Retinoid X receptor α downregulation is required for tail and caudal spinal cord regeneration in the adult newt

Some adult vertebrate species,such as newts,axolotls and zebrafish,have the ability to regenerate their central nervous system（CNS）.However,the factors that establish a permissive CNS environment for correct morphological and functional regeneration in these species are not well understood.Recent evidence supports a role for retinoid signaling in the intrinsic ability of neurons,in these regeneration-competent species,to regrow after CNS injury.Previously,we demonstrated that a specific retinoic acid receptor（RAR）subtype,RARβ,mediates the effects of endogenous retinoic acid（RA）on neuronal growth and guidance in the adult newt CNS after injury.Here,we now examine the expression of the retinoid X receptor RXRα（a potential heterodimeric transcriptional regulator with RARβ）,in newt tail and spinal cord regeneration.We show that at 21 days post-amputation（dpa）,RXRαis expressed at temporally distinct periods and in non-overlapping spatial domains compared to RARβ.Whereas RARβprotein levels increase,RXRαproteins level decrease by 21 dpa.A selective agonist for RXR,SR11237,prevents both this downregulation of RXRαand upregulation of RARβand inhibits tail and caudal spinal cord regeneration.Moreover,treatment with a selective antagonist for RARβ,LE135,inhibits regeneration with the same morphological consequences as treatment with SR11237.Interestingly,LE135 treatment also inhibits the normal downregulation of RXRαin tail and spinal cord tissues at 21 dpa.These results reveal a previously unidentified,indirect regulatory feedback loop between these two receptor subtypes in regulating the regeneration of tail and spinal cord tissues in this regeneration-competent newt.

Transcriptomic analysis of spinal cord regeneration after injury in Cynops orientalis

Cynops orientalis(C.orientalis)has a pronounced ability to regenerate its spinal cord after injury.Thus,exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cord regeneration.In this study,we established a model of spinal cord thoracic transection injury in C.orientalis,which is an endemic species in China.We performed RNA sequencing of the contused axolotl spinal cord at two early time points after spinal cord injury–during the very acute stage(4 days)and the subacute stage(7 days)–and identified differentially expressed genes;additionally,we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses,at each time point.Transcriptome sequencing showed that 13,059 genes were differentially expressed during C.orientalis spinal cord regeneration compared with uninjured animals,among which 4273 were continuously downregulated and 1564 were continuously up-regulated.Down-regulated genes were most enriched in the Gene Ontology term“multicellular organismal process”and in the ribosome pathway at 10 days following spinal cord injury.We found that multiple genes associated with energy metabolism were down-regulated and multiple genes associated with the lysosome were up-regulated after spinal cord injury,indicating the importance of low metabolic activity during wound healing.Immune response-associated pathways were activated during the early acute phase(4 days),while the expression of extracellular matrix proteins such as glycosaminoglycan and collagen,as well as tight junction proteins,was lower at 10 days post-spinal cord injury than 4 days post-spinal cord injury.However,compared with 4 days post-injury,at 10 days post-injury neuroactive ligand-receptor interactions were no longer down-regulated,up-regulated differentially expressed genes were enriched in pathways associated with cancer and the cell cycle,and SHH,VIM,and Sox2 were prominently up-regulated.Immunofluorescence staining showed that glial fibrillary acidic protein was up-regulated in axolotl ependymoglial cells after injury,similar to what is observed in mammalian astrocytes after spinal cord injury,even though axolotls do not form a glial scar during regeneration.We suggest that low intracellular energy production could slow the rapid amplification of ependymoglial cells,thereby inhibiting reactive gliosis,at early stages after spinal cord injury.Extracellular matrix degradation slows cellular responses,represses the expression of neurogenic genes,and reactivates a transcriptional program similar to that of embryonic neuroepithelial cells.These ependymoglial cells act as neural stem cells:they migrate and proliferate to repair the lesion and then differentiate to replace lost glial cells and neurons.This provides the regenerative microenvironment that allows axon growth after injury.

Targeted tissue engineering:hydrogels with linear capillary channels for axonal regeneration after spinal cord injury

Spinal cord injury（SCI）frequently results in the permanent loss of function below the level of injury due to the failure of axonal regeneration in the adult mammalian central nervous system（CNS）.The limited intrinsic growth capacity of adult neurons,a lack of growth-promoting factors and the multifactorial inhibitory microenvironment around the lesion site contribute to the lack of axonalregeneration. Strategies such as transplantation of cells,

Perspectives on “the biology of spinal cord regeneration success and failure”

In our recently co-authored Physiological Reviews manuscript entitled ＂The biology of regeneration failure and success after spinal cord injury＂ （Tran et al., 2018b）, we sought to provide a comprehensive and up-to-date de- scription of how the glial scar develops following spinal cord injury （SCI） to chronically inhibit axon regeneration.

Manipulating extrinsic and intrinsic obstacles to axonal regeneration after spinal cord injury

Spinal cord injury（SCI）is a traumatic event that can lead to permanent motor and sensory deficits.After the initial trauma,axons of surviving neurons rapidly retract.While there may be a small degree of abortive sprouting,virtually all attempts at robust regrowth across the lesion site ultimately fail.Thus,neurons below the level of the injury are permanently disconnected from their normal input,resulting in persistent loss of function.

Distribution of paired immunoglobulin-like receptor B in the nervous system related to regeneration difficulties after unilateral lumbar spinal cord injury

Paired immunoglobulin-like receptor B（Pir B） is a functional receptor of myelin-associated inhibitors for axonal regeneration and synaptic plasticity in the central nervous system, and thus suppresses nerve regeneration. The regulatory effect of Pir B on injured nerves has received a lot of attention. To better understand nerve regeneration inability after spinal cord injury, this study aimed to investigate the distribution of Pir B（via immunofluorescence） in the central nervous system and peripheral nervous system 10 days after injury. Immunoreactivity for Pir B increased in the dorsal root ganglia, sciatic nerves, and spinal cord segments. In the dorsal root ganglia and sciatic nerves, Pir B was mainly distributed along neuronal and axonal membranes. Pir B was found to exhibit a diffuse, intricate distribution in the dorsal and ventral regions. Immunoreactivity for Pir B was enhanced in some cortical neurons located in the bilateral precentral gyri. Overall, the findings suggest a pattern of Pir B immunoreactivity in the nervous system after unilateral spinal transection injury, and also indicate that Pir B may suppress repair after injury.

The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway

Rho-associated kinase（ROCK） is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase（ERK） signaling pathway,but its effect on microglial migration was unknown.Therefore,in this study,we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord,and we examined the underlying mechanisms.The microglia were treated with Y27632,fasudil and/or the ERK inhibitor U0126.Cellular morphology was observed by immunofluorescence.Transwell chambers were used to assess cell migration.ERK levels were measured by incell western blot assay.Y27632 and fasudil increased microglial migration,and the microglia were irregularly shaped and had many small processes.These inhibitors also upregulated the levels of phosphorylated ERK protein.The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil.These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.

Rho A/Rho kinase in spinal cord injury

A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury process involving inflammation, oxidation, excitotoxicity, and cell death. During the secondary injury, many signal pathways are activated and play important roles in mediating the pathogenesis of spinal cord injury. Among them, the Rho A/Rho kinase pathway plays a particular role in mediating spinal degeneration and regeneration. In this review, we will discuss the role and mechanism of Rho A/Rho kinase-mediated spinal cord pathogenesis, as well as the potential of targeting Rho A/Rho kinase as a strategy for promoting both neuroprotection and axonal regeneration.

Salvianolic acid B protects the myelin sheath around injured spinal cord axons

Salvianolic acid B,an active pharmaceutical compound present in Salvia miltiorrhiza,exerts a neuroprotective effect in animal models of brain and spinal cord injury.Salvianolic acid B can promote recovery of neurological function;however,its protective effect on the myelin sheath after spinal cord injury remains poorly understood.Thus,in this study,in vitro tests showed that salvianolic acid B contributed to oligodendrocyte precursor cell differentiation,and the most effective dose was 20 μg/m L.For in vivo investigation,rats with spinal cord injury were intraperitoneally injected with 20 mg/kg salvianolic acid B for 8 weeks.The amount of myelin sheath and the number of regenerating axons increased,neurological function recovered,and caspase-3 expression was decreased in the spinal cord of salvianolic acid B-treated animals compared with untreated control rats.These results indicate that salvianolic acid B can protect axons and the myelin sheath,and can promote the recovery of neurological function.Its mechanism of action is likely to be associated with inhibiting apoptosis and promoting the differentiation and maturation of oligodendrocyte precursor cells.

Outcomes of bowel program in spinal cord injury patients with neurogenic bowel dysfunction

In this study, we aimed to determine gastrointestinal problems associated with neurogenic bowel dysfunction in spinal cord injury patients and to assess the efficacy of bowel program on gastrointestinal problems and the severity of neurogenic bowel dysfunction. Fifty-five spinal cord injury patients were included in this study. A bowel program according to the characteristics of neurogenic bowel dysfunction was performed for each patient. Before and after bowel program, gastrointestinal problems（constipation, difficult intestinal evacuation, incontinence, abdominal pain, abdominal distension, loss of appetite, hemorrhoids, rectal bleeding and gastrointestinal induced autonomic dysreflexia） and bowel evacuation methods（digital stimulation, oral medication, suppositories, abdominal massage, Valsalva maneuver and manual evacuation） were determined. Neurogenic bowel dysfunction score was used to assess the severity of neurogenic bowel dysfunction. At least one gastrointestinal problem was identified in 44（80%） of the 55 patients before bowel program. Constipation（56%, 31/55） and incontinence（42%, 23/55） were the most common gastrointestinal problems. Digital rectal stimulation was the most common method for bowel evacuation, both before（76%, 42/55） and after（73%, 40/55） bowel program. Oral medication, enema and manual evacuation application rates were significantly decreased and constipation, difficult intestinal evacuation, abdominal distention, and abdominal pain rates were significantly reduced after bowel program. In addition, mean neurogenic bowel dysfunction score was decreased after bowel program. An effective bowel program decreases the severity of neurogenic bowel dysfunction and reduces associated gastrointestinal problems in patients with spinal cord injury.

Neuroprotective effects of electroacupuncture on early- and late-stage spinal cord injury

Previous studies have shown that the neurite growth inhibitor Nogo-A can cause secondary neural damage by activating Rho A. In the present study, we hypothesized that electroacupuncture promotes neurological functional recovery after spinal cord injury by inhibiting Rho A expression. We established a rat model of acute spinal cord injury using a modification of Allen＇s method. The rats were given electroacupuncture treatment at Dazhui（Du14）, Mingmen（Du4）, Sanyinjiao（SP6）, Huantiao（GB30）, Zusanli（ST36） and Kunlun（BL60） acupoints with a sparsedense wave at a frequency of 4 Hz for 30 minutes, once a day, for a total of 7 days. Seven days after injury, the Basso, Beattie and Bresnahan（BBB） locomotor scale and inclined plane test scores were significantly increased, the number of apoptotic cells in the spinal cord tissue was significantly reduced, and Rho A and Nogo-A m RNA and protein expression levels were decreased in rats given electroacupuncture compared with rats not given electroacupuncture. Four weeks after injury, pathological tissue damage in the spinal cord at the site of injury was alleviated, the numbers of glial fibrillary acidic protein- and neurofilament 200-positive fibers were increased, the latencies of somatosensory-evoked and motor-evoked potentials were shortened, and their amplitudes were increased in rats given electroacupuncture. These findings suggest that electroacupuncture treatment reduces neuronal apoptosis and decreases Rho A and Nogo-A m RNA and protein expression at the site of spinal cord injury, thereby promoting tissue repair and neurological functional recovery.

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis,thereby exerting a neuroprotective effect.In the present study,we established a rat model of spinal cord contusion injury using the modified Allen＇s method.Three hours after injury,senegenin（30 mg/g） was injected into the tail vein for 3 consecutive days.Senegenin reduced the size of syringomyelic cavities,and it substantially reduced the number of apoptotic cells in the spinal cord.At the site of injury,Bax and Caspase-3 m RNA and protein levels were decreased by senegenin,while Bcl-2 m RNA and protein levels were increased.Nerve fiber density was increased in the spinal cord proximal to the brain,and hindlimb motor function and electrophysiological properties of rat hindlimb were improved.Taken together,our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.

Effects of decompression joint Governor Vessel electro-acupuncture on rats with acute upper cervical spinal cord injury

Decompression is the major therapeutic strategy for acute spinal cord injury,but there is some debate about the time window for decompression following spinal cord injury.An important goal and challenge in the treatment of spinal cord injury is inhibiting or reversing secondary injury.Governor Vessel electroacupuncture can improve symptoms of spinal cord injury by inhibiting cell apoptosis and improving the microenvironment of the injured spinal cord.In this study,Governor Vessel electroacupuncture combined with decompression at different time points was used to treat acute spinal cord injury.The rat models were established by inserting a balloon catheter into the atlanto-occipital space.The upper cervical spinal cord was compressed for 12 or 48 hours prior to decompression.Electroacupuncture was conducted at the acupoints Dazhui（GV14） and Baihui（GV 20）（2 Hz,15 minutes） once a day for 14 consecutive days.Compared with decompression alone,hind limb motor function recovery was superior after decompression for 12 and 48 hours combined with electroacupuncture.However,the recovery of motor function was not significantly different at 14 days after treatment in rats receiving decompression for 12 hours.Platelet-activating factor levels and caspase-9 protein expression were significantly reduced in rats receiving electroacupuncture compared with decompression alone.These findings indicate that compared with decompression alone,Governor Vessel electroacupuncture combined with delayed decompression（48 hours） is more effective in the treatment of upper cervical spinal cord injury.Governor Vessel electroacupuncture combined with early decompression（12 hours） can accelerate the recovery of nerve movement in rats with upper cervical spinal cord injury.Nevertheless,further studies are necessary to confirm whether it is possible to obtain additional benefit compared with early decompression alone.

Time representation of mitochondrial morphology and function after acute spinal cord injury

Changes in mitochondrial morphology and function play an important role in secondary damage after acute spinal cord injury. We recorded the time representation of mitochondrial morphology and function in rats with acute spinal cord injury. Results showed that mitochondria had an irregular shape, and increased in size. Mitochondrial cristae were disordered and mitochondrial membrane rupture was visible at 2–24 hours after injury. Fusion protein mitofusin 1 expression gradually increased, peaked at 8 hours after injury, and then decreased to its lowest level at 24 hours. Expression of dynamin-related protein 1, amitochondrial fission protein, showed the opposite kinetics. At 2–24 hours after acute spinal cord injury, malondialdehyde content, cytochrome c levels and caspase-3 expression were increased, but glutathione content, adenosine triphosphate content, Na＋-K＋-ATPase activity and mitochondrial membrane potential were gradually reduced. Furthermore, mitochondrial morphology altered during the acute stage of spinal cord injury. Fusion was important within the first 8 hours, but fission played a key role at 24 hours. Oxidative stress was inhibited, biological productivity was diminished, and mitochondrial membrane potential and permeability were reduced in the acute stage of injury. In summary, mitochondrial apoptosis is activated when the time of spinal cord injury is prolonged.

Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury

Propofol has been shown to exert neuroprotective effects on the injured spinal cord.However,the effect of propofol on the blood-spinal cord barrier（BSCB） after ischemia/reperfusion injury（IRI） is poorly understood.Therefore,we investigated whether propofol could maintain the integrity of the BSCB.Spinal cord IRI（SCIRI） was induced in rabbits by infrarenal aortic occlusion for 30 minutes.Propofol,30 mg/kg,was intravenously infused 10 minutes before aortic clamping as well as at the onset of reperfusion.Then,48 hours later,we performed histological and m RNA/protein analyses of the spinal cord.Propofol decreased histological damage to the spinal cord,attenuated the reduction in BSCB permeability,downregulated the m RNA and protein expression levels of matrix metalloprotease-9（MMP-9） and nuclear factor-κB（NF-κB）,and upregulated the protein expression levels of occludin and claudin-5.Our findings suggest that propofol helps maintain BSCB integrity after SCIRI by reducing MMP-9 expression,by inhibiting the NF-κB signaling pathway,and by maintaining expression of tight junction proteins.

Effect of glial cells on remyelination after spinal cord injury

Remyelination plays a key role in functional recovery of axons after spinal cord injury.Glial cells are the most abundant cells in the central nervous system.When spinal cord injury occurs,many glial cells at the lesion site are immediately activated,and different cells differentially affect inflammatory reactions after injury.In this review,we aim to discuss the core role of oligodendrocyte precursor cells and crosstalk with the rest of glia and their subcategories in the remyelination process.Activated astrocytes influence proliferation,differentiation,and maturation of oligodendrocyte precursor cells,while activated microglia alter remyelination by regulating the inflammatory reaction after spinal cord injury.Understanding the interaction between oligodendrocyte precursor cells and the rest of glia is necessary when designing a therapeutic plan of remyelination after spinal cord injury.

Electroacupuncture at Dazhui(GV14) and Mingmen(GV4) protects against spinal cord injury:the role of the Wnt/β-catenin signaling pathway

Electroacupuncture at Dazhui（GV14） and Mingmen（GV4） on the Governor Vessel has been shown to exhibit curative effects on spinal cord injury; however, the underlying mechanism remains poorly understood. In this study, we established rat models of spinal cord injury using a modified Allen＇s weight-drop method. Ninety-nine male Sprague-Dawley rats were randomly divided into three equal groups： sham（only laminectomy）, SCI（induction of spinal cord injury at T10）, and EA（induction of spinal cord injury at T10 and electroacupuncture intervention at GV14 and GV4 for 20 minutes once a day）. Rats in the SCI and EA groups were further randomly divided into the following subgroups： 1-day（n = 11）, 7-day（n = 11）, and 14-day（n = 11）. At 1, 7, and 14 days after electroacupuncture treatment, the Basso, Beattie and Bresnahan locomotor rating scale showed obvious improvement in rat hind limb locomotor function, hematoxylin-eosin staining showed that the histological change of injured spinal cord tissue was obviously alleviated, and immunohistochemistry and western blot analysis showed that Wnt1, Wnt3 a, β-catenin immunoreactivity and protein expression in the injured spinal cord tissue were greatly increased compared with the sham and SCI groups. These findings suggest that electroacupuncture at GV14 and GV4 upregulates Wnt1, Wnt3 a, and β-catenin expression in the Wnt/β-catenin signaling pathway, exhibiting neuroprotective effects against spinal cord injury.

Buyang Huanwu decoction up-regulates Notch1 gene expression in injured spinal cord

Expression of genes in the Notch signaling pathway is altered in the injured spinal cord, which indicates that Notch participates in repair after spinal cord injury. Buyang Huanwu decoction, a traditional Chinese herbal preparation, can promote the growth of nerve cells and nerve fibers; however, it is unclear whether Buyang Huanwu decoction affects the Notch signaling pathway in injured spinal cord. In this study, a rat model was established by injuring the T10 spinal cord. At 2 days after injury, rats were intragastrically administered 2 m L of 0.8 g/m L Buyang Huanwu decoction daily until sacrifice. Real-time reverse transcription polymerase chain reaction analysis demonstrated that at 7, 14 and 28 days after injury, the expression of Notch1 was increased in the Buyang Huanwu decoction group compared with controls. These findings confirm that Buyang Huanwu decoction can promote the expression of Notch1 in rats with incomplete spinal cord injury, and may indicate a mechanism to promote the repair of spinal cord injury.

Tanshinone ⅡA improves functional recovery in spinal cord injury-induced lower urinary tract dysfunction

Tanshinone ⅡA, extracted from Salvia miltiorrhiza Bunge, exerts neuroprotective effects through its anti-inflammatory, anti-oxidative and anti-apoptotic properties. This study intravenously injected tanshinone ⅡA 20 mg/kg into rat models of spinal cord injury for 7 consecutive days. Results showed that tanshinone ⅡA could reduce the inflammation, edema as well as compensatory thickening of the bladder tissue, improve urodynamic parameters, attenuate secondary injury, and promote spinal cord regeneration. The number of hypertrophic and apoptotic dorsal root ganglion（L6–S1） cells was less after treatment with tanshinone ⅡA. The effects of tanshinone ⅡA were similar to intravenous injection of 30 mg/kg methylprednisolone. These findings suggested that tanshinone ⅡA improved functional recovery after spinal cord injury-induced lower urinary tract dysfunction by remodeling the spinal pathway involved in lower urinary tract control.

Fine motor skill training enhances functional plasticity of the corticospinal tract after spinal cord injury

Following central nervous system injury, axonal sprouts form distal to the injury site and extend into the denervated area, reconstructing neural circuits through neural plasticity. How to facilitate this plasticity has become the key to the success of central nervous system repair. It remains controversial whether fine motor skill training contributes to the recovery of neurological function after spinal cord injury. Therefore, we established a rat model of unilateral corticospinal tract injury using a pyramidal tract cutting method. Horizontal ladder crawling and food ball grasping training procedures were conducted 2 weeks before injury and 3 days after injury. The neurological function of rat forelimbs was assessed at 1, 2, 3, 4, and 6 weeks after injury. Axon growth was observed with biotinylated dextran amine anterograde tracing in the healthy corticospinal tract of the denervated area at different time periods. Our results demonstrate that compared with untrained rats, functional recovery was better in the forelimbs and forepaws of trained rats. The number of axons and the expression of growth associated protein 43 were increased at the injury site 3 weeks after corticospinal tract injury. These findings confirm that fine motor skill training promotes central nervous system plasticity in spinal cord injury rats.