Spinal Cord Sections Research Articles

Tail spasms in rat spinal cord injury: Changes in interneuronal connectivity

Uncontrolled muscle spasms often develop after spinal cord injury. Structural and functional maladaptive changes in spinal neuronal circuits below the lesion site were postulated as an underlying mechanism but remain to be demonstrated in detail. To further explore the background of such secondary phenomena, rats received a complete sacral spinal cord transection at S2 spinal level. Animals progressively developed signs of tail spasms starting 1week after injury. Immunohistochemistry was performed on S3/4 spinal cord sections from intact rats and animals were sacrificed 1, 4 and 12weeks after injury.We found a progressive decrease of cholinergic input onto motoneuron somata starting 1week post-lesion succeeded by shrinkage of the cholinergic interneuron cell bodies located around the central canal. The number of inhibitory GABAergic boutons in close contact with Ia afferent fibers was greatly reduced at 1week after injury, potentially leading to a loss of inhibitory control of the Ia stretch reflex pathways. In addition, a gradual loss and shrinkage of GAD65 positive GABAergic cell bodies was detected in the medial portion of the spinal cord gray matter. These results show that major structural changes occur in the connectivity of the sacral spinal cord interneuronal circuits below the level of transection. They may contribute in an important way to the development of spastic symptoms after spinal cord injury, while reduced cholinergic input on motoneurons is assumed to result in the rapid exhaustion of the central drive required for the performance of locomotor movements in animals and humans.

Transient receptor potential vanilloid 1 mediates nerve growth factor‐induced bladder hyperactivity and noxious input

To explore the role of transient receptor potential vanilloid 1 (TRPV1) in the excitatory effects of chronic administration of nerve growth factor (NGF) on bladder-generated sensory input and reflex activity. To explore new therapeutic targets for bladder dysfunction. Wild-type (WT) and TRPV1 knockout (KO) mice received daily intraperitoneal injections of NGF (1 µg/10 g) or saline for a period of 4 days, during which time thermal sensitivity was evaluated daily. On the 5th day, mice were anaesthetized and cystometries were performed. The frequency, amplitude and area under the curve (AUC) of bladder reflex contractions were determined. c-Fos expression was evaluated on L6 spinal cord sections of WT and TRPV1 KO mice treated with saline or chronic NGF by immunohistochemistry. TrkA receptor staining intensity was determined in L6 spinal cord sections and respective dorsal root ganglia of WT and TRPV1 KO mice. Repeated administration of NGF induced thermal hypersensitivity in WT but not in TRPV1 KO mice. The frequency of bladder contractions of saline-treated WT and TRPV1 KO mice was similar, the values respectively being 0.45 ± 0.12/min and 0.46 ± 0.16/min. Treatment with NGF enhanced bladder reflex activity in WT mice to 1.23 ± 0.41/min (P < 0.05). In NGF-treated KO mice, the frequency of bladder contractions was 0.60 ± 0.05/min. Irrespective of treatment, no differences were observed in the amplitude of bladder contractions of WT and TRPV1 KO mice. The AUC was significantly increased in NGF-treated WT-mice, when compared with saline-treated WT-mice. No changes were found in AUC of saline-treated and NGF-treated TRPV1 KO mice. Chronic administration of NGF resulted in a significant increase of spinal c-Fos expression in WT mice (P < 0.05 vs KO animals), but not in TRPV1 KO animals. TrkA expression was similar in WT and TRPV1 KO mice. NGF-induced bladder overactivity and noxious input depend on the interaction of NGF with TRPV1. The lack of bladder overactivity in TRPV1 KO mice treated with NGF does not represent loss of TrkA expression. TRPV1 is essential for NGF-driven bladder dysfunction and represents a bottleneck target in bladder pathologies associated with NGF up-regulation.

Mitotic spindle orientation can direct cell fate and bias Notch activity in chick neural tube.

Inheritance of apical membrane is proposed to maintain vertebrate neural stem cell proliferation. However, evidence for this is contradictory. Using direct clonal analysis and live imaging in chick neural tube, we show that divisions that separate apical and basal components generate an apical daughter, which becomes a neuron, and a basal daughter, which rapidly re-establishes apico-basal polarity and divides again. Using a recently described real-time reporter of Notch activity, we confirm progenitor status and demonstrate that division orientation can influence Notch signalling. In addition, we reveal loss of apical complex proteins on neuronal differentiation onset, suggesting that removal of this inherited complex is part of the neuronal differentiation mechanism. These findings reconcile contradictory data, link asymmetric division to Notch signalling dynamics and identify apical complex loss as a new step towards neuronal differentiation.

Application of intraoperative spinal ultrasonography in thoracic spinal decompressive operations

To investigate the feasibility and clinical value of intraoperative ultrasonography (IOUS) in thoracic spinal decompression surgery. Ten patients with confirmed thoracic spinal stenosis underwent thoracic spinal decompression in our center from August 2009 to December 2010. The appearance of the compressed section of spinal cord was observed with IOUS. Before and after the decompression operation, the diameters of dural sac and the spinal cord were recorded respectively. The location and nature of the compression-causing mass were confirmed. IOUS clearly showed the shape of the normal and the compressed sections of dural sac and spinal cord. In the 14 thoracic spinal cord sections of these 10 patients, the anteroposterior diameter, horizontal diameter, and their ratio were bigger than those before decompression. The values of anteroposterior diameter and anteroposterior/horizontal diameter ratio showed significant differences(the P value of dural sac anteroposterior diameter comparison was 0.008, which of spinal cord was 0.007; the P values of these two structures ratio comparison were both 0.002 before and after decompression), while the horizontal diameter presented no significant differences (the P values of both structures were 0.270 and 0.195 respectively before and after decompression). IOUS can clearly show the morphological changes of the dural sac and spinal cord before and after the decompression. In addition, it helps surgeons to locate and specify the nature of the compression-causing mass on the ventral side of dural sac. Furthermore, IOUS can suggest whether the decompression is sufficient in a real-time manner.

Effects of Spinal Cord Section on Inspiratory Pressure Generation During High Frequency Spinal Cord Stimulation (HF‐SCS)

ObjectiveHF‐SCS is a novel and more physiologic method of inspiratory muscle activation which involves activation of spinal cord pathways. The specific pathways by which inspiratory motoneurons are activated by this method however are unknown. The aim of the present study was to evaluate the potential role of upper cervical inspiratory neurons on the observed responses.MethodsIn 6 anesthetized and spinalized (C1 level) dogs, HF‐SCS was applied at T2 level. Airway pressure generation (P) was monitored as an index of the degree of inspiratory muscle activation before and following sequential spinal sections at the C4 (to eliminate upper cervical propriospinal neurons) and subsequently the C8 level.ResultsDuring HF‐SCS, P at FRC was 68±6 cmH2O. Sequential bilateral dorsal columns (DC), lateral funiculi (LF) and complete section (CS) at C4, resulted in no significant changes in P (69±5, 67±5 and 65±7 cmH2O respectively, NS). While DC section at C8 resulted in no changes in P (63±5 cmH2O), LF section resulted in significant reduction in P to 18±3 cmH2O (p&lt;0.05). Subsequent CS resulted in no further reduction in P (14±1 cmH2O, NS).ConclusionDuring upper thoracic HF‐SCS, activation of the phrenic motoneuron pools does not involve upper cervical propriospinal inputs. The phrenic motoneuron pools are activated via spinal pathways located bilaterally in the lateral funiculi.Support: NIH‐NINDS (R01NS064157).

Intrathecal Clonidine in the Neonatal Rat

Neuraxial clonidine is used for perioperative analgesia in children of all ages. Preclinical studies in the postnatal rat allow comparison of the relative toxicity and safety of spinal analgesics throughout postnatal development. Rat pups aged 3, 7, or 21 postnatal (P) days were briefly anesthetized for intrathecal injections of saline or clonidine. At each age, the maximum tolerated, antinociceptive (increased hindlimb mechanical withdrawal threshold) and antihyperalgesic (hindpaw carrageenan inflammation) doses were determined. Lumbar spinal cord sections were assessed for apoptosis and cell death (histology, activated caspase-3 immunohistochemistry, Fluoro-Jade C staining), histopathology (hematoxylin and eosin staining), and increased glial reactivity (microglial and astrocytic markers). P3 intrathecal ketamine sections served as positive controls. In additional groups, thermal latency and mechanical withdrawal threshold were measured at P35. Intrathecal clonidine produces age- and dose-dependent analgesia in rat pups. Maximal doses of clonidine did not alter the degree or distribution of apoptosis or increase glial reactivity in the neonatal spinal cord. No spinal histopathology was seen 1 or 7 days after injection at any age. Intrathecal clonidine did not produce persistent changes in reflex sensitivity to mechanical or thermal stimuli at P35. Intrathecal clonidine in the postnatal rat did not produce signs of spinal cord toxicity, even at doses much larger than required for analgesia. The therapeutic ratio (maximum tolerated dose/antihyperalgesic dose) was >300 at P3, >30 at P7, and >10 at P21. These data provide additional information to inform the clinical choice of spinal analgesic drug in early life.

Effect of free NgR-modified bone marrow stromal cell transplantation on axon regeneration after spinal cord injury

Objective To evaluate the effect of free NgR-modified bone marrow stromal cell (BMSC) transplantation on axon regeneration in rats after spinal cord injury. Methods Genes encoding free NgR protein were cloned and transduced into BMSCs at passage 3 using a lentivirus vector.Indirect immunofluorescence was used to detect the expression of free NgR protein.Meanwhile a spinal cord contusion model was established in 36 adult Sprague-Dawley rats at the T10 segment.The rats were then divided randomly into an experimental group and a control group.NgR + BMSCs were transplanted into the injured site 1 week post-trauma in the experimental group.BMSCs were also transplanted at the same time into the control group.Expression of free NgR at the injury site was detected by immunohistochemical staining at 1 week post-transplantation.The functional recovery of both groups was evaluated at 4 and 6 weeks post-transplantation.Longitudinal sections of the spinal cord were studied for axon regeneration using horseradish peroxidase staining. Results Expression of free NgR was found in the cell plasma of BMSCs by indirect immunofluorescence post-transfection.Positive immunohistochemical staining for NgR was found at the transplant site in the experimental group 1 week post-transplantation.Better axon plasticity could be observed in the experimental group.The Basso-Beattie-Bresnahan scoring of the experimental group was significantly higher than that of the controls at both observation times. Conclusions Free NgR-modified BMSCs can prompt injured axons to regenerate and thus to promote the recovery of neurological function.This might provide a new strategy to treat spinal cord injury. Key words: NgR protein; Bone marrow stromal cells; Axon regeneration; Spinal cord injury; Rehabilitation

Efficient Down-Regulation of Glia Maturation Factor Expression in Mouse Brain and Spinal Cord

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.

Confirmed case of encephalitis caused by Murray Valley encephalitis virus infection in a horse

A 5-year-old Australian stock horse in Monto, Queensland, Australia, developed neurological signs and was euthanized after a 6-day course of illness. Histological examination of the brain and spinal cord revealed moderate to severe subacute, nonsuppurative encephalomyelitis. Sections of spinal cord stained positively in immunohistochemistry with a flavivirus-specific monoclonal antibody. Reverse transcription polymerase chain reaction assay targeting the envelope gene of flavivirus yielded positive results from brain, spinal cord, cerebrospinal fluid, and facial nerve. A flavivirus was isolated from the cerebrum and spinal cord. Nucleotide sequences obtained from amplicons from both tissues and virus isolated in cell culture were compared with those in GenBank and had 96-98% identity with Murray Valley encephalitis virus. The partial envelope gene sequence of the viral isolate clustered into genotype 1 and was most closely related to a previous Queensland isolate.

Olesoxime delays muscle denervation, astrogliosis, microglial activation and motoneuron death in an ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The pathology is mimicked to a striking degree in transgenic mice carrying familial ALS-linked SOD1 gene mutations. Olesoxime (TRO19622), a novel neuroprotective and reparative compound identified in a high-throughput screen based on motoneuron (MN) survival, delays disease onset and improves survival in mutant SOD1G93A mice, a model for ALS. The present study further analyses the cellular basis for the protection provided by olesoxime at the neuromuscular junctions (NMJ) and the spinal cord. Studies were carried out at two disease stages, 60 days, presymptomatic and 104 days, symptomatic. Cohorts of wild type and SOD1G93A mice were randomized to receive olesoxime-charged food pellets or normal diet from day 21 onward. Analysis showed that olesoxime initially reduced denervation from 60 to 30% compared to SOD1G93A mice fed with control food pellets while at the symptomatic stage only a few NMJs were still preserved. Immunostaining of cryostat sections of the lumbar spinal cord with VAChT to visualize MNs, GFAP for astrocytes and Iba1 for microglial cells showed that olesoxime strongly reduced astrogliosis and microglial activation and prevented MN loss. These studies suggest that olesoxime exerts its protective effect on multiple cell types implicated in the disease process in SOD1G93A mice, slowing down muscle denervation, astrogliosis, microglial activation and MN death. A Phase 3 clinical study in ALS patients will determine whether olesoxime could be beneficial for the treatment of ALS.

AnIn VivoCanine Study to Assess Granulomatous Responses in the MedStream Programmable Infusion System™ and the SynchroMed II Infusion System®

A rare, but consistently reported complication of intrathecal (IT) delivery of opioids via programmable implantable infusion pumps is aseptic granuloma formation around the tip of the IT catheter. In the current study, the incidence and severity of IT granuloma formation was assessed for the MedStream Programmable Infusion and SynchroMed II Infusion pumps when delivering saline, baclofen, or morphine at varying concentrations and daily doses. Randomized study with six groups: 1) MedStream with 0.9% saline; 2) SynchroMed II with 0.9% saline; 3) MedStream with morphine sulfate; 4) SynchroMed II with morphine sulfate; 5) MedStream with baclofen; or 6) SynchroMed II with baclofen. The groups receiving morphine received either low or high concentrations (6 or 12.5 mg/mL) delivered in rates resulting in low to high (1.25-12 mg/day) daily doses. Animals receiving baclofen began at low dose/low concentration and underwent stepwise increases in concentration and dose to a maximum of 2 mg/mL/day. Animals receiving saline received a constant flow rate through either a silicone or a polyurethane IT catheter. The study was set as in vivo laboratory experiment. 52 (21-32 kg) canines. Delivering IT saline, baclofen, or morphine through SynchroMed II and MedStream Programmable Infusion pumps. The incidence and severity of IT catheter tip granuloma formation was assessed with paraffin-embedded spinal cord sections by a pathologist without knowledge of treatment and compared between drug type, drug concentration, daily dose, and pump type. Granuloma formation occurred only in animals receiving morphine, with zero incidence in baclofen or saline infusion, regardless of catheter type. Granuloma incidence increased with increasing morphine concentration or daily dose (one [12.5%] low dose/low concentration, 3 [37.5%] low dose/high concentrations, 4 [50%] high dose/low concentration, and 7 [100%] high dose/high concentration). A granulomatous response to morphine was observed in 10 (63%) animals with SynchroMed II pumps and 5 (33%) animals with MedStream pumps, P = 0.156. In this in vivo canine model of IT drug delivery, catheter tip granuloma formation was associated with higher concentrations and daily doses of morphine infusion while none were seen with baclofen or saline, and was not associated with catheter type. For both pumps, granulomas were only produced in the presence of morphine infusion. These results suggest that the MedStream Programmable Infusion System has a granuloma safety profile at least equivalent to that of the SynchroMed II pump.

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Disk Injury in Rats Produces Persistent Increases in Pain-Related Neuropeptides in Dorsal Root Ganglia and Spinal Cord Glia but Only Transient Increases in Inflammatory Mediators

Immunohistological analysis in an injured intervertebral disk (IVD) model. To elucidate and compare in rats the behavior of the sensory nervous system and inflammatory mediators in experimentally injured IVDs. Multiple human and animal studies have verified the presence of sensory nerve fibers in IVDs or investigated the behavior of inflammatory mediators in injured IVDs, but no in vivo study to date has examined the relationship between the 2. Eight-week-old female rats were used. In the disk-injured group, L5/L6 disks were injured with a 24-gauge needle; simultaneously, the neurotracer Fluoro-gold was injected into the L5/L6 IVD. The L5/L6 IVD dorsal root ganglia (DRGs) from the L1 to L6 levels, and the spinal cord was resected at several time points after surgery. Nerve growth factor, tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in the IVDs were quantified using enzyme-linked immunosorbent assay. DRGs were immunostained for calcitonin gene-related peptide, and spinal cord sections were immunostained for ionized calcium-binding adaptor molecule-1 and glial fibrillary acidic protein. Nerve growth factor, and TNF-α levels (through 1 week) and IL-6 levels (through 4 days) were significantly higher in the disk-injured group than in the noninjured group (P < 0.05). However, starting at 2 weeks (nerve growth factor and TNF-α) or 1 week (IL-6), the differences in inflammatory mediator levels between the 2 groups no longer were significant. In contrast, the percentage of calcitonin gene-related peptide-immunoreactive neurons among Fluoro-gold-labeled DRG neurons, and the numbers of ionized calcium-binding adaptor molecule-1-immunoreactive microglia and glial fibrillary acidic protein-immunoreactive astrocytes in the spinal dorsal horn remained significantly higher in the injured group than in the noninjured group at all-time points (P < 0.05). Disk injury in rats produces persistent increases in neuropeptides in DRGs and glia in the spinal cord, but only transient increases in inflammatory mediators in IVDs.

Nociceptive stimuli enhance anesthetic-induced neuroapoptosis in the rat developing brain

Anesthetic-induced neurodegeneration in the developing brain has been well documented. However, the experiments carried out so far do not include surgical conditions. This proof of concept study was designed to investigate the impact of nociceptive stimuli on anesthetic induced neuroapoptosis in the rat developing brain. Separate cohorts of 7-day-old Sprague–Dawley rat pups were randomly assigned to six groups: Naïve (room air); Anesthesia alone (70% nitrous oxide and 0.75% isoflurane for 6 h); Formalin injection alone (subcutaneous injection with 10 μL 5% formalin into the left hind paw); Anesthesia + formalin injection; Surgical incision (to the left hind paw) alone; Anesthesia + surgical incision. Apoptosis (Caspase-3) and neuronal activation (c-Fos) in the brain and spinal cord section, and cortical TNF-α and IL-1β were measured with in situ immunostaining and western blot respectively. Cognition was tested using Trace Fear conditioning 40 days after the insult. Prolonged anesthesia caused widespread apoptosis in the central nervous system compared to naïve animals. Nociceptive stimulation with formalin (F) or surgical incision (S) increased the injury in the brain cortex (F: 60% or S: 40% increase) and spinal cord (F: 80% vs S: 50% increase) respectively. Both nociceptive stimuli further augmented cognitive impairment induced by the anesthetics when assessed 40 days later. The activated pain pathway and the increased expression of the pro-inflammatory cytokine, IL-1β, in the cortex may be responsible for the enhanced neuroapoptosis. Nociceptive stimulation and prolonged anesthesia produced significantly more apoptosis than prolonged anesthesia alone when administered to neonates during the synaptogenic period.

Spinal direct current stimulation modulates the activity of gracile nucleus and primary somatosensory cortex in anaesthetized rats

Afferent somatosensory activity from the spinal cord has a profound impact on the activity of the brain. Here we investigated the effects of spinal stimulation using direct current, delivered at the thoracic level, on the spontaneous activity and on the somatosensory evoked potentials of the gracile nucleus, which is the main entry point for hindpaw somatosensory signals reaching the brain from the dorsal columns, and of the primary somatosensory cortex in anaesthetized rats. Anodal spinal direct current stimulation (sDCS) increased the spontaneous activity and decreased the amplitude of evoked responses in the gracile nucleus, whereas cathodal sDCS produced the opposite effects. At the level of the primary somatosensory cortex, the changes in spontaneous activity induced by sDCS were consistent with the effects observed in the gracile nucleus, but the changes in cortical evoked responses were more variable and state dependent. Therefore, sDCS can modulate in a polarity-specific manner the supraspinal activity of the somatosensory system, offering a versatile bottom-up neuromodulation technique that could potentially be useful in a number of clinical applications.

Single, slice-specific z-shim gradient pulses improve T2*-weighted imaging of the spinal cord

T2*-weighted imaging of the spinal cord suffers from signal dropouts that hamper blood-oxygenation-level-dependent functional magnetic resonance imaging (fMRI). They are due to field inhomogeneities caused by the different magnetic susceptibilities of the vertebrae and the intervertebral disks that vary periodically along the cord and, thus, cannot be compensated appropriately with conventional (constant) shimming. In this study, a single, slice-specific gradient pulse ("z-shim") is applied in echo-planar imaging of axial sections in order to compensate for the corresponding through-slice signal dephasing without affecting the acquisition time, i.e. the temporal resolution. Based on a reference acquisition sampling a range of compensation moments, the value yielding the maximum signal amplitude within the spinal cord is determined for each slice. Severe N/2 ghosting for larger compensation moments is avoided by applying the gradient pulse after the corresponding reference echoes. Furthermore, first-order flow compensation in the slice direction of both the slice-selection and the z-shim gradient pulse considerably reduces signal fluctuations in the cerebro-spinal fluid surrounding the spinal cord, i.e. would minimize ringing artifacts in fMRI. Phantom and in vivo experiments show the necessity to use slice-specific compensation moments in the presence of local susceptibility differences. Measurements performed in a group of 24 healthy volunteers at 3T demonstrate that this approach improves T2*-weighted imaging of axial sections of the cervical spinal cord by (i) increasing the signal intensity (overall by about 20%) and (ii) reducing signal intensity variations along the cord (by about 80%). Thus, it may help to improve the feasibility and reliability of fMRI of the spinal cord.

Optimization of Immunosuppressive Therapy for Spinal Grafting of Human Spinal Stem Cells in a Rat Model of ALS

Previous rodent studies employing monotherapy or combined immunosuppressive regimens have demonstrated a variable degree of spinal xenograft survival in several spinal neurodegenerative models including spinal ischemia, trauma, or amyotrophic lateral sclerosis (ALS). Accordingly, the characterization of optimal immunosuppressive protocols for the specific neurodegenerative model is critical to ensure reliable assessment of potential long-term therapeutic effects associated with cell replacement. In the present study we characterized the survival of human spinal stem cells when grafted into the lumbar spinal cords of a rodent model of ALS, SOD1 (G93A) male and female rats (60-67 days old). Four different immunosuppressive protocols were studied: i) FK506 (q12h); ii) FK506 (qd) + mycophenolate (PO; q12h, up to 7 days postop); iii) FK506 (qd) + mycophenolate (IP; q12h, up to 7 days postop); and iv) FK506 (qd) + mycophenolate (IP; qd, up to 7 days postop). Three weeks after cell grafting the number of surviving human cells was then systematically assessed. The highest density of grafted cells was seen in animals treated with FK506 (qd) and mycophenolate (IP; qd; an average 915 ± 95 grafted cells per spinal cord section). The majority of hNUMA-positive cells colocalized with doublecortin (DCX) immunoreactivity. DCX-positive neurons showed extensive axodendritic sprouting toward surrounding host neurons. In addition, migrating grafted cells were identified up to 500 μm from the graft. In animals treated with FK506 (q12h), FK506 (qd) + mycophenolate (PO; q12h) or FK506 (qd) + mycophenolate (IP; q12h), 11.8 ± 3.4%, 61.2 ± 7.8%, and 99.4 ± 8.9% [expressed as percent of the FK506 (qd) and mycophenolate (IP; qd)] cell survival was seen, respectively. In contrast to animals treated with a combination of FK506 + mycophenolate, robust CD4/8 immunoreactivity was identified in the vicinity of the injection tract in animals treated with FK506 only. These data suggest that a combined, systemically delivered immunosuppression regimen including FK506 and mycophenolate can significantly improve survival of human spinal stem cells after intraspinal transplantation in SOD1 (G93A) rats.

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T3-S1 level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.

Potent suppression of stretch reflex activity after systemic or spinal delivery of tizanidine in rats with spinal ischemia-induced chronic spastic paraplegia

Spasticity and rigidity are serious complications associated with spinal traumatic or ischemic injury. Clinical studies show that tizanidine (Tiz) is an effective antispasticity agent; however, the mechanism of this effect is still not clear. Tiz binds not only to α2-adrenoreceptors (AR) but also to imidazoline (I) receptors. Both receptor systems (AR+I) are present in the spinal cord interneurons and α-motoneurons. The aim of the present study was to evaluate the therapeutic potency of systematically or spinally (intrathecally [IT]) delivered Tiz on stretch reflex activity (SRA) in animals with ischemic spasticity, and to delineate supraspinal or spinal sites of Tiz action. Animals were exposed to 10 min of spinal ischemia to induce an increase in SRA. Increase in SRA was identified by simultaneous increase in recorded electromyography (EMG) activity and ankle resistance measured during computer-controlled ankle dorsiflexion (40°/3 s) in fully awake animals. Animals with increased SRA were divided into several experimental subgroups and treated as follows: (i) Tiz administered systemically at the dose of 1 mg kg(-1), or IT at 10 μg or 50 μg delivered as a single dose; (ii) treatment with systemic Tiz was followed by the systemic injection of vehicle, or by nonselective AR antagonist without affinity for I receptors; yohimbine (Yoh), α2A AR antagonist; BRL44408 (BRL), α2B AR antagonist; ARC239 (ARC), nonselective AR and I(1) receptor antagonist; efaroxan (Efa), or nonselective AR and I(2) receptor antagonist; idazoxan (Ida); (iii) treatment with IT Tiz was followed by the IT injection of selective α2A AR antagonist; atipamezole (Ati). In a separate group of spastic animals the effect of systemic Tiz treatment (1 mg/kg) or isoflurane anesthesia on H-reflex activity was also studied. Systemic and/or IT treatment with Tiz significantly suppressed SRA. This Tiz-mediated anti-SRA effect was reversed by BRL (5 mg kg(-1)), Efa (1 mg kg(-1)), and Ida (1 mg kg(-1)). No reversal was seen after Yoh (3 mg kg(-1)) or ARC (5 mg kg(-1)) treatment. Anti-SRA induced by IT Tiz (50 μg) was reversed by IT injection of Ati (50 μg). Significant suppression of H-reflex was measured after systemic Tiz treatment (1 mg/kg) or isoflurane (2%) anesthesia, respectively. Immunofluorescence staining of spinal cord sections taken from animals with spasticity showed upregulation of α2A receptor in activated astrocytes. These data suggest that α2A AR and I receptors, but not α2B AR, primarily mediate the Tiz-induced antispasticity effect. This effect involves spinal and potentially supraspinal sites and likely targets α2A receptor present on spinal neurons, primary afferents, and activated astrocytes. Further studies using highly selective antagonists are needed to elucidate the involvement of specific subtypes of the AR and I receptors in the antispasticity effect seen after Tiz treatment.

Differential Involvement of Optineurin in Amyotrophic Lateral Sclerosis With or Without &lt;emph type="ital"&gt;SOD1&lt;/emph&gt; Mutations

Mutations in optineurin have recently been linked to amyotrophic lateral sclerosis (ALS). To determine whether optineurin-positive skeinlike inclusions are a common pathologic feature in ALS, including SOD1 -linked ALS. Clinical case series. Academic referral center. We analyzed spinal cord sections from 46 clinically and pathologically diagnosed ALS cases and ALS transgenic mouse models overexpressing ALS-linked SOD1 mutations G93A or L126Z. We observed optineurin-immunoreactive skeinlike inclusions in all the sporadic ALS and familial ALS cases without SOD1 mutation, but not in cases with SOD1 mutations or in transgenic mice overexpressing the ALS-linked SOD1 mutations G93A or L126Z. The data from this study provide evidence that optineurin is involved in the pathogenesis of sporadic ALS and non- SOD1 familial ALS, thus supporting the hypothesis that these forms of ALS share a pathway that is distinct from that of SOD1-linked ALS.