Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury

The effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders was examined to demonstrate if spinal cord injury (SCI) modulates the basal or evoked release of ATP from bladder urothelium and whether intravesical botulinum toxin A (BTX-A) administration inhibits urothelial ATP release, a measure of sensory nerve activation. A Ussing chamber was used to isolate and separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Following spinal cord injury, resting urothelial release of ATP was ninefold higher than that of normal rats. Botulinum toxin A instillation did not significantly affect the resting release of ATP after spinal cord injury. Evoked ATP release following hypoosmotic stimulation was significantly higher in chronic spinal cord injured compared to normal rat bladders. However, botulinum toxin A treatment markedly reduced ATP release in spinal cord injured bladders by 53% suggesting that ATP release by mechanoreceptor stimulation, as opposed to basal release, occurs by exocytotic mechanisms. In contrast, there was no significant difference in basal or evoked ATP release from bladder serosa following spinal cord injury. Moreover, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side after spinal cord injury, suggesting that its effects were confined to the urothelial side of the bladder preparation. In summary: (1) increased release of ATP from the urothelium of spinal cord injured bladders may contribute to the development of bladder hyperactivity and, (2) mechanoreceptor stimulated vesicular ATP release, as opposed to basal non-vesicular release of ATP, is significantly inhibited in spinal cord injured bladders by intravesical instillation of botulinum toxin A. These results may have important relevance in our understanding of the mechanisms underlying plasticity of bladder afferent pathways following SCI.     

Origin of new glial cells in intact and injured adult spinal cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.     

Botulinum toxin type A normalizes alterations in urothelial ATP and NO release induced by chronic spinal cord injury

The purpose of this paper was to simultaneously examine changes in urothelial ATP and NO release in normal and spinal cord injured animals as well as in spinal cord injured animals treated with botulinum toxin type A (BoNT-A). Furthermore we correlated changes in transmitter release with functional changes in bladder contraction frequency, and determined the effects of BoNT-A on bladder efferent nerve function. Normal and spinal cord injured rat bladders were injected on day 0 with either vehicle (saline containing bovine serum albumin) or BoNT-A. On day 2, in vitro neurotransmitter release and bladder strip contractility studies as well as in vivo cystometrographic studies were conducted. Resting ATP release was significantly enhanced following spinal cord injury (i.e. 57% increase, p<0.05) and was unaffected by BoNT-A treatment. SCI increased hypoosmotic evoked urothelial ATP release by 377% (p<0.05). BoNT-A treatment reduced evoked ATP release in SCI bladders by 83% (p<0.05). In contrast, hypoosmotic stimulation induced NO release was significantly inhibited following SCI (i.e. 50%, p<0.05) but recovered in SCI rats treated with BoNT-A (i.e. 195% increase in NO release in SCI-BTX-treated rats compared to SCI controls, p<0.01). Changes in urothelial transmitter release coincided with a significant decrease in non-voiding bladder contraction frequency (i.e. 71%, p<0.05) in SCI-BTX rats compared to SCI rats. While no difference was measured between neurally evoked contractile amplitude between SCI and SCI-BTX animals, atropine (1 microM) inhibited contractile amplitude to a greater extent (i.e. 76%, p<0.05) in the SCI-BTX group compared to the SCI group. We hypothesize that alterations in the ratio of excitatory (i.e. ATP) and inhibitory (i.e. NO) urothelial transmitters promote bladder hyperactivity in rat bladders following SCI that can be reversed, to a large extent, by treatment with BoNT-A.     

高压氧对大鼠脊髓损伤后局部炎症因子的影响

目的:观测高压氧对脊髓损伤大鼠运动功能恢复的作用及机制研究。方法:采用改良Allen’S法制作大鼠不完全脊髓损伤模型。45只SD大鼠,随机分为3组(n=15):假手术组,脊髓损伤对照组和高压氧治疗组。分别于治疗后1、2、3及4周,利用BBB评分法对大鼠进行运动功能评分。应用ELISA法测定手术后14 d大鼠损伤脊髓组织中肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、γ干扰素(IFN-γ)的含量。结果:成功建立大鼠不完全性脊髓损伤模型,运动功能评分显示高压氧治疗组和模型组差异显著。ELISA结果显示脊髓损伤后脊髓组织中炎症因子含量显著升高,而高压氧处理后,炎症因子含量明显降低。结论:高压氧可通过降低损伤脊髓局部的炎症反应,达到改善脊髓损伤的作用。     

Early exercise in spinal cord injured rats induces allodynia through TrkB signaling

Rehabilitation is important for the functional recovery of patients with spinal cord injury. However, neurological events associated with rehabilitation remain unclear. Herein, we investigated neuronal regeneration and exercise following spinal cord injury, and found that assisted stepping exercise of spinal cord injured rats in the inflammatory phase causes allodynia. Sprague-Dawley rats with thoracic spinal cord contusion injury were subjected to assisted stepping exercise 7 days following injury. Exercise promoted microscopic recovery of corticospinal tract neurons, but the paw withdrawal threshold decreased and C-fibers had aberrantly sprouted, suggesting a potential cause of the allodynia. Tropomyosin-related kinase B (TrkB) receptor for brain-derived neurotrophic factor (BDNF) was expressed on aberrantly sprouted C-fibers. Blocking of BDNF-TrkB signaling markedly suppressed aberrant sprouting and decreased the paw withdrawal threshold. Thus, early rehabilitation for spinal cord injury may cause allodynia with aberrant sprouting of C-fibers through BDNF-TrkB signaling.     

Glutamate-stimulated ATP release from spinal cord astrocytes is potentiated by substance P

ATP has recently emerged as a key molecule mediating pathological pain. The aim of this study was to examine whether spinal cord astrocytes could be a source of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from these astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamate-induced inward currents, but did not cause such currents alone. When glutamate was applied alone it acted exclusively through alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors to stimulate Ca(2+) influx-dependent ATP release. However, when substance P was co-applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. Activation of neurokinin receptor subtypes, protein kinase C and phospholipases A(2), C and D were needed for substance P to bring about its effects. These results suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that release substance P and glutamate.     

Plasma glutamine concentration in spinal cord injured patients

Glutamine, a non-essential amino acid, is the most important source of energy for macrophages and lymphocytes. Reduction in its plasma concentration is related with loss of immune function, as leukocyte proliferation and cytokine production. It is well known that glutamine is largely produced by the skeletal muscle which is severely compromised as a consequence of the paralysis due to the damage of the spinal cord. In spinal cord injury (SCI) patients, infections, such as pneumonia and sepsis in general, are a major cause of morbidity and mortality. In comparison with the control group, a 54% decrease in plasma glutamine concentration was observed as well as a decrease in the production of TNF and IL-1 by peripheral blood mononuclear cells cultivated for 48 h in SCI patients. Therefore, we propose that a decrease in plasma glutamine concentration is an important contributor to the immunosuppression seen in SCI patients.     

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.     

Acute and chronic tactile sensory testing after spinal cord injury in rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia(3). Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.     

Blood pressure regulation in neurally intact human vs. acutely injured paraplegic and tetraplegic patients during passive tilt

We investigated autonomic control of cardiovascular function in able-bodied (AB), paraplegic (PARA), and tetraplegic (TETRA) subjects in response to head-up tilt following spinal cord injury. We evaluated spectral power of blood pressure (BP), baroreflex sensitivity (BRS), baroreflex effectiveness index (BEI), occurrence of systolic blood pressure (SBP) ramps, baroreflex sequences, and cross-correlation of SBP with heart rate (HR) in low (0.04-0.15 Hz)- and high (0.15-0.4 Hz)-frequency regions. During tilt, AB and PARA effectively regulated BP and HR, but TETRA did not. The numbers of SBP ramps and percentages of heartbeats involved in SBP ramps and baroreflex sequences increased in AB, were unchanged in PARA, and declined in TETRA. BRS was lowest in PARA and declined with tilt in all groups. BEI was greatest in AB and declined with tilt in all groups. Low-frequency power of BP and the peak of the SBP/HR cross-correlation magnitude were greatest in AB, increased during tilt in AB, remained unchanged in PARA, and declined in TETRA. The peak cross-correlation magnitude in HF decreased with tilt in all groups. Our data indicate that spinal cord injury results in decreased stimulation of arterial baroreceptors and less engagement of feedback control as demonstrated by lower 1) spectral power of BP, 2) number (and percentages) of SBP ramps and barosequences, 3) cross-correlation magnitude of SBP/HR, 4) BEI, and 5) changes in delay between SBP/HR. Diminished vasomotion and impaired baroreflex regulation may be major contributors to decreased orthostatic tolerance following injury.     

Oral mucosa stem cells alleviates spinal cord injury-induced neurogenic bladder symptoms in rats

Background

Spinal cord injury (SCI) deteriorates various physical functions, in particular, bladder problems occur as a result of damage to the spinal cord. Stem cell therapy for SCI has been focused as the new strategy to treat the injuries and to restore the lost functions. The oral mucosa cells are considered as the stem cells-like progenitor cells. In the present study, we investigated the effects of oral mucosa stem cells on the SCI-induced neurogenic bladder in relation with apoptotic neuronal cell death and cell proliferation.

Results

The contraction pressure and the contraction time in the urinary bladder were increased after induction of SCI, in contrast, transplantation of the oral mucosa stem cells decreased the contraction pressure and the contraction time in the SCI-induced rats. Induction of SCI initiated apoptosis in the spinal cord tissues, whereas treatment with the oral mucosa stem cells suppressed the SCI-induced apoptosis. Disrupted spinal cord by SCI was improved by transplantation of the oral mucosa stem cells, and new tissues were increased around the damaged tissues. In addition, transplantation of the oral mucosa stem cells suppressed SCI-induced neuronal activation in the voiding centers.

Conclusions

Transplantation of oral mucosa stem cells ameliorates the SCI-induced neurogenic bladder symptoms by inhibiting apoptosis and by enhancing cell proliferation. As the results, SCI-induced neuronal activation in the neuronal voiding centers was suppressed, showing the normalization of voiding function.     

Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: effect of botulinum toxin A

The effects of mechanoreceptor stimulation and subsequent ATP release in cyclophosphamide evoked chronic bladder inflammation was examined to demonstrate: (1) whether inflammation modulates ATP release from bladder urothelium and (2) whether intravesical botulinum toxin A administration inhibits urothelial ATP release, a measure of sensory nerve activation. ATP release was measured from rat bladders in a Ussing chamber, an apparatus that allows one to separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Cystometry was utilized to correlate changes in ATP release with alterations in the frequency of voiding and non-voiding bladder contractions, in vivo measures of bladder afferent activity. The resting urothelial release of ATP was not significantly affected by either cyclophosphamide or botulinum toxin A treatment. However, evoked ATP release following hypoosmotic stimulation was significantly increased (i.e. 94%) in chronic cyclophosphamide treated bladder urothelium compared to control bladders. In addition, botulinum toxin A treatment significantly reduced hypoosmotic shock induced ATP release in cyclophosphamide treated animals by 69%. Cystometry revealed that cyclophosphamide and botulinum toxin A treatments altered non-voiding (i.e. cyclophosphamide increased, botulinum toxin A decreased) but not voiding contraction frequency suggesting that alterations in urothelial ATP release selectively diminished underlying bladder C-fiber nerve activity. Finally, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side implying that its effects were confined to the urothelial side of the bladder preparation.     

Change in muscarinic modulation of transmitter release in the rat urinary bladder after spinal cord injury

Muscarinic facilitation of 14C-ACh release from post-ganglionic parasympathetic nerve terminals was studied in bladder strips prepared from spinal intact (SI) and spinal cord transected (SCT) rats. The spinal cord was transected at the lower thoracic spinal segments 3 weeks prior to the experiments. Using non-facilitatory stimulation (2 Hz) the release of ACh in spinal intact rats did not change in the presence of a non-specific muscarinic antagonist, atropine (100 nM), an M(1) specific antagonist (pirenzepine, 50 nM) or an M(1)-M(3) specific antagonist (4-DAMP, 5 nM). However, during a facilitatory stimulation paradigm (10 Hz or 40 Hz, 100 shocks) atropine and pirenzepine, but not 4-DAMP inhibited the release of ACh in bladders from spinal intact rats, indicating an M(1) receptor-mediated facilitation. In spinal cord transected rats, 2 Hz stimulation-induced release was significantly inhibited by atropine or 4-DAMP but not by pirenzepine indicating that a pre-junctional facilitatory mechanism mediated via M(3) muscarinic receptors could be induced by a non-facilitatory stimulation paradigm after spinal injury. In bladders of spinal cord transected rats, 10 Hz stimulation-evoked release of ACh was also inhibited by atropine and 4-DAMP (5 nM) but not by pirenzepine (50 nM). These results indicate that pre-junctional muscarinic receptors at cholinergic nerve endings in the bladder change after chronic spinal cord injury. It appears that low affinity M(1) muscarinic receptors are replaced by high affinity M(3) receptors. This change in modulation of ACh release may partly explain the bladder hyperactivity after chronic spinal cord injury.     

Response of chemokine antagonists to inflammation in injured spinal cord

Inflammation is a primary reaction to infection, allergic disorders, autoimmune diseases, and mechanical injury. The goal of an inflammatory response is to rapidly respond to noxious stimuli, such as trauma or pathogen, with a controlled amplification of cellular activation to eliminate, control, or wall off the triggering agent. Although the inflammatory response is necessary for resolution of the pathogenic event, by stander or collateral tissue damage is caused by the toxic nature of many of its by-products. It is characterized by the infiltration of leukocytes into the affected area. Chemokines and their receptors play an essential role as mediators of leukocyte infiltration. In most cases this response is so vigorous that its control, especially in the central nervous system, would inhibit recovery. The benefits of anti-inflammatory therapy based on interference with the chemokine system has been established in animal models and is being pursued with chemokine antibodies and receptor antagonists. Prolonged treatment with a broad-spectrum chemokine antagonist, vMIPII, has been shown to reduce the rate of infiltration of monocytes into injured rat spinal cord and promote survival.