Horn Of Lumbar Spinal Cord Research Articles

Magnetic Stimulation of Gigantocellular Reticular Nucleus with Iron Oxide Nanoparticles Combined Treadmill Training Enhanced Locomotor Recovery by Reorganizing Cortico-Reticulo-Spinal Circuit.

Gigantocellular reticular nucleus (GRNs) executes a vital role in locomotor recovery after spinal cord injury. However, due to its unique anatomical location deep within the brainstem, intervening in GRNs for spinal cord injury research is challenging. To address this problem, this study adopted an extracorporeal magnetic stimulation system to observe the effects of selective magnetic stimulation of GRNs with iron oxide nanoparticles combined treadmill training on locomotor recovery after spinal cord injury, and explored the possible mechanisms. Superparamagnetic iron oxide (SPIO) nanoparticles were stereotactically injected into bilateral GRNs of mice with moderate T10 spinal cord contusion. Eight-week selective magnetic stimulation produced by extracorporeal magnetic stimulation system (MSS) combined with treadmill training was adopted for the animals from one week after surgery. Locomotor function of mice was evaluated by the Basso Mouse Scale, Grid-walking test and Treadscan analysis. Brain MRI, anterograde virus tracer and immunofluorescence staining were applied to observe the tissue compatibility of SPIO in GRNs, trace GRNs' projections and evaluate neurotransmitters' expression in spinal cord respectively. Motor-evoked potentials and H reflex were collected for assessing the integrity of cortical spinal tract and the excitation of motor neurons in anterior horn. (1) SPIO persisted in GRNs for a minimum of 24 weeks without inducing apoptosis of GRN cells, and degraded slowly over time. (2) MSS-enabled treadmill training dramatically improved locomotor performances of injured mice, and promoted cortico-reticulo-spinal circuit reorganization. (3) MSS-enabled treadmill training took superimposed roles through both activating GRNs to drive more projections of GRNs across lesion site and rebalancing neurotransmitters' expression in anterior horn of lumbar spinal cord. These results indicate that selective MSS intervention of GRNs potentially serves as an innovative strategy to promote more spared fibers of GRNs across lesion site and rebalance neurotransmitters' expression after spinal cord injury, paving the way for the structural remodeling of neural systems collaborating with exercise training, thus ultimately contributing to the reconstruction of cortico-reticulo-spinal circuit.

Sex-specific characteristics of cells expressing the cannabinoid 1 receptor in the dorsal horn of the lumbar spinal cord.

It is becoming increasingly clear that robust sex differences exist in the processing of acute and chronic pain in both rodents and humans. However, the underlying mechanism has not been well characterized. The dorsal horn of the lumbar spinal cord is the fundamental building block of ascending and descending pain pathways. It has been shown that numerous neurotransmitter and neuromodulator systems in the spinal cord, including the endocannabinoid system and its main receptor, the cannabinoid 1 receptor (CB1 R), play vital roles in processing nociceptive information. Our previous findings have shown that CB1 R mRNA is widely expressed in the brain in sex-dependent patterns. However, the sex-, lamina-, and cell-type-specific characteristics of CB1 R expression in the spinal cord have not been fully described. In this study, the CB1 R-iCre-EGFP mouse strain was generated to label and identify CB1 R-positive (CB1 RGFP ) cells. We reported no sex difference in CB1 R expression in the lumbar dorsal horn of the spinal cord, but a dynamic distribution within superficial laminae II and III in female mice between estrus and nonestrus phases. Furthermore, the cell-type-specific CB1 R expression pattern in the dorsal horn was similar in both sexes. Over 50% of CB1 RGFP cells were GABAergic neurons, and approximately 25% were glycinergic and 20-30% were glutamatergic neurons. The CB1 R-expressing cells also represented a subset of spinal projection neurons. Overall, our work indicates a highly consistent distribution pattern of CB1 RGFP cells in the dorsal horn of lumbar spinal cord in males and females.

Inhibitor of DNA binding 2 accelerates nerve regeneration after sciatic nerve injury in mice.

Inhibitor of DNA binding 2 (Id2) can promote axonal regeneration after injury of the central nervous system. However, whether Id2 can promote axonal regeneration and functional recovery after peripheral nerve injury is currently unknown. In this study, we established a mouse model of bilateral sciatic nerve crush injury. Two weeks before injury, AAV9-Id2-3×Flag-GFP was injected stereotaxically into the bilateral ventral horn of lumbar spinal cord. Our results showed that Id2 was successfully delivered into spinal cord motor neurons projecting to the sciatic nerve, and the number of regenerated motor axons in the sciatic nerve distal to the crush site was increased at 2 weeks after injury, arriving at the tibial nerve and reinnervating a few endplates in the gastrocnemius muscle. By 1 month after injury, extensive neuromuscular reinnervation occurred. In addition, the amplitude of compound muscle action potentials of the gastrocnemius muscle was markedly recovered, and their latency was shortened. These findings suggest that Id2 can accelerate axonal regeneration, promote neuromuscular reinnervation, and enhance functional improvement following sciatic nerve injury. Therefore, elevating the level of Id2 in adult neurons may present a promising strategy for peripheral nerve repair following injury. The study was approved by the Experimental Animal Ethics Committee of Jinan University (approval No. 20160302003) on March 2, 2016.

Anti‐apoptotic and Neuroregenerative Effects of Soluble Protein Fraction of the Epidermal Secretion from the Arabian Gulf Catfish following Sciatic Nerve Crush Injury in Rats

Crush injuries (axonotmesis) occur from an acute traumatic compression of the nerve resulting in many different degrees of neural damage. Significant cell death and axon degeneration occur as a result of this damage, leading to permanent functional deficits. Previous clinical trials showed that catfish skin preparations (CSP) have an anti‐inflammatory and healing properties for diabetic foot ulcers, chronic back pain, and other neurological disorders. This study was designed to investigate the anti‐apoptotic effect of soluble protein fraction (SPF)‐Fraction C (FC) derived from CSP on the sciatic nerve crush injury. Assessment of peripheral axonal regeneration recovery and the neuroprotective properties of SPF‐FC on the spinal neurons was done following nerve injury and SPF‐FC treatment. Male Wistar rats (weight 200–250g) were randomly assigned into five groups: (I) SHAM, (II) CRUSH, (III) CRUSH+(1.5mg/kg) SPF‐FC, (IV) CRUSH+(3mg/kg) SPF‐FC, and (V) CRUSH+(4.5mg/kg) SPF‐FC. Rats underwent sciatic nerve crush surgery, followed by treatment with SPF‐FC administered intraperitoneally (IP) for two weeks, and sacrificed at the end of the fourth week. All animals were assessed for sensory and motor neurobehavioral tests throughout the four weeks. Peripheral axonal regeneration was assessed through whole mount staining technique of sciatic nerve using axonal markers (S100, NF, and MBP) and cell proliferation marker (Ki67). The neuroprotective properties of SPF‐FC treatment on the spinal cord neurons were assessed using Cresyl violet staining and immunohistochemistry of NeuN, while the apoptotic pathway was assessed using Western blot, immunohistochemistry, and TUNEL techniques. The results of this study showed that IP administration of different SPF doses significantly (p<0.05–0.001) improved the sensory and motor neurobehavioral functions recovery of the nerve‐injured groups. Visualization of sciatic nerve through whole mount staining revealed significant (p<0.000) increase in the expression levels of axonal regeneration recovery with CRUSH+(4.5mg/kg) SPF‐FC treatment. Moreover, the CRUSH+(4.5mg/kg) SPF‐FC treatments have neuroprotective effects on spinal cord neurons as was shown in Cresyl violet stained sections and NeuN‐immunoreactive neurons. Our results after employing WB, IHC, and TUNEL for the apoptotic pathway revealed that SPF‐FC treatments reduce neuronal cell death resulting from sciatic nerve crush injury. The data suggest that SPF‐FC reduces sensory and motor neurobehavioral deficits and enhances axonal regeneration recovery. Also, SPF‐FC alleviates the apoptotic and neurodegenerative modifications in spinal cord neurons. Therefore, SPF‐FC protects spinal neurons after peripheral nerve injury.Support or Funding InformationThis work was funded by the college of graduate studies, Kuwait university.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stimulating Frequency-dependent Analgesic Effect of Electroacupuncture via Down-regulating Expression of VEGF in Dorsal Horns of Lumbar Spinal Cord in Post Herpetic Neuralgia Rats

To observe the effect of different frequencies of electroacupuncture (EA) stimulation on pain threshold (PT) and expression of vascular endothelial growth factor (VEGF) in dorsal horns (DHs) of the lumbar spinal cord in resiniferatoxin (RTX)-induced post-herpetic neuralgia (PHN) rats, so as to reveal its mechanism in alleviating PHN. Male SD rats were randomized into control, model, 2 Hz-EA, 15 Hz-EA, 100 Hz-EA and sham EA groups (n=16 in each). The PHN model was induced by a single intraperitoneal injection of RTX (250 μg/kg), and rats of the control group received intraperitoneal injection of the same dose of vehicle (10% Tween 80, 10% alcohol and 0.9% NaCl). Rats of EA treatment groups received EA stimulation (2 Hz, 15 Hz or 100 Hz, 1 mA) at the left "Huantiao" (GB 30) and "Yanglingquan" (GB 34) for 30 min, once every other day for 35 days, starting from 1 week after RTX injection. For sham control, acupuncture needles were inserted ipsilaterally into GB 30 and GB 34 for 30 min without electrical stimulation or manual needle manipulation. The mechanical allodynia was quantified with Von Frey filaments. The expression of mRNA and protein of VEGF in the DHs of lumbar spinal cord 4-6 segments (sampled under light microscope) was detected by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. A single RTX injection gradually induced tactile allodynia (significant reduction of the mechanical PT) within 3 weeks relevant to the control group (P<0.01). EA applied to GB 30 and GB 34 at 2 Hz and 15 Hz, but not 100 Hz, significantly decreased the tactile allodynia after the treatment (2 Hz from 2 weeks on and 15 Hz from 3 weeks on) in RTX-treated rats (P<0.05). RTX administration increased the mRNA and protein expression of VEGF in the lumbar spinal cord compared with the control group (P<0. 05). Moreover, 2 Hz, but not 15 Hz and 100 Hz EA significantly reduced VEGF mRNA and protein expression(P<0.05). The expression of both VEGF mRNA and protein was negatively correlated with mechanical PT in RTX-induced PHN rats. EA at 2 Hz can significantly reduce VEGF expression in the lumbar spinal cord DHs of PHN rats, which is possibly in part related to its effect in alleviating the mechanical allodynia. Our study suggests that 2 Hz EA is the best stimulation frequency for relieving PHN.

Targeting macrophage and microglia activation with colony stimulating factor 1 receptor inhibitor is an effective strategy to treat injury-triggered neuropathic pain

IntroductionNeuropathic pain is a debilitating condition. The importance of neuroimmune interactions in neuropathic pain has been evidenced by the involvement of different immune cells in peripheral and central sensitization of pathological pain. Macrophages and microglia are the most abundant immune cells activated in injured nerves and spinal cord, respectively. Several lines of evidence showed that macrophage/microglia survival, activation, proliferation, and differentiation require the involvement of macrophage-colony stimulating factor. In this study, we investigated whether blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling can be effective in relieving neuropathic pain.Materials and methodsPartial sciatic nerve ligation was performed in mice to induce neuropathic pain behavior. Mice were orally treated with a selective colony stimulating factor 1 receptor inhibitor, PLX5622, daily in both preventive (two days prior to surgery until D14 post-partial sciatic nerve ligation) and reversal paradigms (D28–D33 post-partial sciatic nerve ligation). Animal neuropathic pain behavior was monitored using von Frey hairs and acetone application. Phenotype of macrophages in injured nerves was analyzed at D3 and D33 post-injury using flow cytometry analysis. The effect of PLX5622 on microglia activation in lumbar spinal cord was further examined by immunohistochemistry using Iba-1 antibody.ResultsSignificant alleviation of both mechanical and cold allodynia was observed in PLX5622-treated animals, both in preventive and reversal paradigms. PLX5622 treatment reduced the total number of macrophages in injured nerves, it appears colony stimulating factor 1 receptor inhibition affected more specifically CD86+ (M1 like) macrophages. Consequently, the expression of various pro-inflammatory cytokines (TNF-α, IL-1β) was reduced. Microglia activation in dorsal horn of lumbar spinal cord following partial sciatic nerve ligation was significantly inhibited with PLX5622 treatment in both preventive and reversal paradigms.ConclusionMacrophages in peripheral nerve and microglia in the spinal cord are required in the generation and maintenance of injury-associated neuropathic pain. Blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling on these myeloid cells along the pain transmission pathway is an effective strategy to alleviate neuropathic pain.

Effects of resveratrol on motor function and anterior horn neuron of spinal cord following acute sciatic nerve compression injury in rats

Objective To investigate the effects of resveratrol (Res) on motor function and the anterior horn neuron of lumbar spinal cord after acute sciatic nerve compression injury in rats. Methods The rat model with acute sciatic nerve compression injury was established in 32 Sprague Dawley (SD) rats, which were randomly divided into four groups: Res group, Dimethyl Sulfoxide (DMSO) group, Normal Saline (NS) group and sham-operation group. Res, DMSO and saline were successively injected by intraperitoneal for 10 days after established crush acute sciatic nerve compression injury model, while sham-operation group was sutured only after exposure to the sciatic nerve. The weight, the change of toe extension angle, and the sciatic functional index (SFI) of rats were observed at the 1st day before operation and the 1st, 3rd, 7th, and 10th days after surgery. The expressions of neuron-specific nuclear protein (NeuN) and microtubule-associate protein 2 (MAP2) were detected by immunofluorescent staining of L4-L6 spinal cord anterior horn on the 10th day after surgery. Results No significant changes were found in the weight of rats among four groups. Compared to the sham, the motor function of the injured limb in Res, DMSO, and NS rats was impaired, and the anterior horn neurons were seriously damaged. But the differences of the change of toe extension and the sciatic functional index of rats were significantly higher in Res group than that of the DMSO group and NS group (P<0.01) at the 3rd, 7th, and 10th days after surgery. The expressions of NeuN and MAP2 in the anterior horn of rat lumbar spinal cord were up-regulated in Res group relative to DMSO and NS, and the number of neurons in the lumbar spinal cord was significantly relieved at the 10th days. Conclusions Res was significant to rat model of acute sciatic nerve injury, which could increase the number of neurons in the anterior horn of the spinal cord and help the recovery of motor function. Key words: Veratrum alkaloids/PD; Sciatic nerve/IN; Motor activity/DE; Anterior horn cells/DE

Astrocytes and Microglia-Mediated Immune Response in Maladaptive Plasticity is Differently Modulated by NGF in the Ventral Horn of the Spinal Cord Following Peripheral Nerve Injury.

Reactive astrocytes and activated microglia are the key players in several pathophysiologic modifications of the central nervous system. We used the spared nerve injury (SNI) of the sciatic nerve to induce glial maladaptive response in the ventral horn of lumbar spinal cord and examine its role in the remodeling of the tripartite synapse plasticity. Imaging the ventral horn revealed that SNI was associated with both an early microglial and astrocytic activation, assessed, respectively, by analysis of Iba1 and GFAP expression. Microglia, in particular, localized peculiarly surrounding the motor neurons somata. Perineuronal astrocytes, which play a key role in maintaining the homeostasis of neuronal circuitry, underwent a substantial phenotypic change following peripheral axotomy, producing reactive gliosis. The gliosis was associated with the reduction of glial aminoacid transporters (GLT1 and GlyT1) and increase of neuronal glutamate transporter EAAC1. Although the expression of GABAergic neuronal marker GAD65/67 showed no change, glutamate increase, as demonstrated by HPLC analysis, shifted the excitatory/inhibitory balance as showed by the net increase of the glutamate/GABA ratio. Moreover, endogenous NGF levels were altered in SNI animals and not restored by the intrathecal NGF administration. This treatment reverted phenotypic changes associated with reactive astrocytosis, but failed to modify microglia activation. These findings on one hand confirm the correlation between gliopathy and maladaptive plasticity of the spinal synaptic circuitry, on the other hand add new data concerning the complex peculiar behavior of different glial cells in neuronal degenerative processes, defining a special role of microglia in sustaining the inflammatory response.

Pannexin 1: A novel participant in neuropathic pain signaling in the rat spinal cord

Pannexin 1 (panx1) is a large-pore membrane channel expressed in many tissues of mammals, including neurons and glial cells. Panx1 channels are highly permeable to calcium and adenosine triphosphatase (ATP); on the other hand, they can be opened by ATP and glutamate, two crucial molecules for acute and chronic pain signaling in the spinal cord dorsal horn, thus suggesting that panx1 could be a key component for the generation of central sensitization during persistent pain. In this study, we examined the effect of three panx1 blockers, namely, 10panx peptide, carbenoxolone, and probenecid, on C-reflex wind-up activity and mechanical nociceptive behavior in a spared nerve injury neuropathic rat model involving sural nerve transection. In addition, the expression of panx1 protein in the dorsal horn of the ipsilateral lumbar spinal cord was measured in sural nerve–transected and sham-operated control rats. Sural nerve transection resulted in a lower threshold for C-reflex activation by electric stimulation of the injured hindpaw, together with persistent mechanical hypersensitivity to pressure stimuli applied to the paw. Intrathecal administration of the panx1 blockers significantly depressed the spinal C-reflex wind-up activity in both neuropathic and sham control rats, and decreased mechanical hyperalgesia in neuropathic rats without affecting the nociceptive threshold in sham animals. Western blotting showed that panx1 was similarly expressed in the dorsal horn of lumbar spinal cord from neuropathic and sham rats. The present results constitute the first evidence that panx1 channels play a significant role in the mechanisms underlying central sensitization in neuropathic pain.

A novel acylaminoimidazole derivative, WN1316, alleviates disease progression via suppression of glial inflammation in ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron degenerative disease. Given that oxidative stress and resulting chronic neuronal inflammation are thought to be central pathogenic, anti-oxidative agents and modulators of neuronal inflammation could be potential therapies for ALS. We report here that the novel small molecular compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316) selectively suppresses oxidative stress-induced cell death and neuronal inflammation in the late-stage ALS mice. WN1316 has high blood-brain-barrier permeability and water solubility, and boosts both neuronal apoptosis inhibitory protein (NAIP) and NF-E2-related factor 2 (Nrf2) which governed glutathione (GSH)-related anti-oxidation pathway protecting motor neurons against oxidative injuries. Post-onset oral administration of low dose (1–100 µg/kg/day) WN1316 in ALS(SOD1H46R) and ALS(SOD1G93A) mice resulted in sustained improved motor function and post onset survival rate. Immunohistochemical analysis revealed less DNA oxidative damage and motor neuronal inflammation as well as repression of both microgliosis and astrocytosis, concomitant down regulation of interleukin-1β and inducible nitric oxide synthase, and preservation of the motoneurons in anterior horn of lumbar spinal cord and skeletal muscle (quadriceps femoris). Thus, WN1316 would be a novel therapeutic agent for ALS.

Elevated Interleukin-8 Enhances Prefrontal Synaptic Transmission in Mice with Persistent Inflammatory Pain

BackgroundInterleukin-8 (IL-8) is known for its roles in inflammation and plays critical roles in the development of pain. Its expression increases in the brain after peripheral inflammation. Prefrontal cortex, including the anterior cingulate cortex (ACC), is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission, however, little is known about the expression of IL-8 and its role in the enhanced ACC synaptic transmission in animals with persistent inflammatory pain.FindingsIn the present study, we examined IL-8 expression in the ACC, somatosensory cortex (SSC), and the dorsal horn of lumbar spinal cord following hind-paw administration of complete Freund's adjuvant (CFA) in mice and its effects on the ACC synaptic transmission. Quantification of IL-8 at protein level (by ELISA) revealed enhanced expression in the ACC and spinal cord during the chronic phases of CFA-induced peripheral inflammation. In vitro whole-cell patch-clamp recordings revealed that IL-8 significantly enhanced synaptic transmission through increased probability of neurotransmitter release in the ACC slice. ACC local infusion of repertaxin, a non-competitive allosteric blocker of IL-8 receptors, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in mice.ConclusionsOur findings suggest that up-regulation of IL-8 in the ACC partly attributable to the enhanced prefrontal synaptic transmission in the mice with persistent inflammatory pain.

The effect of olfactory ensheathing cell transplantation combined with walking training on neurofunction recovery in rats after spinal cord contusion

Objective To explore the effect of olfactory ensheathing cell （OEC） transplantation combined with walking training on neurofunction recovery in rats after spinal cord contusion. Methods Forty adult female rats aged （75 ± 1 ） days were subjected to experimental spinal cord contusion at the T10 level using a New York University impactor at a height of 25 mm. They were then divided into 4 groups： （ 1 ） an OEC transplantation combined with walking training （OEC-walking training） group, （2） an OEC transplantation （OEC） group, （3） a walking training combined with Dulbecco＇s modified Eagle medium injection （DMEM） （walking training-DMEM） group, and （4） aDMEM injection （SCI-DMEM） group. The OEC transplants and DMEM injections were performed 2 weeks post-injury. Walking training began at the 7th day post-injury and consisted of daily sessions （once daily, 5 days a week for 10 weeks） of quadrupedal treadmill training, starting from 15 min and gradually increasing to 30 min daily, at speeds starting from 3 m/min and gradually increasing in accordance to the condition of the rats. Locomotor function recovery of the rats＇ hindlimbs was evaluated weekly using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale.The expression of tyrosine hydroxylase （ TH ） was detected in the injured region of the lumbar spinal cord. Results The BBB scores of rats in the OEC-walking training group and the walking training-DMEM group improved significantly from the 4th week post-injury compared to the SCI-DMEM injection group. Rats in the OEC transplantation group had a significant improvement in BBB scores at the 5th to 8th weeks post-injury. At the end of the 11th week post-injury, the average BBB scores were 13.14 ± 0.24 in the OEC-walking training group, 11. 64 ± 0.56 in the OEC transplantation group, 12.29 ±0.64 in the walking training-DMEM group and 11.07 ± 0.84 in the SCI-DMEM group.The OEC-walking training group scored significantly higher than the other 3 groups. Although the number of TH-positive neurons in the lumbar spinal cord was not significantly different among the groups, the morphology of TH-positiveneurons in the OEC-walking training group and the walking training-DMEM group was different from those in the OEC transplantation group and the SCI-DMEM group. Conclusions OEC transplantation combined with walking training can effectively promote the functional recovery of the hindlimb. The plasticity of the descending TH system and of motoneurons of the ventral horn of the lumbar spinal cord might mediate the changes. Key words: Spinal cord contusion; Olfactory ensheathing cells transplantation; Walking training; Neurofunction; Tyrosine hydroxylase

Changes of mitochondrial ultrastructures and function in central nervous tissue of hens treated with tri-ortho-cresyl phosphate (TOCP)

Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, is capable of producing organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animals. The mechanism of OPIDN has not been fully understood. The present study has been designed to evaluate the role of mitochondrial dysfunctions in the development of OPIDN. Adult hens were treated with 750 mg/kg·bw TOCP by gavage and control hens were given an equivalent volume of corn oil. On day 1, 5, 15, 21 post-dosing, respectively, hens were anesthetized by intraperitoneal injection of sodium pentobarbital and perfused with 4% paraformaldehyde. The cerebral cortex cinerea and the ventral horn of lumbar spinal cord were dissected for electron microscopy. Another batch of hens were randomly divided into three experimental groups and control group. Hens in experimental groups were, respectively, given 185, 375, 750 mg/kg·bw TOCP orally and control group received solvent. After 1, 5, 15, 21 days of administration, they were sacrificed and the cerebrum and spinal cord dissected for the determination of the mitochondrial permeability transition (MPT), membrane potential (Δψ(m)) and the activity of succinate dehydrogenase. Structural changes of mitochondria were observed in hens' nervous tissues, including vacuolation and fission, which increased with time post-dosing. MPT was increased in both the cerebrum and spinal cord, with the most noticeable increase in the spinal cord. Δψ(m) was decreased in both the cerebrum and spinal cord, although there was no significant difference in the three treated groups and control group. The activity of mitochondrial succinate dehydrogenase assayed by methyl thiazolyl tetrazolium (MTT) reduction also confirmed mitochondrial dysfunctions following development of OPIDN. The results suggested mitochondrial dysfunction might partly account for the development of OPIDN induced by TOCP.

The lumbar spinal cord glial cells actively modulate subcutaneous formalin induced hyperalgesia in the rat

We investigated the response and relationship of glial cells and neurons in lumbar spinal cord to hyperalgesia induced by the unilateral subcutaneous formalin injection into the hindpaw of rats. It was demonstrated that Fos/NeuN immunoreactive (-IR) neurons, glial fibrillary acidic protein (GFAP)-IR astrocytes and OX42-IR microglia were distributed in dorsal horn of lumbar spinal cord, predominantly in the superficial layer. In the time-course studies, GFAP-IR astrocytes were firstly detected, OX42-IR microglia were sequentially observed, Fos/NeuN-IR neurons were found slightly late. Immunoelectron microscopy studies established that many heterotypic gap junctions (HGJs), which consisting of Cx43-IR astrocytic process on one side and Cx32-IR dendrite on the other side, were present in superficial layer of dorsal horn. Ninety-one HGJs were found in 100 areas of experimental rats and occupied 91%, while only 39% HGJs were found in control rats. In experimental rats pretreated with intrathecal (i.t.) application of the carbenoxolone (a gap junction blocker) or fluorocitrate (a glial metabolic inhibitor), the paw withdrawal thermal latency was prolonged than those application of the sterile saline (i.t.). It suggests that spinal cord glial cells may play an important role for modulation of hyperalgesia induced by noxious stimuli through HGJs which located between astrocytes and neurons.

Spinal cord neuronal apoptosis induced by triorthocresyl phosphate poisoning in hens

To investigate the role of neuronal apoptosis in organophosphorus poisoning-induced delayed neuropathy (OPIDN) and its dynamic pathological changes. To establish OPIDN animal model, triorthocresyl phosphate (TOCP)was given to hens with a single dose (1 000 mg/kg, im). Changes of neuropathology, number of neurons and apoptotic cells in the third lumbar spinal cord were observed by HE, Nissl and TUNEL methods 3, 5, 7, 10, 14, 18 days after injection. The hens showed OPIDN typical signs (progressive ataxia and hypotonia) about 9 days after TOCP exposure. HE staining revealed dark red nucleus in neurons of anterior horn of lumbar spinal cord 5 days after exposure, but this phenomenon disappeared 18 days later. Nissl method showed that the number of neurons in anterior horn of spinal cord decreased [from (82 +/- 4) cell/mm(2) to (66 +/- 6) cell/mm(2)]. TUNEL positive cells began to appear [(22 +/- 2) cell/mm(2)] 5 days after TOCP exposure, and reached the peak [(27 +/- 3) cell/mm(2)] 7 days later, and disappeared 18 days later. Neuronal apoptosis in anterior horn of spinal cord of hens appeared in OPIDN, suggesting that cellular apoptosis may play an important role in the pathogenesis of OPIDN.

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels.

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

Peripheral Nerve Injury and CGRP Receptor Antagonist Modulate RCP-1 Expression in Primary Sensory Neurons and in Neurons of the Spinal Cord

Calcitonin gene-related peptide (CGRP) receptors appear to be rather unique among G-protein receptor superfamily since they may require accessory proteins to be functional. For example, a CGRP receptor clone behaves as a CGRP1 receptor subtype in presence of the RAMP-1 accessory protein but as an adrenomedullin receptor when coexpressed with RAMP-2. Another putative protein bearing no sequence homology to the RAMPs has been cloned and named CGRP receptor component or RCP. This protein is involved in the signal transduction pathway of the CGRP receptors. Previously, using highly specific RCP-1 antibody, we immunocytochemically demonstrated the localization of RCP-1 immunoreactivity in the brain and peripheral nervous system of the rat. The aim of this study was to determine if RCP-1 expression in primary sensory neurons and spinal motoneurons could be modulated by partial sciatic nerve injury, a model for neuropathic pain, or by intrathecal administration of CGRP8-37, a CGRP receptor antagonist. We found that partial sciatic nerve injury reduced RCP-1 expression in neurons of both dorsal and ventral horns of the lumbar spinal cord. Interestingly, following intrathecal daily injection of CGRP8-37 (4 µg/day for 7 days), RCP-1 immunoreactive axons and neurons were significantly increased in both dorsal and ventral horns of the rat lumbar spinal cord. These data suggest that RCP-1 expression in the primary sensory neurons and spinal motoneurons is subject to modulation by peripheral nerve injury as well as CGRP receptor activation. It could be used as a reliable marker to represent CGRP receptor. Supported by grant from CIHR.

Transsynaptic changes of neurons and associated microglial reaction in the spinal cord of rats following middle cerebral artery occlusion

This study investigated transsynaptic neuronal damage and microglial reaction in the spinal cord contralateral to focal cerebral ischaemia in rats induced by permanent occlusion of the right middle cerebral artery (MCA). Three and five days after MCA occlusion, some neurons in the dorsal horn of lumbar spinal cord underwent degeneration and they appeared to be engulfed by reactive microglia; on the other hand, the ventral horn neurons remained ultrastructurally intact. It is suggested that the neuronal degeneration in the dorsal horn was attributed to deafferentation of the local neurons following ischaemic lesion of the corticospinal neurons which are the main source of afferent inputs.

Brainstem neurons with projecting axons to both phrenic and abdominal motor nuclei: A double fluorescent labeling study in the cat

The distribution of retrogradely doubly labeled brainstem neurons were analyzed in the cat after injection of two different fluorescent markers into the phrenic and abdominal motor nuclei. Diamidino Yellow (DY) was first injected either ipsilaterally or bilaterally into the ventral horn of lumbar spinal cord, and then Fast Blue (FB) into the right ventral horn of cervical spinal cord. Doubly labeled neurons were mainly found in the caudal ventrolateral medulla (retroambiguus region), in the dorsomedial and dorsolateral regions of the nucleus of the tractus solitarius (NTS) and in the raphe nuclei. In addition, doubly labeled neurons were found in the parabrachial and Kölliker-Fuse nuclei. Our results give anatomical evidence that pontine and medullary neurons are the source of a common pathway to both phrenic and abdominal motor nuclei. These neurons might be involved in strain efforts for expulsion such as vomiting or defecation.

Differential distribution of (Na,K)-ATPase α isoform mRNAs in the peripheral nervous system

mRNA transcripts for 3 isoforms of the α subunit of (Na,K)-ATPase have been peviously identified in the nervous system (designated α1, α2 and α3). In order to study the localization and expression of the different α isoforms in the peripheral nervous system, we prepared probes from the unique 3′ untranslated region of α1 cDNA, and from the translated region of α3 cDNA. These probes were used in dot blot and in situ hybridization assays of rat spinal cord, dorsal root ganglia (DRG), and sciatic nerve. Within the ventral horn of lumbar spinal cord, α1 mRNA was found in a discrete set of laterally placed motor neurons, while α3 was found in all the identified neurons of the spinal cord, including those motor neurons containing α1. In the lumbar DRG, α3 was uniformly distributed in DRG neurons, while α1 was abundant in some neurons but little or none was found in other neurons. Satellite cells contained neither isoform. Schwann cells in sciatic nerve were labeled with the α1 probe in a perinuclear distribution, but contained no detectable α3. Dot blot analysis showed α1 and α3 in spinal cord and DRG, but only α1 in peripheral nerve. These results imply that: (1) the riboprobes are able to distinguish α1 from α3 isoform mRNAs under in situ hybridization conditions, (2) both α1 and α3 isoforms of (Na,K)-ATPase are found in neurons, (3) some motor and sensory neurons contain abundant α3 with little or no detectable α1, while others contain abundant α1 and α3, (4) no neurons in the lumbar spinal cord or DRG contain detectable α1 in the absence of α3, and (5) Schwann cells contain the α1 isoform but no detectable α3 isoform mRNA.