strength and control of flexor and extensor muscle activity in neurologically impaired animals. Additionally, these measures may be useful as a means to quantify training intensity or dose-related functional outcomes of automated training.     

伸长细胞移植对大鼠脊髓损伤后运动功能及运动诱发电位的影响

目的:研究伸长细胞是否可以促进成年大鼠脊髓损伤后传导束再生。方法:采用Wistar大鼠脊髓T8全横断模型,移植传代培养的伸长细胞,以未移植脊髓损伤组为对照,观察两组损伤后第12周末BBB评分,损伤平面以下红核-脊髓运动诱发电位,和横断部位组织学染色结果。结果:第12周末伸长细胞移植组红核脊髓运动诱发电位总峰值显著高于对照组(MD=133.2μV,P0.01),峰潜伏期较对照组缩短(MD=0.061ms,P=0.040);第12周末伸长细胞移植组BBB评分显著高于对照组(MD=5.0000,P0.01);第12周末脊髓横断部位HE染色显示伸长细胞移植组脊髓损伤处结构较完整。结论:伸长细胞移植可以促进大鼠脊髓损伤后神经传导的恢复。     

Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: Long-term survival and functional recovery in canines

Science China Life Sciences - Spinal cord injury (SCI), especially complete transected SCI, leads to loss of cells and extracellular matrix and functional impairments. In a previous study, we...     

Transplantation of adult spinal cord tissue: Transection spinal cord repair and potential clinical translation

Science China Life Sciences -     

Neurotrophin expressions in neural stem cells grafted acutely to transected spinal cord of adult rats linked to functional improvement

It is well known that neural stem cells (NSC) could promote the repairment after spinal cord injury, but the underlying mechanism remains to be elucidated. This study showed that the transplantation of NSC significantly improved hindlimb locomotor functions in adult rats subjected to transection of the spinal cord. Biotin dextran amine tracing together with the stimulus experiment in motor sensory area showed that little CST regeneration existed and functional synaptic formation in the injury site. Immunocytochemistry and RT-PCR demonstrated the secretion of NGF, BDNF, and NT-3 by NSC in vitro and in vivo, respectively. However, only mRNA expression of BDNF and NT-3 but not NGF in injury segment following NSC transplantation was upregulated remarkably, while caspase-3, a crucial apoptosis gene, was downregulated simultaneously. These provided us a clue that the functional recovery was correlated with the regulation of BDNF, NT-3, and caspase-3 in spinal cord transected rats following NSC transplantation.     

Implantation of embryonic neocortex and spinal cord into injured peripheral nerve of adult rats

Spinal cord and cerebral cortex of 14-day-old embryos of Wistar rats were implanted into the sciatic nerve of mature rats in order to study dynamics of the development of neuronal and neuroglial elements in ectopic sites. By means of light and electron microscopy it has been stated that the implanted nerve cells of the cortex and spinal cord survive during 5 month and differentiate from neuroepithelial cells and neuroblasts up to young and mature neurons. It was found that thirty days after operation the spinal cord implants contained myelinated nerve fibers and numerous synapses. The data obtained suggest that the implants of fetal spinal cord are more favorable for regeneration of the injured nervous stems than the cerebral cortex.     

Transplantation of neural stem cells into the spinal cord after injury

Thanks to advances in the stem cell biology of the central nervous system (CNS), the previously inconceivable regeneration of the damaged CNS is approaching reality. The availability of signals to induce the appropriate differentiation of the transplanted and/or endogenous neural stem cells (NSCs) as well as the timing of the transplantation are important for successful functional recovery of the damaged CNS. Because the immediately post-traumatic microenvironment of the spinal cord is in an acute inflammatory stage, it is not favorable for the survival and differentiation of NSC transplants. On the other hand, in the chronic stage after injury, glial scars form in the injured site that inhibit the regeneration of neuronal axons. Thus, we believe that the optimal timing of transplantation is 1-2 weeks after injury.     

Activated satellite cells fail to restore myonuclear number in spinal cord transected and exercised rats

In this study, possible mechanisms underlying soleus muscleatrophy after spinal cord transection and attenuation of atrophy withcycling exercise were studied. Adult female Sprague-Dawley rats weredivided into three groups; in two groups the spinal cord was transectedby a lesion at T₁₀. One group wastransected and killed 10 days later, and another group was transectedand exercised for 5 days starting 5 days after transection. The third group served as an uninjured control. All animals received acontinuous-release 5'-bromo-2'-deoxyuridine pellet 10 daysbefore they were killed. Transection alone and transection withexercise lead to activation of satellite cells, but only the exercisegroup showed a trend toward an increase in the number of proliferatingsatellite cells. In all cases the number of activated satellite cellswas significantly higher than the number that divided. Although thenumber of cells undergoing proliferation increased with exercise, noincrease in fusion of satellite cells into muscle fibers was apparent. Spinal cord transection resulted in a 25% decrease in myonuclear number, and exercise was not associated with a restoration of myonuclear number. The number of apoptotic nuclei was increased aftertransection, and exercise attenuated this increase. However, thedecrease in apoptotic nuclei with exercise did not significantly affectmyonuclear number. We conclude that apoptotic nuclear loss likelycontributes to loss of nuclei during muscle atrophy associated withspinal cord transection and that exercise can maintain muscle mass, atleast in the short term, without restoration of myonuclear number.

     

Mesenchymal stem cell graft improves recovery after spinal cord injury in adult rats through neurotrophic and pro-angiogenic actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue.     

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5?cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.     

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5 cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.     

Deletion of mammalian sterile 20-like kinase 1 attenuates neuronal loss and improves locomotor function in a mouse model of spinal cord trauma

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.     

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats.

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric alpha-actin, cardiac alpha-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.     

Transplantation of human embryonal nervous tissue to the spinal cord of mature rats

Pieces of the wall obtained from the anterior cerebral bladder of human embryos at the age of 8-10 weeks can survive in the spinal cord of mature animals. In the transplant, unlike the normal embryonal histogenesis, neuroepithelial cells make groups of rosellas. The differentiation process of cells of the human nervous tissue transplant can be followed in the rat spinal cord without any immune suppression up to the end of the 2d month of development. During the 3d month the transplant neuroblasts perish as a result of the immune reaction.