Cord Transection Research Articles

Improved Recovery of Complete Spinal Cord Transection by a Plasma-Modified Fibrillar Scaffold.

Complete spinal cord injury causes an irreversible disruption in the central nervous system, leading to motor, sensory, and autonomic function loss, and a secondary injury that constitutes a physical barrier preventing tissue repair. Tissue engineering scaffolds are presented as a permissive platform for cell migration and the reconnection of spared tissue. Iodine-doped plasma pyrrole polymer (pPPy-I), a neuroprotective material, was applied to polylactic acid (PLA) fibers and implanted in a rat complete spinal cord transection injury model to evaluate whether the resulting composite implants provided structural and functional recovery, using magnetic resonance (MR) imaging, diffusion tensor imaging and tractography, magnetic resonance spectroscopy, locomotion analysis, histology, and immunofluorescence. In vivo, MR studies evidenced a tissue response to the implant, demonstrating that the fibrillar composite scaffold moderated the structural effects of secondary damage by providing mechanical stability to the lesion core, tissue reconstruction, and significant motor recovery. Histologic analyses demonstrated that the composite scaffold provided a permissive environment for cell attachment and neural tissue guidance over the fibers, reducing cyst formation. These results supply evidence that pPPy-I enhanced the properties of PLA fibrillar scaffolds as a promising treatment for spinal cord injury recovery.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased,and bladder MAGL was decreased after MAGL inhibitor treatment. MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

The Left-Right Side-Specific Neuroendocrine Signaling from Injured Brain: An Organizational Principle.

A neurological dogma is that the contralateral effects of brain injury are set through crossed descending neural tracts. We have recently identified a novel topographic neuroendocrine system (T-NES) that operates via a humoral pathway and mediates the left-right side-specific effects of unilateral brain lesions. In rats with completely transected thoracic spinal cords, unilateral injury to the sensorimotor cortex produced contralateral hindlimb flexion, a proxy for neurological deficit. Here, we investigated in acute experiments whether T-NES consists of left and right counterparts and whether they differ in neural and molecular mechanisms. We demonstrated that left- and right-sided hormonal signaling is differentially blocked by the δ-, κ- and µ-opioid antagonists. Left and right neurohormonal signaling differed in targeting the afferent spinal mechanisms. Bilateral deafferentation of the lumbar spinal cord abolished the hormone-mediated effects of the left-brain injury but not the right-sided lesion. The sympathetic nervous system was ruled out as a brain-to-spinal cord-signaling pathway since hindlimb responses were induced in rats with cervical spinal cord transections that were rostral to the preganglionic sympathetic neurons. Analysis of gene-gene co-expression patterns identified the left- and right-side-specific gene co-expression networks that were coordinated via the humoral pathway across the hypothalamus and lumbar spinal cord. The coordination was ipsilateral and disrupted by brain injury. These findings suggest that T-NES is bipartite and that its left and right counterparts contribute to contralateral neurological deficits through distinct neural mechanisms, and may enable ipsilateral regulation of molecular and neural processes across distant neural areas along the neuraxis.

Effect of robotic gait training on muscle and bone characteristics in spinal cord transected rats.

Osteoporosis and loss of muscle mass are secondary issues with spinal cord injury. Robotic gait training has provided evidence of increasing bone density and muscle mass, but its effect on bone strength is undetermined. The purpose of this study was to determine the effect of a 6-week robotic locomotion training program on skeletal muscle mass and bone characteristics. Twelve female Sprague-Dawley rats received a mid-thoracic spinal cord transection at 5 days old and at 3 weeks old were assigned to a Control or Trained Group. The Trained Group performed 5-min sessions on the Rat Stepper 5 days a week for 6 weeks with 90% of body weight supported. At the end of the 6 weeks, body mass was obtained and right femurs and four lower extremity muscles were harvested. Femur bone mineral density was measured with DXA and mechanical characteristics of the femur were determined via 3-point bending testing. Independent t-tests, effects sizes and percent differences were computed between the two groups (p < 0.05). The Trained Group had significantly larger normalized femur mass (p = 0.007) and normalized soleus muscle mass (p = 0.033) when compared to the Control Group. There was a medium or large effect size with the Trained Groups' femurs having larger mass, bone mineral density, rupture loads, cortical wall thickness, shaft cross sectional area, soleus mass, normalized gastrocnemius mass, and smaller shaft inner diameters compared to the Control Group. These changes may contribute to decreasing osteoporosis and fracture risk in those with spinal cord injuries.

Inner and outer penetrating spinal cord injuries lead to distinct overground walking in mice

Spinal cord injury (SCI) is a devastating mechanical trauma. Although locomotion of model animals that mimic contusion SCI was actively examined, locomotion after penetrating SCI caused by sharp objects was not extensively studied. Severity of walking difficulty after partial transection of the spinal cord including penetrating SCI likely depends on the regions affected. Therefore, we compared beam walking and overground walking between mice after penetrating SCI at inner spinal cord region and mice with the injury at the outer region. Mice with the both penetrating SCIs did not display changes in beam walking. When appearance and movements of hindlimbs during overground walking was rated using Basso Mouse Scale for locomotion (BMS), however, mice with inner penetrating SCI showed low score shortly after the SCI. However, the score became high at later time points, as seen in contusion SCI mice. By contrast, BMS score did not decrease shortly after the outer penetrating SCI. However, the score became low 3 weeks after the SCI. As quantitative values during overground walking, movement duration in an open field were shorter at 1 day after the two penetrating SCIs. However, slower moving speed and fewer number of movement at 1 day were specific to mice with inner and outer penetrating SCIs, respectively. Moreover, BMS score was correlated with walking distance in open field only in mice with inner penetrating SCI. Thus, inner and outer penetrating SCI cause difficulty in overground walking with different severity and progress.

Vof16-miR-185-5p-GAP43 network improves the outcomes following spinal cord injury via enhancing self-repair and promoting axonal growth.

Self-repair of spinal cord injury (SCI) has been found in humans and experimental animals with partial recovery of neurological functions. However, the regulatory mechanisms underlying the spontaneous locomotion recovery after SCI are elusive. This study was aimed at evaluating the pathological changes in injured spinal cord and exploring the possible mechanism related to the spontaneous recovery. Immunofluorescence staining was performed to detect GAP43 expression in lesion site after spinal cord transection (SCT) in rats. Then RNA sequencing and gene ontology (GO) analysis were employed to predict lncRNA that correlates with GAP43. LncRNA smart-silencing was applied to verify the function of lncRNA vof16 invitro, and knockout rats were used to evaluate its role in neurobehavioral functions after SCT. MicroRNA sequencing, target scan, and RNA22 prediction were performed to further explore the underlying regulatory mechanisms, and miR-185-5p stands out. A miR-185-5p site-regulated relationship with GAP43 and vof16 was determined by luciferase activity analysis. GAP43-silencing, miR-185-5p-mimic/inhibitor, and miR-185-5p knockout rats were also applied to elucidate their effects on spinal cord neurite growth and neurobehavioral function after SCT. We found that a time-dependent increase of GAP43 corresponded with the limited neurological recovery in rats with SCT. CRNA chip and GO analysis revealed lncRNA vof16 was the most functional in targeting GAP43 in SCT rats. Additionally, silencing vof16 suppressed neurite growth and attenuated the motor dysfunction in SCT rats. Luciferase reporter assay showed that miR-185-5p competitively bound the same regulatory region of vof16 and GAP43. Our data indicated miR-185-5p could be a detrimental factor in SCT, and vof16 may function as a ceRNA by competitively binding miR-185-5p to modulate GAP43 in the process of self-recovery after SCT. Our study revealed a novel vof16-miR-185-5p-GAP43 regulatory network in neurological self-repair after SCT and may underlie the potential treatment target for SCI.

GPNMB Modulates Autophagy to Enhance Functional Recovery After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) severely affects the quality of life and autonomy of patients, and effective treatments are currently lacking. Autophagy, an essential cellular metabolic process, plays a crucial role in neuroprotection and repair after SCI. Glycoprotein non-metastatic melanoma protein B (GPNMB) has been shown to promote neural regeneration and synapse reconstruction, potentially through the facilitation of autophagy. However, the specific role of GPNMB in autophagy after SCI is still unclear. In this study, we utilized the spinal cord transection method to establish SCI rats model and overexpressed GPNMB using adenoviral vectors. We assessed tissue damage using hematoxylin and eosin (H&E) and Nissl staining, and observed cell apoptosis using TUNEL staining. We evaluated the inflammatory response by measuring inflammatory factors using enzyme-linked immunosorbent assay (ELISA). In addition, we measured reactive oxygen species (ROS) levels using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and assessed oxidative stress levels by measuring malondialdehyde (MDA) and glutathione (GSH) using ELISA. To evaluate autophagy levels, we performed immunofluorescence staining for the autophagy marker Beclin-1 and conducted Western blot analysis for autophagy-related proteins. We also assessed limb recovery through functional evaluation. Meanwhile, we induced cell injury using lipopolysaccharide (LPS) and added an autophagy inhibitor to verify the impact of GPNMB on SCI through autophagy modulation. The results demonstrated that GPNMB alleviated the inflammatory response, reduced oxidative stress levels, inhibited cell apoptosis, and promoted autophagy following SCI. Inhibiting autophagy reversed the effects of GPNMB. These findings suggest that GPNMB promotes neural injury repair after SCI, potentially through attenuating the inflammatory response, reducing oxidative stress, and inhibiting cell apoptosis.

Adipose tissue derived stem cell secretome induces motor and histological gains after complete spinal cord injury in Xenopus laevis and mice

Mesenchymal stem cell-based therapies have been studied for spinal cord injury (SCI) treatment due to their paracrine action upon damaged tissues. MSCs neuroregenerative role may relate to the contents of their secretome in anti-inflammatory cytokines and growth-permissive factors. We propose using the secretome of MSCs isolated from the adipose tissue—adipose tissue-derived stem cells (ASCs) as a cell-free based therapy for SCI. In vivo studies were conducted in two SCI models, Xenopus laevis and mice, after complete spinal cord transection. Our results on both models demonstrated positive impacts of ASC secretome on their functional recovery which were correlated with histopathological markers of regeneration. Furthermore, in our mice study, secretome induced white matter preservation together with modulation of the local and peripheral inflammatory response. Altogether, these results demonstrate the neuroregenerative and potential for inflammatory modulation of ASC secretome suggesting it as a good candidate for cell-free therapeutic strategies for SCI.

Mesenchymal Stem Cells Combined With Electroacupuncture Treatment Regulate the Subpopulation of Macrophages and Astrocytes to Facilitate Axonal Regeneration in Transected Spinal Cord.

Herein, we investigated whether mesenchymal stem cells (MSCs) transplantation combined with electroacupuncture (EA) treatment could decrease the proportion of proinflammatory microglia/macrophages and neurotoxic A1 reactive astrocytes and inhibit glial scar formation to enhance axonal regeneration after spinal cord injury (SCI). Adult rats were divided into 5 groups after complete transection of the spinal cord at the T10 level: a control group, a nonacupoint EA (NA-EA) group, an EA group, an MSC group, and an MSCs+EA group. Immunofluorescence labeling, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blots were performed. The results showed that MSCs+EA treatment reduced the proportion of proinflammatory M1 subtype microglia/macrophages, but increased the differentiation of anti-inflammatory M2 phenotype cells, thereby suppressing the mRNA and protein expression of proinflammatory cytokines (tumor necrosis factor-α and IL-1β) and increasing the expression of an anti-inflammatory cytokine (interleukin [IL]-10) on days 7 and 14 after SCI. The changes in expression correlated with the attenuated neurotoxic A1 reactive astrocytes and glial scar, which in turn facilitated the axonal regeneration of the injured spinal cord. In vitro, the proinflammatory cytokines increased the level of proliferation of astrocytes and increased the expression levels of C3, glial fibrillary acidic protein, and chondroitin sulfate proteoglycan. These effects were blocked by administering inhibitors of ErbB1 and signal transducer and activator of transcription 3 (STAT3) (AG1478 and AG490) and IL-10. These findings showed that MSCs+EA treatment synergistically regulated the microglia/macrophage subpopulation to reduce inflammation, the formation of neurotoxic A1 astrocytes, and glial scars. This was achieved by downregulating the ErbB1-STAT3 signal pathway, thereby providing a favorable microenvironment conducive to axonal regeneration after SCI.

Metabolism in the large intestine wall after injury of the thoracic spinal cord (experimental study)

Ailm: to assess metabolic processes in the serous layer of the large intestine in the acute period of spinal injury.Materials and Methods: the experiment was carried out on laboratory animals — Wistar rats (n = 20). Spinal injury was simulated by complete transection of the spinal cord at the level of Th5-Th6 vertebrae. Metabolic changes in the intestinal wall were assessed before injury, 3 and 24 hours after spinal cord transection. Metabolism was assessed in vivo using fluorescence time-resolved macroimaging technology (macroFLIM) by autofluorescence in the spectral channel of the metabolic cofactor NAD(P)H.Results: a significant (p = 0.041) increase in the mean fluorescence lifetime (τm) by 12% and the lifetime of the long component (τ2) of the NAD(P)H cofactor by 13% (p = 0.008) was detected 24 hours after injury. MacroFLIM showed an increase in the intensity of metabolism in the large intestine wall.Conclusion: for the first time in an in vivo experiment, it has been shown that the acute period of spinal injury is accompanied by an increase in metabolic activity in the tissues of the large intestine. The revealed phenomenon confirms the role of the large intestine in compensatory reactions to spinal injury and open up new possibilities for therapy in the acute period of spinal cord injury.

Combined RhoA morpholino and ChABC treatment protects identified lamprey neurons from retrograde apoptosis after spinal cord injury.

Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo. We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo. Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.

Sprouting of afferent and efferent inputs to pelvic organs after spinal cord injury.

Neural plasticity occurs within the central and peripheral nervous systems after spinal cord injury (SCI). Although central alterations have extensively been studied, it is largely unknown whether afferent and efferent fibers in pelvic viscera undergo similar morphological changes. Using a rat spinal cord transection model, we conducted immunohistochemistry to investigate afferent and efferent innervations to the kidney, colon, and bladder. Approximately 3-4 weeks after injury, immunostaining demonstrated that tyrosine hydroxylase (TH)-labeled postganglionic sympathetic fibers and calcitonin gene-related peptide (CGRP)-immunoreactive sensory terminals sprout in the renal pelvis and colon. Morphologically, sprouted afferent or efferent projections showed a disorganized structure. In the bladder, however, denser CGRP-positive primary sensory fibers emerged in rats with SCI, whereas TH-positive sympathetic efferent fibers did not change. Numerous CGRP-positive afferents were observed in the muscle layer and the lamina propria of the bladder following SCI. TH-positive efferent inputs displayed hypertrophy with large diameters, but their innervation patterns were sustained. Collectively, afferent or efferent inputs sprout widely in the pelvic organs after SCI, which may be one of the morphological bases underlying functional adaptation or maladaptation.

PEG-chitosan (Neuro-PEG) induced restoration of motor function after complete transection of the dorsal spinal cord in swine. A pilot study

Background: Spinal cord injury (SCI) remains an unmet medical need. Recently, fusogens, such as polyethylene glycol (PEG), have been proven effective in restoring sensorimotor function after complete transection of the spinal cord at different levels and in different species. Here, we report on the use of a PEG-chitosan combo in a different animal model (swine). Methods: Five Hungarian Mangalica pigs were subjected to complete transection of the thoracic cord (T7-9). Three animals were treated with locally injected PEG-chitosan (Neuro-PEG) gel; two acted as controls. PEG-600 was also injected intra- and post-operatively intravenously. Animals were submitted to rehabilitation, including electrical myostimulation. Results were assessed after 60 days using the Individual Limb Motor Score, the Porcine Thoracic Spinal Cord Injured Behavioral Scale, and the modified motor Basso, Beattie, and Bresnahan scale; sensory and sphincter functions were also assessed. Animals underwent in vivo spinal cord tracing with DiI. Immunofluorescence histology included NF-200, DAPI, and a fluorochrome-conjugated secondary antibody. Results: Starting on postoperative day (POD) 2, neuro-PEG-treated animals evinced the first signs of recovery, and on POD 60, they could all support their weight and were mobile. Controls never recovered any useful function. Fluorescence microscopy in the experimental group revealed axons passing through the site of injury, while degenerative post-traumatic changes were noted in controls. Conclusion: Neuro-PEG affords sensorimotor recovery after complete spinal cord transection. This opens the door to human experimentation, including trials of spinal cord transplantation.

Chitosan/PEG-mediated spinal cord fusion after complete dorsal transection in rabbits – functional results at 30 days

Background: The aim was to study functional recovery in experimental animals (rabbits) with transected spinal cords treated with a combination of photo-cross-linked chitosan in a homogeneous mixture with polyethylene glycol (PEG-chitosan). Methods: 20 rabbits (n = 10 experimental and n = 10 controls) were submitted to complete spinal cord transection at T9. The experimental group received an intraoperative injection of PEG-chitosan. Neurological recovery was assessed using the modified Basso, Beattie, and Bresnahan scale. Results: In the experimental group, partial recovery of movements, sensory function, and sphincter control were all observed by postoperative day 30. Paraplegia and anesthesia persisted in the control group; 4 controls died versus none in the test group. Conclusion: PEG-chitosan is a candidate for neurological restoration after spinal paralysis.

Functional Hydrogel Co-Remolding Migration and Differentiation Microenvironment for Severe Spinal Cord Injury Repair.

Spinal cord injury (SCI) activates nestin+ neural stem cells (NSCs), which can be regarded as potential seed cells for neuronal regeneration. However, the lesion microenvironment seriously hinders the migration of the nestin+ cells to the lesion epicenter and their differentiation into neurons to rebuild neural circuits. In this study, a photosensitive hydrogel scaffold is prepared as drug delivery carrier. Genetically engineered SDF1α and NT3 are designed and the scaffold is binary modified to reshape the lesion microenvironment. The binary modified scaffold can effectively induce the migration and neuronal differentiation of nestin+ NSCs in vitro. When implanted into a rat complete SCI model, many of the SCI-activated nestin+ cells migrate into the lesion site and give rise to neurons in short-term. Meanwhile, long-term repair results also show that implantation of the binary modified scaffold can effectively promote the maturation, functionalization and synaptic network reconstruction of neurons in the lesion site. In addition, animals treated with binary scaffold also showed better improvement in motor functions. The therapeutic strategy based on remolding the migration and neuronal differentiation lesion microenvironment provides a new insight into SCI repair by targeting activated nestin+ cells, which exhibits excellent clinical transformation prospects.

Novel phenotype characterization utilizing electrical impedance myography signatures in murine spinal cord injury neurogenic bladder models

Neurogenic bladder (NB) affects people of all ages. Electric impedance myography (EIM) assesses localized muscle abnormalities. Here, we sought to investigate whether unique detrusor EIM signatures are present in NB due to spinal cord injury (SCI). Twenty-eight, 8–10 weeks old, C57BL/6J female mice were studied. Twenty underwent spinal cord transection; 8 served as controls. Cohorts were euthanized at 4 and 6 weeks after spinal cord transection. Each bladder was measured in-situ with EIM with applied frequencies of 1 kHz to 10 MHz, and then processed for molecular and histologic study. SCI mice had greater bladder-to-body weight ratio (p < 0.0001), greater collagen deposition (p = 0.009), and greater smooth-muscle-myosin-heavy-chain isoform A/B ratio (p < 0.0001). Compared with the control group, the SCI group was associated with lower phase, reactance, and resistance values (p < 0.01). Significant correlations (p < 0.001) between bladder-to-body weight ratios and EIM measurements were observed across the entire frequency spectrum. A severely hypertrophied phenotype was characterized by even greater bladder-to-body weight ratios and more depressed EIM values. Our study demonstrated distinct EIM alterations in the detrusor muscle of mice with NB due to SCI. With further refinement, EIM may offer a potential point-of-care tool for the assessment of NB and its response to treatment.

Sex differences in lower urinary tract function in mice with or without spinal cord injury.

We examined sex differences of lower urinary tract function and molecular mechanisms in mice with and without spinal cord injury (SCI). SCI was induced by Th8-9 spinal cord transection in male and female mice. We evaluated cystometrograms (CMG) and electromyography (EMG) of external urethral sphincter (EUS) at 6 weeks after SCI in spinal intact (SI) and SCI mice. The mRNA levels of Piezo2 and TRPV1 were measured in L6-S1 dorsal root ganglia (DRG). Protein levels of nerve growth factor (NGF) in the bladder mucosa was evaluated using an enzyme-linked immunosorbent assay. Sex differences were found in the EUS behavior during voiding as voiding events in female mice with or without SCI occurred during EUS relaxation periods without EUS bursting activity whereas male mice with or without SCI urinated during EUS bursting activity in EMG recordings. In both sexes, SCI decreased voiding efficiency along with increased tonic EUS activities evident as reduced EUS relaxation time in females and longer active periods of EUS bursting activity in males. mRNA levels of Piezo2 and TRPV1 of DRG in male and female SCI mice were significantly upregulated compared with SI mice. NGF in the bladder mucosa showed a significant increase in male and female SCI mice compared with SI mice. However, there were no significant differences in Piezo2 or TRPV1 levels in DRG or NGF protein levels in the bladder mucosa between male and female SCI mice. We demonstrated that female and male mice voided during EUS relaxation and EUS bursting activity, respectively. Also, upregulation of TRPV1 and Piezo2 in L6-S1 DRG and NGF in the bladder could be involved in SCI-induced lower urinary tract dysfunction in both sexes of mice.

The protein kinase A signaling pathway mediates the effect of electroacupuncture on excessive contraction of the bladder detrusor in a rat model of neurogenic bladder.

Neurogenic bladder (NB) is a form of neurological bladder dysfunction characterized by excessive contraction of the bladder detrusor. Protein kinase A (PKA) signaling is involved in the contraction of the detrusor muscle. To investigate whether PKA signaling mediates the effect of electroacupuncture (EA) on the excessive contraction of the bladder detrusor in NB. Sixty rats were randomly divided into control, sham, NB, NB + EA, and NB + EA + H89 (a PKA receptor antagonist) groups. The modified Hassan Shaker spinal cord transection method was used to generate a NB model. After EA intervention for one week, urodynamic tests were used to evaluate bladder function, hematoxylin and eosin staining was conducted to assess morphological changes, enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentration of PKA, and Western blotting was conducted to measure the protein levels of phosphorylated myosin light chain kinase (p-MLCK)/p-MLC. The results showed that NB resulted in morphological disruption, impairment of urodynamics, and decreases in the concentration of PKA and the protein levels of p-MLCK/p-MLC. EA reversed the changes induced by NB dysfunction. However, the improvement in urodynamics and the increases in the concentration of PKA and the protein levels of p-MLCK/p-MLC were inhibited by H89. Our findings indicate that the PKA signaling pathway mediates the beneficial effect of EA on excessive contraction of the bladder detrusor in a rat model of NB.

Effect of electroacupuncture on urodynamics and Raf/MEK/ERK signaling pathway in spinal cord tissue of rats with detrusor hyperreflexia after suprasacral spinal cord injury.

To observe the effect of electroacupuncture (EA) on urodynamics and Raf/MEK/ERK signaling pathway in spine cord tissue of rats after suprasacral spinal cord injury (SSCI), so as to explore its possible mechanism in improving bladder function in rats with detrusor hyperreflexia after SSCI. Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at "Ciliao" (BL32), "Zhongji" (CV3), "Sanyinjiao" (SP6) and "Dazhui" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot. Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group. EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.

Effect of Yiyuan moxibustion on urodynamics and TRPV4/ATP/P2X5 signal pathway of bladder tissue in rats with neurogenic bladder after sacral cord injury.

To observe the effect of Yiyuan moxibustion on urodynamics and the expressions of transient receptor potential vanilloid 4 (TRPV4), adenosine triphosphate (ATP), tyrosine protein kinase KIT (C-Kit) and adenosine triphosphate receptor P2X5 in bladder tissue of rats with detrusor reflex-free neurogenic bladder (NB) after sacral cord injury (SCI), so as to explore its mechanism in promoting the recovery of urination function of NB rats. Female SD rats were randomly divided into sham operation, model, Yiyuan moxibustion, Yiyuan moxibustion+inhibitor (combination) and inhibitor groups, with 12 rats in each group. The model of detruser reflex-free NB after sacral SCI was established by modified Hassan Shaker spinal cord transection method. The behavioral score of Basso Beasttie Bresnahan (BBB) and urodynamic indexes were used to evaluate the model of rats after operation. Fifteen days after modeling, Yiyuan moxibustion was applied to "Shenque" (CV8) and "Guanyuan" (CV4) for 20 min, once daily for 14 days. Rats of the inhibitor and combination groups were given intravesical instillation of HC067047 (1 mL, 1 μmol/L, 30 min). After the interventions, urodynamics was used to evaluate the bladder function of rats. HE staining was used to observe the morphology of bladder tissue. ATP content in bladder tissue was detected by colorimetric method. The positive expression rates of C-Kit and their receptor P2X5 in bladder tissue were observed by immunofluorescence double labeling method, and TRPV4, C-Kit, and P2X5 protein expression levels in bladder tissue were detected by Western blot. Compared with the sham operation group, the maximum bladder capacity and bladder compliance of rats in the model group were increased (P<0.01), the leak point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and the protein expression levels of TRPV4, C-Kit, P2X5 in bladder tissue were decreased (P<0.01). In comparison with the model and combination groups, the Yiyuan moxibustion group showed a decrease in maximum bladder capacity and bladder compliance (P<0.01), an increase in leakage point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and TRPV4, C-Kit, and P2X5 protein expression levels (P<0.01, P<0.05)；However, these indicators showed opposite trends in the inhibitor group (P<0.01, P<0.05). Yiyuan moxibustion can improve the urodynamics and bladder function in rats with bladder detrusor nonreflective after SCI, which may be related to its effect in activating the TRPV4 channel in bladder tissue, promoting the release of ATP from bladder epithelium, thus increasing the expression of bladder Cajal interstitial cells and their purinergic P2X5 receptors.