cyclic-gmp and Spinal-Cord-Injuries

In the current report, we summarize our findings related to the involvement of nitric oxide (NO) in the pathology of spinal cord trauma. We initially studied the distribution of nitric oxide synthase (NOS)-immunolabeled and/or nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd; which is highly colocalized with NOS)-stained somata and fibers in the spinal cord of the rabbit. Segmental and laminar distribution of NADPHd-stained neurons in the rabbit revealed a large number of NADPHd-stained neurons in the spinal cord falling into six categories, N1-N6, while others could not be classified. Large numbers of NADPHd-stained neurons were identified in the superficial dorsal horn and around the central canal. Four morphologically distinct kinds of NADPHd-stained axons 2.5-3.5 microm in diameter were identified throughout the white matter in the spinal cord. Moreover, a massive occurrence of axonal NADPHd-staining was detected in the juxtagriseal layer of the ventral funiculus along the rostrocaudal axis. The prominent NADPHd-stained fiber bundles were identified in the mediobasal and central portion of the ventral funiculus. The sulcomarginal fasciculus was found in the basal and medial portion of the ventral funiculus in all cervical and thoracic segments. Since the discovery that NO may act as a neuronal transmitter, an increasing interest has focused on its ability to modulate synaptic function. NO passes through cell membranes without specific release or uptake mechanisms inducing changes in signal-related functions by several means. In particular, the activation of the soluble guanylyl cyclases (sGC), the formation of cyclic guanosine 3',5'-monophosphate (cGMP) and the action of cGMP-dependent protein kinases has been identified as the main signal transduction pathways of NO in the nervous system including spinal cord. It is known that the intracellular level of cGMP is strictly controlled by its rate of synthesis via guanylyl cyclases (GC) and/or by the rate of its degradation via 3',5'-cyclic nucleotide phosphodiesterases (PDE). GC can be divided into two main groups, i.e., the membrane-bound or particular guanylyl cyclase (pGC) and the cytosolic or sGC. In the spinal cord, the activation of pGC has only been demonstrated for natriuretic peptides, which stimulate cGMP accumulation in GABA-ergic structures in laminae I-III of the rat cervical spinal cord. These neurons are involved in controlling the action of the locomotor circuit. In view of

Topics: Animals; Cyclic GMP; Humans; Immunohistochemistry; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase Type I; Signal Transduction; Spinal Cord Injuries

In Iranian folkloric medicine, Bupleurum falcatum L. (Chinese Thoroughwax) has been used as a selective analgesic remedy for several centuries.. The current research was conducted to explore the anti-nociceptive and anti-allodynic action of Bupleurum falcatum L. roots essential oil (BFEO) in Swiss mice.. Formalin-induced paw licking (FIPL) model was applied for exploring of BFEO antinociceptive effects (neurogenic or inflammatory pain). The involvements of L-arginine-NO-cGMP-KATP channel pathway and several receptors such as opioid, peroxisome proliferator-activated (PPA), cannabinoid, transient receptor potential vanilloid, and adrenergic receptors were assesses to detect the anti-nociceptive activity of BFEO. Cervical spinal cord contusion (CSC) paradigm was employed for induction of neuropathic pain.. BFEO (100 mg/kg), in the FIPL model, produced significant antinociception compared to the control mice (p < 0.01). Furthermore, L-arginine, methylene blue, glibenclamide, naloxonazine, GW9662, and SR141716A pre-treatments restored the BFEO anti-nociceptive effects (p < 0.05) in the FIPL (second phase) test (p < 0.05). Intraperitoneal administration of saikosaponin A (one of the main constituents of BFEO) partially alleviated (p < 0.05) pain in FIPL test. Likewise, in CSC mice, the von Frey assay exhibited that BFEO could alter mechanical allodynia.. Finally, it seems that, in male mice, BFEO has both anti-allodynic and anti-nociceptive effects. The present data also suggest activating the L-arginine-NO-cGMP-KATP channel pathway as well as interaction of opioid, PPA, and cannabinoid receptors in the BFEO anti-nociceptive activities. These results also propose that BFEO could effectively attenuate allodynia in CSC mice.

Topics: Animals; Arginine; Bupleurum; Cyclic GMP; Formaldehyde; Hyperalgesia; Iran; Male; Medicine, Traditional; Mice; Nitric Oxide; Oils, Volatile; Pain; Phytotherapy; Plant Oils; Plant Roots; Potassium Channels; Spinal Cord Injuries

Nitric oxide (NO) is an important regulator of vasodilation and angiogenesis in the central nervous system (CNS). Signaling initiated by the membrane receptor CD47 antagonizes vasodilation and angiogenesis by inhibiting synthesis of cyclic guanosine monophosphate (cGMP). We recently found that deletion of CD47 led to significant functional locomotor improvements, enhanced angiogenesis, and increased epicenter microvascular perfusion in mice after moderate contusive spinal cord injury (SCI). We tested the hypothesis that improving NO/cGMP signaling within the spinal cord immediately after injury would increase microvascular perfusion, angiogenesis, and functional recovery, with an acute, 7-day administration of the cGMP phosphodiesterase 5 (PDE5) inhibitor sildenafil. PDE5 expression is localized within spinal cord microvascular endothelial cells and smooth muscle cells. While PDE5 antagonism has been shown to increase angiogenesis in a rat embolic stroke model, sildenafil had no significant effect on angiogenesis at 7 days post-injury after murine contusive SCI. Sildenafil treatment increased cGMP concentrations within the spinal cord and improved epicenter microvascular perfusion. Basso Mouse Scale (BMS) and Treadscan analyses revealed that sildenafil treatment had no functional consequence on hindlimb locomotor recovery. These data support the hypothesis that acutely improving microvascular perfusion within the injury epicenter by itself is an insufficient strategy for improving functional deficits following contusive SCI.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Capillaries; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelial Cells; Female; Hindlimb; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Immunohistochemistry; Locomotion; Mice; Mice, Inbred C57BL; Microcirculation; Nitric Oxide; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Recovery of Function; Regional Blood Flow; Sildenafil Citrate; Spinal Cord Injuries; Sulfones

During postnatal development large amplitude spontaneous activity of the neonatal rat bladder changes to a low amplitude adult pattern of activity that leads to improved storage function. Previously, we have shown that spontaneous activity in neonatal rat bladder strips is inhibited by activation of the nitric oxide (NO)-cGMP signaling pathway. In the present experiments we determined if this inhibitory pathway is altered during postnatal development or spinal cord injury.. Baseline tone and amplitude and frequency of spontaneous contractions were measured in bladder strips from male or female neonatal (days 10-21), juvenile (days 24-39) and adult female spinal cord intact or chronic spinal cord injured Sprague-Dawley rats.. The inhibitory effects of an NO donor (SNAP) and a PDE-5 inhibitor (zaprinast) on spontaneous activity of bladder strips decreased during postnatal development, while an inhibitory effect of 8-bromo-cGMP, which was blocked by a protein kinase G inhibitor, was detected at all ages tested. However, the effect of NO-cGMP signaling to reduce baseline tone emerged during postnatal development. The inhibition induced by the NO donor was blocked by an inhibitor of soluble guanylyl cyclase (sGC). Chronic spinal cord injury (cSCI), which causes the re-emergence of a neonatal-like pattern of spontaneous activity, did not restore sensitivity to NO-mediated inhibition in adult rat bladders.. These data indicate that while cGMP signaling inhibits activity in young and adult bladders as well as after cSCI, there is a developmental decrease in the sensitivity of bladder to NO-mediated inhibition.

Topics: Age Factors; Animals; Animals, Newborn; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Female; Guanylate Cyclase; Male; Nitric Oxide; Nitric Oxide Donors; Phosphodiesterase 5 Inhibitors; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Spinal Cord Injuries; Urinary Bladder; Urodynamics

To understand the pathomechanisms of spinal cord injuries will be a prerequisite to develop efficient therapies. By investigating acute lesions of spinal cord white matter in anesthetized mice with fluorescently labeled microglia and axons using in vivo two-photon laser-scanning microscopy (2P-LSM), we identified the messenger nitric oxide (NO) as a modulator of injury-activated microglia. Local tissue damages evoked by high-power laser pulses provoked an immediate attraction of microglial processes. Spinal superfusion with NO synthase and guanylate cyclase inhibitors blocked these extensions. Furthermore, local injection of the NO-donor spermine NONOate (SPNO) or the NO-dependent second messenger cGMP induced efficient migration of microglial cells toward the injection site. High-tissue levels of NO, achieved by uniform superfusion with SPNO and mimicking extended tissue damage, resulted in a fast conversion of the microglial shape from ramified to ameboid indicating cellular activation. When the spinal white matter was preconditioned by increased, ambient ATP (known as a microglial chemoattractant) levels, the attraction of microglial processes to local NO release was augmented, whereas it was abolished at low levels of tissue ATP. Because both signaling molecules, NO and ATP, mediate acute microglial reactions, coordinated pharmacological targeting of NO and purinergic pathways will be an effective mean to influence the innate immune processes after spinal cord injury.

Topics: Acute Disease; Adenosine Triphosphate; Animals; Axons; Cell Movement; Cell Polarity; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Mice; Mice, Transgenic; Microglia; Nerve Fibers, Myelinated; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Signal Transduction; Spermine; Spinal Cord; Spinal Cord Injuries

1. Nitric oxide (NO) participates, at least in part, to the establishment and maintenance of pain after nerve injury. Therefore, drugs that target the NO/cGMP signaling pathway are of interest for the treatment of human neuropathic pain. Various compounds endowed with NO-releasing properties modulate the expression and function of inducible nitric oxide synthase (iNOS), the key enzyme responsible for sustained NO production under pathological conditions including neuropathic pain. 2. With this background, we synthesized a new chemical entity, [1-(aminomethyl)cyclohexane acetic acid 3-(nitroxymethyl)phenyl ester] NCX8001, which has a NO-releasing moiety bound to gabapentin, a drug currently used for the clinical management of neuropathic pain. We examined the pharmacological profile of this drug with respect to its NO-releasing properties in vitro as well as to its efficacy in treating neuropathic pain conditions (allodynia) consequent to experimental sciatic nerve or spinal cord injuries. 3. NCX8001 (1-30 microm) released physiologically relevant concentrations of NO as it induced a concentration-dependent activation of soluble guanylyl cyclase (EC(50)=5.6 microm) and produced consistent vasorelaxant effects in noradrenaline-precontracted rabbit aortic rings (IC(50)=1.4 microm). 4. NCX8001, but not gabapentin, counteracted in a concentration-dependent fashion lipopolysaccharide-induced overexpression and function of iNOS in RAW264.7 macrophages cell line. Furthermore, NCX8001 also inhibited the release of tumor necrosis factor alpha (TNFalpha) from stimulated RAW264.7 cells. 5. NCX8001 (28-280 micromol x kg(-1), i.p.) reduced the allodynic responses of spinal cord injured rats in a dose-dependent fashion while lacking sedative or motor effects. In contrast, gabapentin (170-580 micromol x kg(-1), i.p.) resulted less effective and elicited marked side effects. 6. NCX8001 alleviated the allodynia-like responses of rats to innocuous mechanical or cold stimulation following lesion of the sciatic nerve. This effect was not shared by equimolar doses of gabapentin. 7. Potentially due to the slow releasing kinetics of NO, NCX8001 alleviated pain-like behaviors in two rat models of neuropathic pain in a fashion that is superior to its parent counterpart gabapentin. This new gabapentin derivative, whose mechanism deserves to be explored further, offers new hopes to the treatment of human neuropathic pain.

Topics: Acetates; Amines; Animals; Aorta, Thoracic; Behavior, Animal; Cyclic GMP; Cyclohexanecarboxylic Acids; Cyclohexanes; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Female; Gabapentin; gamma-Aminobutyric Acid; Humans; Injections, Intraperitoneal; Lipopolysaccharides; Macrophages; Male; Mice; Muscle, Smooth; Nitrates; Nitric Oxide; Pain; Pain Measurement; PC12 Cells; Peripheral Nerve Injuries; Peripheral Nerves; Rabbits; Randomized Controlled Trials as Topic; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Spinal Cord Injuries; Tumor Necrosis Factor-alpha; Vasodilation

The heme oxygenase (HO) isozymes catalyze oxidation of the heme molecule to biliverdin and carbon monoxide (CO) with the release of chelated iron. Presently, we have defined, for the first time, propensity for site of injury-directed induction of isozymes--the stress-inducible isozyme, HO-1, responds distal (below) and the glucocorticoid (GC)-inducible HO-2 responds proximal (above) to the site of injury. We have also shown that reactive iron (Fe3+) and cGMP staining spatially resemble that of HO-1; which, in turn, colocalizes in motor neurons with transcription factors: Fas-associated protein containing death domain (FADD), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and p53. Spinal cord injury (SCI) was inflicted by clip compression for 30 min, and analyses were carried out after 4 h or 16 h. When compared with spinal cord segments proximal to the site of injury, northern blot analysis showed remarkably higher levels of HO-1 mRNA distal (below) to the site of injury at both time points. In contrast, HO-2 mRNA levels were elevated proximal (above) to the site of injury and more prominently at 16 h post SCI. Immunohistochemical analyses were carried out using 2 x 5 mm segments above and below the compression site. When compared with segments above the site of injury, the intensity of HO-1 immunostaining and the number of HO-1 positive neurons in the ventral horn motor neurons were prominently increased in segments below the injury. Western blot analysis confirmed the observations. HO-2 protein was mapped to the ventral horn motor neurons, oligodendrocytes, the Clarke's nucleus neurons and the ependymal cells. When compared with segments below the site of injury, neuronal HO-2 staining intensity was increased above the site of injury, and most notably at 16 h. These observations were also confirmed by western blotting and HO activity measurements. Tissue Fe3+ and cGMP staining were increased and prominently mapped below the site of injury, where cGMP colocalized with HO-1 in the nucleus of the motor neurons. Also, a site of injury-directed pattern of induction of FADD, TRAIL, and p53 immunoreactivity, and a widespread colocalization of the oncogenes with HO-1 protein, were found within motor neurons below the level of injury. We forward the hypothesis that HO-1 and HO-2 have different roles in the defense mechanisms of the injured nervous system. We hypothesize that HO-1 protects against further damage by contributing to controlle

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Carrier Proteins; Cyclic GMP; Disease Models, Animal; Disease Progression; Enzyme Induction; Fas-Associated Death Domain Protein; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Iron; Membrane Glycoproteins; Membrane Proteins; Mice; Neurons; Spinal Cord; Spinal Cord Injuries; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Experiments were carried out to explore the possible role played by the nitric oxide (NO) system in the organum vasculosum laminae terminalis (OVLT) of rat brain in arterial pressure regulation. Intracerebroventricular (ICV) or intra-OVLT administration of NO donors such as hydroxylamine, sodium nitro-prusside or s-nitro-acetylpenicillamine caused an up to 55 mmHg decrease in blood pressure (BP) but an increase in NO release (measured by porphyrin/nafion coated carbon fibre electrodes in combination with voltammetry) in the OVLT. In contrast, ICV or intra-OVLT administration of N(G)-nitro-L-arginine methyl ester (L-NAME; a constitutive NO synthase inhibitor) caused an up to 45 mmHg increase in BP but a fall in NO release in the OVLT. Compared with the BP responses induced by ICV injection of NO donors or NO synthase inhibitors, the OVLT route of injection required a much lower dose of NO donors or NO synthase inhibitors to produce a similar BP effect. The depressor effects induced by ICV or intra-OVLT administration of NO donors were attenuated by pretreatment with intra-OVLT injection of methylene blue (an inhibitor of guanylate cyclase), haemoglobin (a NO scavenger), L-NAME or spinal transection. On the other hand, the L-NAME-induced pressor effects were attenuated by pretreatment with intra-OVLT injection of L-arginine or spinal transection. The data suggest that activation of cyclic GMP-dependent NO synthase in the OVLT of rat brain causes cyclic GMP-dependent decreases in arterial pressure via inhibiting the sympathetic efferent activity.

Topics: Animals; Arginine; Blood Pressure; Brain; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Hydroxylamine; Male; Methylene Blue; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Rats; Rats, Sprague-Dawley; Sodium Azide; Spinal Cord Injuries

A spinal cord injury was produced in Wistar rats by extradural compression of the cord with a Sugita aneurysm clip for 5 seconds. During a 2-week observation period following the injury, the tissue norepinephrine (NE), dopamine (DA), and serotonin (5-HT) concentrations decreased uniformly at and below the injured site. The chemical denervation of NE or 5-HT neurons produced by the intraspinal injection of 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT) 2 weeks before the injury did not cause a marked difference in the extent of hemorrhagic necrosis of the spinal cord after trauma as compared to control animals without pretreatment. In the rats pretreated with 6-OHDA, NE was decreased to less than 30% of control (non-pretreated) values, and, beginning at 5 days after injury, motor performance (assessed quantitatively with the inclined-plane method) was significantly improved compared to results in the non-pretreated control rats. The rats pretreated with 5,7-DHT showed no change from control animals. Spinal cord samples from non-pretreated control animals obtained at the injury site 30 minutes after the compression injury showed a marked decrease in the activity of synaptosomal Na+-K+-ATPase (adenosine triphosphatase) of about 50%, and an increase in both thiobarbituric acid reaction substance (about 170%) and cyclic guanine monophosphate (about 150%). The NE-denervated rats showed no significant changes in these three parameters. The results indicated that NE released after crush injury may impair the neuronal cell membrane around the lesion site by induction of lipid peroxidation. The possible mechanisms by which released NE may alter membrane function are discussed.

Topics: Animals; Biogenic Amines; Cyclic GMP; Dopamine; Hydroxydopamines; Lipid Peroxides; Male; Norepinephrine; Oxidopamine; Rats; Rats, Inbred Strains; Serotonin; Sodium-Potassium-Exchanging ATPase; Spinal Cord; Spinal Cord Injuries; Synaptosomes

Pain-alleviating effects of epidural spinal cord stimulation (ESCS) were assessed in patients with chronic intractable pain including cancer pain, and some aspects concerning its mechanisms were discussed. The temporary ESCS with percutaneously inserted electrodes was employed in 105 patients, and the implantable systems for long-term use in 19 patients. The ESCS had satisfactory effects especially in patients suffering from cancer pain, causalgia, facial and nape pain. The conditioning stimuli applied to the cervical dorsal cord exhibited interactions with the segmentally evoked spinal cord potential (SCP). The N1 wave of the SCP was inhibited up to 120 ms, while the P2 wave was facilitated for more than 100 ms, suggesting that the presynaptic inhibitory action at the dorsal horn is responsible for mechanism of the ESCS. CSF concentration of norepinephrine was significantly decreased by the ESCS therapy, indicating the existence of the relationship between norepinephrinergic descending inhibitory system and the ESCS in pain-alleviating mechanism.

Topics: Adult; Aged; Cerebral Palsy; Cyclic GMP; Electric Stimulation Therapy; Electrodes, Implanted; Female; Humans; Male; Middle Aged; Neoplasms; Neuralgia; Norepinephrine; Pain, Intractable; Pain, Postoperative; Spinal Cord; Spinal Cord Diseases; Spinal Cord Injuries

Topics: Animals; Blood Pressure; Cats; Cyclic GMP; Dose-Response Relationship, Drug; Female; Free Radicals; Injections, Intravenous; Lipid Peroxides; Male; Methylprednisolone; Methylprednisolone Hemisuccinate; Sodium-Potassium-Exchanging ATPase; Spinal Cord Injuries; Time Factors