neuropeptide-y and Spinal-Cord-Injuries

In strong contrast to limited repair within the mammalian central nervous system, the spinal cord of adult zebrafish is capable of almost complete recovery following injury. Understanding the mechanism underlying neural repair and functional recovery in zebrafish may lead to innovative therapies for human spinal cord injury (SCI). Since neuropeptide Y (NPY) plays a protective role in the pathogenesis of several neurological diseases, in the present study, we evaluated the effects of NPY on neuronal repair and subsequent recovery of motor function in adult zebrafish following SCI. Real-time quantitative PCR (qRT-PCR), in situ hybridization and immunostaining for NPY revealed decreased NPY expression at 12 hours (h), 6 and 21 days (d) after SCI. Double-immunostaining for NPY and islet-1, a motoneuron marker, showed that NPY was expressed in spinal cord motoneurons. Morpholino (MO) treatment for suppressing the expression of NPY inhibited supraspinal axon regrowth and locomotor recovery, in which double-staining for proliferating cell nuclear antigen (PCNA) and islet-1 showed a reduction in motoneuron proliferation. Similarly, a downregulated mRNA level of Y1 receptor of NPY (NPY1R) was also detected at 12 h, 6 and 21 d after injury. Immunostaining for NPY and in situ hybridization for NPY1R revealed that NPY1R was co-localized with NPY. Collectively, the results suggest that NPY expression in motoneurons promotes descending axon regeneration and locomotor recovery in adult zebrafish after SCI, possibly by regulating motoneuron proliferation through activation of NPY1R.

Topics: Animals; Female; Gene Expression; Male; Motor Neurons; Neuropeptide Y; Recovery of Function; Spinal Cord Injuries; Zebrafish; Zebrafish Proteins

Patients with spinal cord injury (SCI) develop chronic pain that severely compromises their quality of life. We have previously reported that progesterone (PG), a neuroprotective steroid, could offer a promising therapeutic strategy for neuropathic pain. In the present study, we explored temporal changes in the expression of the neuropeptides galanin and tyrosine (NPY) and their receptors (GalR1 and GalR2; Y1R and Y2R, respectively) in the injured spinal cord and evaluated the impact of PG administration on both neuropeptide systems and neuropathic behavior. Male rats were subjected to spinal cord hemisection at T13 level, received daily subcutaneous injections of PG or vehicle, and were evaluated for signs of mechanical and thermal allodynia. Real time PCR was used to determine relative mRNA levels of neuropeptides and receptors, both in the acute (1day) and chronic (28days) phases after injury. A significant increase in Y1R and Y2R expression, as well as a significant downregulation in GalR2 mRNA levels, was observed 1day after SCI. Interestingly, PG early treatment prevented Y1R upregulation and resulted in lower NPY, Y2R and GalR1 mRNA levels. In the chronic phase, injured rats showed well-established mechanical and cold allodynia and significant increases in galanin, NPY, GalR1 and Y1R mRNAs, while maintaining reduced GalR2 expression. Animals receiving PG treatment showed basal expression levels of galanin, NPY, GalR1 and Y1R, and reduced Y2R mRNA levels. Also, and in line with previously published observations, PG-treated animals did not develop mechanical allodynia and showed reduced sensitivity to cold stimulation. Altogether, we show that SCI leads to considerable changes in the spinal expression of galanin, NPY and their associated receptors, and that early and sustained PG administration prevents them. Moreover, our data suggest the participation of galaninergic and NPYergic systems in the plastic changes associated with SCI-induced neuropathic pain, and further supports the therapeutic potential of PG- or neuropeptide-based therapies to prevent and/or treat chronic pain after central injuries.

Topics: Animals; Galanin; Gene Expression Regulation; Humans; Neuralgia; Neuropeptide Y; Neuropeptides; Pain Measurement; Progesterone; Rats; Receptor, Galanin, Type 1; Receptor, Galanin, Type 2; RNA, Messenger; Spinal Cord; Spinal Cord Injuries

Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α in vitro, we selected shRNA3 to construct LV-TNF-shRNA3 for in vivo study. We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain.

Topics: Activating Transcription Factor 3; Animals; Cell Death; Disease Models, Animal; Ganglia, Spinal; Gene Expression Regulation; Gene Silencing; Genetic Therapy; Genetic Vectors; Interleukin-6; Lentivirus; Male; Mice; Mice, Inbred C57BL; Neuralgia; Neurons; Neuropeptide Y; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Spinal Cord Injuries; Tumor Necrosis Factor-alpha

CVD (cardiovascular disease) represents a leading cause of mortality in chronic SCI (spinal cord injury). Several component risk factors are observed in SCI; however, the underlying mechanisms that contribute to these risks have not been defined. Central and peripheral chronic inflammation is associated with metabolic dysfunction and CVD, including adipokine regulation of neuroendocrine and cardiac function and inflammatory processes initiated by the innate immune response. We use female C57 Bl/6 mice to examine neuroendocrine, cardiac, adipose and pancreatic signaling related to inflammation and metabolic dysfunction in response to experimentally induced chronic SCI. Using immuno-histochemical, -precipitation, and -blotting analysis, we show decreased POMC (proopiomelanocortin) and increased NPY (neuropeptide-Y) expression in the hypothalamic ARC (arcuate nucleus) and PVN (paraventricular nucleus), 1-month post-SCI. Long-form leptin receptor (Ob-Rb), JAK2 (Janus kinase)/STAT3 (signal transducer and activator of transcription 3)/p38 and RhoA/ROCK (Rho-associated kinase) signaling is significantly increased in the heart tissue post-SCI, and we observe the formation and activation of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome in VAT (visceral adipose tissue) and pancreas post-SCI. These data demonstrate neuroendocrine signaling peptide alterations, associated with central inflammation and metabolic dysfunction post-SCI, and provide evidence for the peripheral activation of signaling mechanisms involved in cardiac, VAT and pancreatic inflammation and metabolic dysfunction post-SCI. Further understanding of biological mechanisms contributing to SCI-related inflammatory processes and metabolic dysfunction associated with CVD pathology may help to direct therapeutic and rehabilitation countermeasures.

Topics: Adipose Tissue; Animals; Carrier Proteins; Female; Hypothalamus; Inflammasomes; Janus Kinase 2; Mice; Mice, Inbred C57BL; Myocardium; Neuropeptide Y; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pro-Opiomelanocortin; Receptors, Leptin; rhoA GTP-Binding Protein; Signal Transduction; Spinal Cord Injuries; STAT3 Transcription Factor

To observe the altered expressions of neuropeptide Y, substance P and vasoactive intestinal peptide in detrusor of SD rats after spinal cord injury and explore the relationship of the above neuropeptides and neurogenic bladder after spine cord injury.. Twenty male clean-grade SD rats, aged 6 weeks, were selected and randomized into spinal cord injury group (n = 10) and control group (n = 10). Rats in spinal cord injury group were smashed at T10 to cause spinal cord incomplete injury model by the weight drop method while laminectomy alone without smashing was administered in control group. At Week 1 post-operation, all rats were assessed by the maximum bladder capacity, bladder compliance and detrusor pressure for the confirmation of spastic bladder. And all detrusor specimens were marked with argentation and immunohistochemistry for the analyses of nerve fibers, neuropeptide Y, substance P and vasoactive intestinal peptide. The results were evaluated with semiquantitative method to observe the contents of nerve fiber and neuropeptides.. At Week 1 post-operation, the mean maximum bladder compactly, mean maximum detrusor pressure and mean compliance in SCI rats was 0.71 ± 0.24 ml, 32.27 ± 3.12 cm H2O and 0.020 ± 0.009 ml/cm H2O versus 2.0 ± 0.4 ml, 21.0 ± 3.0 cm H2O and 0.090 ± 0.020 ml/cm H2O in normal control group respectively. And the differences were statistically significant (P < 0.01). Meanwhile, the mean content of nerve fibers of neurogenic bladder decreased markedly than that of normal control group (2.58 ± 0.13 vs 5.65 ± 0.26). As compared with the normal control group, the expressions of neuropeptide Y, substance P and vasoactive intestinal peptide (mean integrated optical density: 3.2 ± 0.5, 1.7 ± 0.4 and 2.1 ± 0.4 respectively) decreased dramatically in SCI rats. And the differences were statistically significant (P < 0.01).. The number of nerve fibers and the content of neuropeptides significantly decrease in neurogenic bladder after spinal cord injury in rats. The reduction of neuropeptides may be correlated with the formation of neurogenic bladder after spinal cord injury.

Topics: Animals; Male; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Substance P; Urinary Bladder; Urinary Bladder, Neurogenic; Vasoactive Intestinal Peptide

To evaluate the role of neuropeptide Y in the detrusor of patients with neurogenic detrusor overactivity (NDO), as it has an important role in the neural regulation of the lower urinary tract by exerting differential effects on the release of cholinergic and adrenergic transmitters via autoinhibition and heterosynaptic interactions.. Detrusor biopsies were obtained from 38 patients; 31 had video-urodynamically verified NDO, caused by meningomyelocele in 17 or spinal cord injury in 14. Seven had stress urinary incontinence (SUI) and this group served as a control. All specimens were fixed, paraffin wax-embedded, sectioned and stained with a monoclonal antibody against neuropeptide Y and a general nerve marker protein-gene-product 9.5 (PGP 9.5). The number of PGP 9.5- and neuropeptide Y-containing nerves was quantified by a standardized evaluation using image-analysis software.. The median (range) number of neuropeptide Y-containing nerves in the neurogenic detrusor, at 0.273 (0.126-0.639) per muscle cell nucleus (MCN), was significantly lower (P = 0.014) than that in patients with SUI, at 0.383 (0.267-0.728). In the neurogenic detrusor the number of PGP 9.5-positive nerves, at 0.278 (0.054-0.641)/MCN was also lower (P = 0.111) than in patients with SUI, at 0.368 (0.258-0.497). The ratio of neuropeptide Y to PGP 9.5 counts per biopsy did not differ between the groups (P = 0.628).. The number of PGP 9.5-positive nerves was not significantly and the number of neuropeptide Y-containing nerves was significantly reduced in patients with NDO. This may have been caused by transynaptic nerve degeneration of the detrusor, as described by in patients with spinal cord injury. As neuropeptide Y inhibits the contractile response of the detrusor the reduction of neuropeptide Y-containing nerves may play a role in the development and persistence of DO.

Topics: Adolescent; Adult; Biopsy, Needle; Child; Female; Humans; Immunohistochemistry; Male; Meningomyelocele; Muscle, Smooth; Nerve Tissue; Neuropeptide Y; Spinal Cord Injuries; Urinary Bladder, Neurogenic

Neuropeptide Y (NPY) was immunohistochemically investigated in the frog spinal cord and dorsal root ganglia after axotomy. In normal ganglia, moderate NPY-like immunoreactivity (NPY-IR) prevailed in large and medium cells. In the spinal cord, the NPY-IR was densest in the dorsal part of the lateral funiculus. Other fibers and neurons NPY-IR were observed in the dorsal and ventral terminal fields and mediolateral band. NPY-IR fibers were also found in the ventral horn and in the ventral and lateral funiculi. The sciatic nerve transection increased the NPY-IR in large and medium neurons of the ipsilateral and contralateral dorsal root ganglia at 3 and 7 days, but no clear change was found at 15 days. In the spinal cord, there was a bilateral increase in the NPY-IR of the dorsal part of the lateral funiculus. In the ipsilateral side, the NPY-IR was increased at 3 and 7 days but was decreased at 15 days. In the contralateral side, a significant reduction at 15 days occurred. These findings seem to favor the role of NPY in the modulation of pain-related information in frogs, suggesting that this role of NPY may have appeared early in vertebrate evolution.

Topics: Animals; Female; Immunohistochemistry; Male; Neurons, Afferent; Neuropeptide Y; Rana catesbeiana; Sciatic Nerve; Spinal Cord; Spinal Cord Injuries

In the present study the immunohistochemical localization of proline-rich peptide, so called galarmin, was examined in the brain structures of intact and galarmin-treated rats. Galarmin (a fragment of neurophysin II C-end glycopeptide) was isolated by A.A. Galoyan and coworkers in 1997, from the neurosecretory granules of bovine neurohypophysis, produced by the hypothalamic magnocellular nuclei. In intact rats galarmin-immunoreactive neurons and nerve fibers were widely distributed in the central nervous system. Single intramuscular injection of galarmin to the rats resulted in the increase of both galarmin-immunoreactivity and the number of galarmin-immunoreactive nerve cells, fibers and capillaries. In control experiments where the antisera against the fragment of immunophilin (a receptor of immunosuppressor macrolide FK-506) and the pancreatic neuropeptide Y were used as the primary antibodies, the significant increase of neuropeptide Y-immunoreactive nerve fibers and immunophilin-positive lymphocytes was revealed in galarmin-treated rats. Based on these results and the data on the motoneurons regeneration in the spinal cord hemisectioned rats given galarmin daily for 3 weeks, galarmin has been suggested to act as an immunomodulator, neurotransmitter and neuroregulator.

Topics: Animals; Brain; Immunohistochemistry; Male; Motor Neurons; Nerve Fibers; Neuropeptide Y; Neurophysins; Peptide Fragments; Proline; Rats; Spinal Cord Injuries

Previously by immunohistochemical technique the distribution of immunophilin 1-15 fragment (IphF) isolated from bovine hypothalamus was examined in various tissues (heart, lung), including immune system organs (spleen and thymus) of intact rats. IphF-like immunoreactivity (IphF-LI) was revealed in several cell types: lymphocytes, monocytes, macrophages and mast cells. In the present study the immunohistochemical localization of IphF was examined in intact rat and frog brains. In rat brain several cell groups concentrated particularly in the supraoptic nucleus (SON) of hypothalamus, medulla oblongata (reticular formation, olives, hypoglossal and facial motor nuclei) and cerebellum (lateral cerebellar nucleus) demonstrated IphF-LI. In frog hypothalamus (SON) the same working dilution (1:5000) of IphF-antiserum revealed very strong immunoreactivity. In the paraventricular nucleus (PVN) IphF-LI varicosities were scattered around the immunonegative cells. The second cell groups showing IphF-LI in the frog brain were gliocytes (mainly the astrocytes). Besides, IphF distribution was investigated in rats subjected to hemisection of spinal cord (SC) with and without administration of proline-rich polypeptide (PRP). PRP was isolated from bovine neurohypophysis neurosecretory granules, produced by magnocellular nuclei of hypothalamus. Hemisection of SC led to changes of IphF distribution in the hypothalamus. In PRP treated animals IphF showed no immunoreactivity. PRP is suggested to act as a neurotransmitter and neuroregulator.

Topics: Animals; Brain; Capillaries; Immunohistochemistry; Immunophilins; Male; Nerve Fibers; Neuropeptide Y; Peptide Fragments; Rats; Rats, Inbred Strains; Spinal Cord; Spinal Cord Injuries

The bladder receives an extensive nerve supply that is predominantly cholinergic, but several putative transmitters are present, some of which are colocalized. Previous studies have shown increased levels of sensory nerves, reduced inhibitory transmitters, and structural and functional changes in the excitatory input in unstable bladder conditions. The present study compared the end-organ nerve supply to the bladder in spinal cord injury (SCI) with uninjured controls. Acetylcholinesterase histochemistry and double-label immunofluorescence were used to investigate neurotransmitter content, with confocal laser scanning microscopy to assess colocalization. Organ bath studies provided functional correlates for the structural changes in the excitatory innervation. Control samples had dense innervation of the detrusor containing a diverse range of transmitters. Hyperreflexic SCI samples showed patchy denervation, and areflexic SCI samples were diffusely denervated. Vasoactive intestinal polypeptide-, neuropeptide Y-, neuronal nitric oxide synthase-, and galanin-immunoreactive nerve fibers were reduced from frequent or moderately frequent to infrequent or very infrequent in SCI. Calcitonin gene-related peptide-immunoreactive fibers were infrequent in controls and SCI samples. Patterns of colocalization were unchanged, but significantly fewer fibers expressed more than one transmitter. The subepithelial plexus was markedly reduced and several of the smaller coarse nerve trunks showed no immunoreactivity to the transmitters assessed. There was no reduction in sensitivity to electrical field stimulation of intrinsic nerves in SCI, but the maximum force generated by each milligram of bladder tissue and the peak force as a proportion of the maximum carbachol contraction were significantly reduced and the responses were protracted. There was no significant functional atropine-resistant neuromuscular transmission in controls or SCI. The reported findings have clinical implications in the management of chronic SCI and development of new treatments.

Topics: Adult; Calcitonin Gene-Related Peptide; Carbachol; Denervation; Electric Stimulation; Female; Humans; In Vitro Techniques; Male; Middle Aged; Muscle, Smooth; Neuropeptide Y; Spinal Cord Injuries; Urinary Bladder; Vasoactive Intestinal Peptide

Spinal cord injury disrupts control of sympathetic preganglionic neurons because bulbospinal input has been lost and the remaining regulation is accomplished by spinal circuits consisting of dorsal root afferent and spinal neurons. Moreover, an initial retraction and regrowth of dendrites of preganglionic neurons in response to deafferentation creates the potential for remodelling of spinal circuits that control them. Although catecholamines and neuropeptide Y are found in descending inputs to the preganglionic neurons, their presence in spinal circuits has not been established. Spinal circuits controlling preganglionic neurons contain substance P but participation of these peptidergic neurons in remodelling responses has not been examined. Therefore, we compared immunoreactivity for the catecholamine-synthesizing enzyme dopamine beta-hydroxylase, for neuropeptide Y and for substance P in the intermediate gray matter of the spinal cord in control rats and in rats seven or fourteen days after transection at the fourth thoracic cord segment. Sympathetic preganglionic neurons were retrogradely labelled by intraperitoneal injection of the tracer FluoroGold. These experiments yielded three original findings. 1) At one and two weeks after cord transection, fibres and terminals immunoreactive for dopamine beta-hydroxylase and neuropeptide Y were consistently found in the intermediolateral cell column in segments caudal to the transection. The area of fibres and terminals containing these immunoreactivities was markedly reduced compared to control rats or to segments rostral to the transection in the spinal rats. 2) Immunoreactivity for substance P was increased after cord transection and the distribution of fibres immunoreactive for this peptide in segments caudal to the transection extended more widely through the intermediate gray matter. These reactions demonstrated a plastic reaction to cord transection by spinal neurons expressing substance P. 3) Dopamine beta-hydroxylase expression was up-regulated in somata within the intermediate gray matter of spinal segments caudal to the transection. The numbers of somata immunoreactive for this enzyme increased six-fold by 14 days after cord transection, compared to the few somata counted in control rats. In conclusion, the presence of a catecholamine synthesizing enzyme and neuropeptides in fibres surrounding sympathetic preganglionic neurons caudal to a cord transection suggests a source of catecholamines and these

Topics: Animals; Autonomic Fibers, Preganglionic; Brain Chemistry; Catecholamines; Dopamine beta-Hydroxylase; Immunohistochemistry; Male; Nerve Fibers; Neurons; Neuropeptide Y; Neuropeptides; Rats; Rats, Wistar; Spinal Cord Injuries; Substance P

Modest increases in urinary bladder pressure result in acute hypertensive episodes in humans with spinal cord lesions above T5. The underlying mechanisms of this condition, referred to as autonomic dysreflexia, are not well understood. The aim of this study was to characterize the contribution of alpha- and beta-adrenoceptors as well as circulating neuropeptide-Y (NPY) to the pressor response to bladder distension in conscious cervical spinal rats. Rats were chronically instrumented with arterial and venous catheters. After 2-3 days, a complete spinal transection (C7) was performed, and the urinary bladder was catheterized: 24 h later, mean arterial pressure (MAP) responses to 5 min bladder distensions (+40) were measured under control conditions and after administration of specific autonomic antagonists. To assess the contribution of alpha and beta adrenergic mechanisms the alpha antagonist prazosin (0.45 mg/kg i.v.) and beta antagonist, propranolol (4 mg/kg i.v.), were administered individually or together. Blood samples were taken before, during and after bladder distension for determination of plasma NPY by radioimmunoassay. The pressor response to bladder distension was approximately 30 mmHg under control conditions. The response was attenuated (-38%), but not abolished, by prazosin. A similar attenuation (-41%) was observed with propranolol. There were no changes in plasma NPY in response to bladder distension. Finally, the pressor response was completely abolished by combined alpha- and beta-adrenergic blockade. These results suggest that autonomic dysreflexia is mediated exclusively by adrenergic receptors in the spinal rat. Moreover, both alpha and beta adrenergic receptors contribute to the pressor response induced by bladder distension in the conscious cervical spinal rat.

Topics: Animals; Autonomic Nervous System Diseases; Blood Pressure; Ganglionic Blockers; Heart Rate; Hexamethonium; Hypertension; Male; Neuropeptide Y; Prazosin; Propranolol; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Spinal Cord Injuries; Sympathetic Nervous System; Urinary Bladder; Urinary Catheterization

Specimens of urethra were obtained from patients with cervical and thoracic spinal cord lesion with detrusor-sphincter dyssynergia and from patients with lower motor neurone lesion with detrusor areflexia, undergoing transurethral sphincterotomy. Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in nerves associated with both the smooth and striated muscle components of the urethral sphincter were studied immunohistochemically and by immunoassay. In patients with detrusor-sphincter dyssynergia following cervical and thoracic spinal cord injury, NPY- and VIP-immunoreactive varicose nerve fibres were seen in both the smooth and striated muscle components of the urethral sphincter. In the smooth muscle, NPY- and VIP-immunoreactive nerves did not appear to have any particular orientation, but in the striated muscle region they were found to run along the length of individual muscle fibres. In patients with detrusor areflexia following lower motor neurone lesion, while the pattern, density and fluorescence intensity of NPY- and VIP-immunoreactive nerves in the smooth muscle of the sphincter mechanism appeared the same as seen in patients with detrusor-sphincter dyssynergia, there was a marked increase in the density of these nerves in the striated muscle region of the sphincter mechanism. Quantitation of the peptides by immunoassay was consistent with the histochemical findings, with significantly higher levels of both NPY and VIP in the striated muscle of patients with lower motor neurone lesion, compared to those with cervical and thoracic spinal cord lesion, p = 0.04. NPY and VIP levels in urethral smooth muscle were in the same range in lower motor neurone lesion patients and cervical and thoracic spinal cord lesion patients. We conclude that there are increased NPY- and VIP-containing fibres in striated muscle of the intrinsic external urethral sphincter in patients with areflexic bladder compared with those with detrusor-sphincter dyssynergia.

Topics: Adult; Enzyme-Linked Immunosorbent Assay; Humans; Male; Muscles; Neurons; Neuropeptide Y; Radioimmunoassay; Spinal Cord Injuries; Urethra; Urinary Bladder, Neurogenic; Vasoactive Intestinal Peptide