Lumbar Spinal Cord Sections Research Articles

Abstract 230: Spinal Cord Injury In A 14 Days Survival Rat Model Of 6 And 8 Min Of Ventricular Fibrillation Cardiac Arrest, Resuscitated With Conventional Chest Compression

Introduction: Impact of global ischemia and reperfusion on brain in resuscitation animal models is well investigated. Investigations on vulnerability of spinal cord in such models are rarely reported. We investigated the spinal cord in a cardiopulmonary resuscitation ventricular fibrillation cardiac arrest (VFCA) rat model and hypothesized that an untreated CA of 6 and 8 min will lead to histological damage and dysfunction in the resuscitated animal′s spinal cord, depending on CA duration. Methods: Adult male Sprague-Dawley rats (350-450 g) were subjected to 6 (n=8) or 8 min (n=14) of VFCA and resuscitated with conventional chest compressions with a thumper device, mechanical ventilation and drugs (epinephrine, bicarbonate, heparin). After defibrillation and ROSC, rats survived for 14 days. The spinal cord was removed from the spine after fixation in formalin and embedded in paraffin. Transverse sections of cervical, thoracic and lumbar spinal cord were stained with hematoxylin and eosin (HE). Immunohistochemistry was performed with antibodies against GFAP for astrocytes and Iba1 for microglia. Sections were evaluated by a pathologist blinded to treatment regimens, and compared to sham animals (n=5). Results: Resuscitated animals showed high mortality; after 6 min CA four animals and after 8 min CA eleven animals died before endpoint. Surviving animals showed focal bilateral symmetrical loss of neurons and distinct gliosis in the gray matter of the thoracic spinal cord level with the central canal. Similar lesions were detected in other segments of the spinal cord, but were located predominantly in the ventral horn of the spinal cord. These lesions were accompanied by dilated myelin sheaths in the white matter. Immunohistochemistry showed activation of astrocytes and microglia in the damaged regions. There were no differences detectable between animals subjected to 6 or 8 min VFCA. Sham animals showed no lesions in the spinal cord. Conclusions: We found consistent histological damage in the spinal cords of rats resuscitated after 6 and 8 min untreated VFCA with CPR after 14 days survival but without clinical symptoms in these animals. However, similar lesions might be cause of paralysis and urinary retention found in animals that died before endpoint.

Altered Spinal Homeostasis and Maladaptive Plasticity in GFAP Null Mice Following Peripheral Nerve Injury

The maladaptive response of the central nervous system (CNS) following nerve injury is primarily linked to the activation of glial cells (reactive gliosis) that produce an inflammatory reaction and a wide cellular morpho-structural and functional/metabolic remodeling. Glial acidic fibrillary protein (GFAP), a major protein constituent of astrocyte intermediate filaments (IFs), is the hallmark of the reactive astrocytes, has pleiotropic functions and is significantly upregulated in the spinal cord after nerve injury. Here, we investigated the specific role of GFAP in glial reaction and maladaptive spinal cord plasticity following sciatic nerve spared nerve injury (SNI) in GFAP KO and wild-type (WT) animals. We evaluated the neuropathic behavior (thermal hyperalgesia, allodynia) and the expression of glial (vimentin, Iba1) and glutamate/GABA system markers (GLAST, GLT1, EAAC1, vGLUT, vGAT, GAD) in lumbar spinal cord sections of KO/WT animals. SNI induced neuropathic behavior in both GFAP KO and WT mice, paralleled by intense microglial reaction (Iba1 expression more pronounced in KO mice), reactive astrocytosis (vimentin increase) and expression remodeling of glial/neuronal glutamate/GABA transporters. In conclusion, it is conceivable that the lack of GFAP could be detrimental to the CNS as it lacks a critical sensor for neuroinflammation and morpho-functional–metabolic rewiring after nerve injury. Understanding the maladaptive morpho-functional changes of glial cells could represent the first step for a new glial-based targeted approach for mechanisms of disease in the CNS.

Chitotriosidase, a biomarker of amyotrophic lateral sclerosis, accentuates neurodegeneration in spinal motor neurons through neuroinflammation

BackgroundCerebrospinal fluid from amyotrophic lateral sclerosis patients (ALS-CSF) induces neurodegenerative changes in motor neurons and gliosis in sporadic ALS models. Search for identification of toxic factor(s) in CSF revealed an enhancement in the level and enzyme activity of chitotriosidase (CHIT-1). Here, we have investigated its upregulation in a large cohort of samples and more importantly its role in ALS pathogenesis in a rat model.MethodsCHIT-1 level in CSF samples from ALS (n = 158), non-ALS (n = 12) and normal (n = 48) subjects were measured using ELISA. Enzyme activity was also assessed (ALS, n = 56; non-ALS, n = 10 and normal-CSF, n = 45). Recombinant CHIT-1 was intrathecally injected into Wistar rat neonates. Lumbar spinal cord sections were stained for Iba1, glial fibrillary acidic protein and choline acetyl transferase to identify microglia, astrocytes and motor neurons respectively after 48 h of injection. Levels of tumour necrosis factor-α and interleukin-6 were measured by ELISA.FindingsCHIT-1 level in ALS-CSF samples was increased by 20-fold and it can distinguish ALS patients with a sensitivity of 87% and specificity of 83.3% at a cut off level of 1405.43 pg/ml. Enzyme activity of CHIT-1 was also 15-fold higher in ALS-CSF and has a sensitivity of 80.4% and specificity of 80% at cut off value of 0.1077989 μmol/μl/min. Combining CHIT-1 level and activity together gave a positive predictive value of 97.78% and negative predictive value of 100%. Administration of CHIT-1 increased microglial numbers and astrogliosis in the ventral horn with a concomitant increase in the levels of pro-inflammatory cytokines. Amoeboid-shaped microglial and astroglial cells were also present around the central canal. CHIT-1 administration also resulted in the reduction of motor neurons.ConclusionsCHIT-1, an early diagnostic biomarker of sporadic ALS, activates glia priming them to attain a toxic phenotype resulting in neuroinflammation leading to motor neuronal death.

Protective Effects of Cerium Oxide Nanoparticles on Oxaliplatin induced Neurotoxicity in Adult Male Albino Rats

Background: Oxaliplatin is a chemotherapeutic agent used for treatment of colorectal cancer. Peripheral neuropathy is a dose limiting toxicity which may persist even after its termination. Oxidative stress may be a direct cause of this neurotoxicity. Cerium oxide nanoparticles (CONPs) are promising antioxidants for applications in medicine as they are well tolerated in biological models. Aim: The present work was designed to study the protective effect of CONPs on neurotoxicity induced by oxaliplatin in adult male albino rats. Materials and methods: 120 rats were divided into 4 groups: Control group, CONPs group (60mg/kg, 5 times/week), Oxaliplatin group (4mg/kg, twice/week) and Oxaliplatin&CONPs group, for 8 weeks. After 4 & 8 weeks, 10 rats from each group were used to measure myelin protein zero (MPZ), malondialdhyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) in the left sciatic nerves. The right sciatic nerves and lumbar spinal cord were used for histopathological study. Results: Oxaliplatin decreased MPZ and induced demyelination and degeneration of sciatic nerve fibers. Also, it increased MDA levels. SOD and GPx activity were increased then decreased. GSH levels were decreased. In the lumbar spinal cord sections, abnormal morphology were seen. CONPs administration increased MPZ and returned normal morphology of sciatic nerve fibers. They decreased MDA levels. SOD was increased after 8 weeks, GPx and GSH not changed. In the lumbar spinal cord sections normal morphology were seen with some hydropic degeneration. Conclusion Oxaliplatin treatment induced hypomyelination in sciatic nerves. CONPs have neuroprotective effect.

Altered transcription of glutamatergic and glycinergic receptors in spinal cord dorsal horn following spinal cord transection is minimally affected by passive exercise of the hindlimbs.

Gene expression is altered following a spinal transection (STx) in both motor and sensory systems. Exercise has been shown to influence gene expression in both systems post-STx. Gene expression alterations have also been shown in the dorsal root ganglia and nociceptive laminae of the spinal cord following either an incomplete spinal cord injury (SCI) or a contusive SCI. However, the effect of STx and exercise on gene expression in spinal cord laminae I-III has not fully been examined. Therefore, the purpose of this study was to determine whether gene expression in laminae I-III is altered following STx and determine whether superimposed passive exercise of the hindlimbs would influence gene expression post-STx in laminae I-III. Laser capture microdissection was used to selectively harvest laminae I-III of lumbar spinal cord sections, and quantitative RT-PCR was used to examine relative expression of 23 selected genes in samples collected from control, STx and STx plus exercise rats. We demonstrate that post-STx, gene expression for metabotropic glutamate receptors 1, 5 and 8 were up-regulated, whereas ionotropic glutamatergic receptor (Glur2) and glycinergic subunit GLRA1 expression was down-regulated. Daily exercise attenuated the down-regulation of Glur2 gene expression in laminae I-III. Our results demonstrate that in a STx model, gene expression is altered in laminae I-III and that although passive exercise influences gene expression in both the motor and sensory systems, it had a minimal effect on gene expression in laminae I-III post-STx.

The Bcl-2 Homology-3 Domain (BH3)-Only Proteins, Bid, DP5/Hrk, and BNip3L, Are Upregulated in Reactive Astrocytes of End-Stage Mutant SOD1 Mouse Spinal Cord.

The molecular mechanisms leading to motor neuron death in amyotrophic lateral sclerosis (ALS) are unknown; however, several studies have provided evidence of a central role for intrinsic apoptosis. Bcl-2 homology-3 domain (BH3)-only proteins are pro-apoptotic members of the Bcl-2 family whose enhanced expression acts as a trigger for the intrinsic apoptotic cascade. Here, we compared the relative expression of BH3-only proteins in the spinal cord of end-stage G93A mutant SOD1 mice to age-matched wild-type (WT) mice. Large alpha motor neurons in lumbar spinal cord sections of both WT and end-stage mutant SOD1 mice stained positively for a number of BH3-only proteins; however, no discernible differences were observed in either the relative intensity of staining or number of BH3-immunoreactive motor neurons between WT and mutant SOD1 mice. On the other hand, we observed significantly enhanced staining for Bid, DP5/Hrk, and BNip3L in GFAP-positive astrocytes only in end-stage G93A mutant SOD1 spinal cord. Staining of additional end-stage G93A mutant SOD1 tissues showed specific upregulation of DP5/Hrk in lumbar spinal cord sections, but not in cerebellum or cortex. Finally, examination of protein expression using western blotting also revealed marked increases in DP5/Hrk and BNip3L in G93A mutant SOD1 lumbar spinal cord lysates compared to WT controls. The upregulation of a specific subset of BH3-only proteins, including Bid, DP5/Hrk, and BNip3L, in reactive astrocytes suggests that these proteins may execute a novel non-apoptotic function within astrocytes to promote ALS disease progression, thus providing a new potential target for therapeutic intervention.

Gain-of-Function Mutation in Ryanodine Receptor Type 1 Modulates Murine Thymocyte Calcium Signaling and Autoimmune Response in Mice

Gain-of-function mutations in gene encoding ryanodine receptor type 1 (RyR1), an endo/sarcoplasmic reticulum Ca2+ release channel, are linked to a variety of skeletal muscular disorders. Other tissues, including cells of the immune system, have been shown to express RyR1. A previous study demonstrated enhanced immune responses in mice with gain-of-function RyR1 mutation (Vukcevic et al., 2013, J Cell Sci, 126: 3485-92). Here we report that after induction of an experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease, the mice carrying gain-of-function RyR1 R163C mutation (R163C mice) displayed an earlier disease onset and augmentation of neurological symptoms compared with wild-type (WT) mice. In vitro studies revealed that splenocytes from the R163C mice have altered Ca2+ signaling compared with those from WT mice, which was revealed by challenging the cells with different concentrations of extracellular Ca2+ and Ca2+ mobilizing agent thapsigargin. No differences were found in the relative abundances of immune cell subtypes in spleens and lymph nodes from R163C and WT mice prior to the onset of the EAE. Nevertheless, immunostaining of the lumbar spinal cord sections after the onset of the clinical symptoms, revealed the larger pro-inflammatory infiltrate and axonal damage in the R163C mice compared with the WT mice, indicating that R163C mutation may affect immune cell trafficking to the CNS. These data indicate that altered Ca2+ handling in R163C mice immune cells may enhance these cells autoimmune responses and that gain-of-function mutations in RYR1 may represent a previously unknown risk factor for autoimmune diseases.

Effects of microRNA-223 on morphine analgesic tolerance by targeting NLRP3 in a rat model of neuropathic pain.

ObjectiveTo investigate the effects of microRNA-223 on morphine analgesic tolerance by targeting NLRP3 in a rat model of neuropathic pain.MethodsOur study selected 100 clean grade healthy Sprague-Dawley adult male rats weighing 200 to 250 g. After establishment of a rat model of chronic constriction injury, these rats were divided into 10 groups (10 rats in each group): the normal control, sham operation, chronic constriction injury, normal saline, morphine, miR-223, NLRP3, miR-223 + morphine, NLRP3 + morphine, and miR-223 + NLRP3 + morphine groups. The real-time quantitative polymerase chain reaction assay, Western blotting, and enzyme-linked immunosorbent assay were used for detecting the mRNA and protein expressions of NLRP3, apoptosis-associated speck-like protein, Caspase-1, Interleukin (IL)-1β, and IL-18 in sections of lumbar spinal cord. Immunohistochemistry was applied for detecting the positive rates of NLRP3, apoptosis-associated speck-like protein, Caspase-1, IL-1β, and IL-18.ResultsThe paw withdrawal threshold and percentage maximum possible effect (%MPE) were higher in chronic constriction injury group when compared with the normal control and sham operation groups. Behavioral tests showed that compared with the chronic constriction injury and normal saline groups, the morphine and miR-223 + morphine groups showed obvious analgesic effects. Expressions of miR-223 in the miR-223, miR-223 + morphine, and miR-223 + NLRP3 + morphine were significantly higher than those in the chronic constriction injury, normal saline, and morphine groups. Compared with chronic constriction injury, normal saline and morphine groups, the mRNA and protein expressions of NLRP3, apoptosis-associated speck-like protein, Caspase-1, IL-1β, and IL-18 were significantly decreased in the miR-223 and miR-223 + morphine groups, while mRNA and protein expressions of NLRP3, apoptosis-associated speck-like protein, Caspase-1, IL-1β, and IL-18 were significantly increased in the NLRP3 and NLRP3 + morphine group.ConclusionOur study provides strong evidence that miR-223 could suppress the activities of NLRP3 inflammasomes (NLRP3, apoptosis-associated speck-like protein, and Caspase-1) to relieve morphine analgesic tolerance in rats by down-regulating NLRP3.

Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation.

BackgroundAstrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation.MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35–55 immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes.ResultsThe spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes.ConclusionsWe described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0348-y) contains supplementary material, which is available to authorized users.

Prenatal effects of retinoic acid on lumbar spinal cord development and liver antioxidants in rats

During embryonic and early postnatal development, retinoic acid (RA) regulates genes that control neuronal differentiation and neurite outgrowth from the neural tube. The effects of high levels of RA on the CNS can be detected via nitric oxide (NO), which plays a crucial role in neural transmission. The aim of the study was to investigate the prenatal influence of high levels of RA on postnatal development of nitrergic structures in lumbar spinal cord and antioxidant status. RA was administered orally at a dose of 10mg/kg body weight to pregnant female Wistar rats during days 8–10 of gestation. Neuronal nitric oxide synthase (nNOS) of lumbar spinal cord sections was processed for visualization via nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry on postnatal day one, day twenty-one and in adults. The results suggest that prenatal administration of high levels of RA is not associated with postnatal morphological changes in nNOS-positive neurons in the rat lumbar spinal cord. An estimation of the activity of enzymes related to the storage of retinoid in the liver showed possible side effects. Suppression and deepening of superoxide dismutase activity persisted into adulthood, and a concurrent downregulation of glutathione reductase was noted. A decrease in reduced glutathione persisted until adulthood when other compensatory mechanisms were probably active to maintain an appropriate level.

EphA4 Receptor Tyrosine Kinase Is a Modulator of Onset and Disease Severity of Experimental Autoimmune Encephalomyelitis (EAE)

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

Role of CB1 and CB2 cannabinoid receptors in the development of joint pain induced by monosodium iodoacetate

Joint pain is a common clinical problem for which both inflammatory and degenerative joint diseases are major causes. The purpose of this study was to investigate the role of CB1 and CB2 cannabinoid receptors in the behavioral, histological, and neurochemical alterations associated with joint pain. The murine model of monosodium iodoacetate (MIA) was used to induce joint pain in knockout mice for CB1 (CB1KO) and CB2 cannabinoid receptors (CB2KO) and transgenic mice overexpressing CB2 receptors (CB2xP). In addition, we evaluated the changes induced by MIA in gene expression of CB1 and CB2 cannabinoid receptors and μ-, δ- and κ-opioid receptors in the lumbar spinal cord of these mice. Wild-type mice, as well as CB1KO, CB2KO, and CB2xP mice, developed mechanical allodynia in the ipsilateral paw after MIA intra-articular injection. CB1KO and CB2KO demonstrated similar levels of mechanical allodynia of that observed in wild-type mice in the ipsilateral paw, whereas allodynia was significantly attenuated in CB2xP. Interestingly, CB2KO displayed a contralateral mirror image of pain developing mechanical allodynia also in the contralateral paw. All mouse lines developed similar histological changes after MIA intra-articular injection. Nevertheless, MIA intra-articular injection produced specific changes in the expression of cannabinoid and opioid receptor genes in lumbar spinal cord sections that were further modulated by the genetic alteration of the cannabinoid receptor system. These results revealed that CB2 receptor plays a predominant role in the control of joint pain manifestations and is involved in the adaptive changes induced in the opioid system under this pain state.

Extracellular signal‐regulated kinase activation in the chronic constriction injury model of neuropathic pain in anaesthetized rats

The role of extracellular signal-regulated kinases (ERKs) in nociception has been explored in the last years. While in spinal cord their activation is frequently correlated with pain or acute noxious stimuli, supraspinally, this association is not so evident and remains unclear. This study aims to evaluate ERK1/2 activation in the spinal cord and brainstem nuclei upon neuropathy and/or an additional mechanical stimulus. Acute noxious mechanical stimulation was applied in the left hindpaw of anaesthetized SHAM-operated and chronic constriction injured (CCI, neuropathic pain model) rats. Other SHAM or CCI rats did not receive any stimulus. Immunohistochemistry against the phosphorylated isoforms of ERK1/2 (pERK1/2) was performed in lumbar spinal cord and brainstem sections to assess ERK1/2 activation. In the spinal cord, stimulation promoted an increase in pERK1/2 expression in the superficial dorsal horn of SHAM rats. No significant effects were caused by CCI alone. At supraspinal level, changes in ERK1/2 activation induced by CCI were observed in A5, locus coeruleus (LC), raphe obscurus (ROb), raphe magnus, dorsal raphe (DRN), lateral reticular and paragigantocellularis nucleus. CCI increased pERK1/2 expression in all these nuclei, with exception of LC, where a significant decrease was verified. Mechanical noxious stimulation of CCI rats decreased pERK1/2 expression in ROb and DRN, but no further changes were detected in either SHAM- or CCI-stimulated animals. ERK1/2 are differentially activated in the spinal cord and in selected brainstem nuclei implicated in nociception, in response to an acute noxious stimulus and/or to a neuropathic pain condition.

Neuropathic pain and reactive gliosis are reversed by dialdehydic compound in neuropathic pain rat models

The role of the purinergic system in the modulation of pain mechanisms suggests that it might be promising target for treating neuropathic pain. In this study we evaluated the effects of two different dialdehydic compounds: a modified stable adenosine (2-[1-(6-amminopurin-9-il)-2-osso-etossi]prop-2-enale, named MED1101), and oxidized ATP (Ox-ATP), in two different neuropathic pain rat models: the sciatic spared nerve injury (SNI) and paclitaxel evoked painful peripheral neuropathy (pPPN). Neuropathic animals were divided in groups as follows: (a) treated with intraperitoneal (i.p.) MED1101 or Ox-ATP for 21 days; (b) receiving vehicle (VEH) and (c) control (CTR) rats. The allodynic and hyperalgesic behavior was investigated by Von Frey filament test and thermal Plantar test, respectively. We evaluated by immunocytochemistry the astrocytic (GFAP) and microglial (Iba1) response on lumbar spinal cord sections. In either experimental models and using either substances, treated animals showed reduced allodynia and thermal hyperalgesia paralleled by a significant reduction of glial reaction in the spinal cord. These data prompt to hypothesize a potential role of dialdehydes as analgesic agent in chronic neuropathic pain and a possible role as anti-gliotic molecules.

Human Neural Stem Cell Replacement Therapy for Amyotrophic Lateral Sclerosis by Spinal Transplantation

BackgroundMutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1G93A animals.Methods/Principal FindingsPresymptomatic SOD1G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.Conclusions/SignificanceThese data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.

The Importance of Preclinical Trial Timing – a Potential Reason for the Disconnect between mouse Studies and Human Clinical Trials in ALS

The Importance of Preclinical Trial Timing – a Potential Reason for the Disconnect between mouse Studies and Human Clinical Trials in <scp>ALS</scp>

Mechanisms of Bv8-induced biphasic hyperalgesia: Increased excitatory transmitter release and expression

Bv8 is a pronociceptive peptide that binds to two G-protein coupled prokineticin receptors, PK-R1 and PK-R2. These receptors are localized in the dorsal horn of the spinal cord and dorsal root ganglia (DRG) of nociceptive neurons in rodents. Systemic administration of Bv8 elicits a biphasic reduction in nociceptive thresholds to thermal and mechanical stimuli. Here, the possibility that Bv8 might directly modulate the expression and release of excitatory transmitters within the early and late phases of hyperalgesia was evaluated. Administration of Bv8 to mouse lumbar spinal cord sections produced a direct, significant and concentration-related release of CGRP. Bv8- or capsaicin-stimulated CGRP release was markedly enhanced in tissues taken from Bv8-pretreated mice during the late, but not the early, phase of hyperalgesia. Pretreatment of rats with protein synthesis inhibitors blocked the expression of the late, but not early, phase of Bv8-induced hyperalgesia. Finally, during the late-phase of hyperalgesia, there was an upregulation of CGRP and substance P immunoreactivity in the rat lumbar dorsal horn and DRG. Such upregulation was prevented by pretreatment with protein synthesis inhibitors. These data suggest that Bv8 induces hyperalgesia by direct release of excitatory transmitters in the spinal cord, consistent with the first phase of hyperalgesia. Additionally, Bv8 elicits a subsequent, protein-synthesis dependent increase in expression and release of excitatory transmitters that may underlie the long-lasting second phase of hyperalgesia. Activation of prokineticin receptors may therefore contribute to persistent hyperalgesia occurring as a consequence of tissue injury further suggesting that these receptors are attractive targets for development of therapeutics for pain treatment.

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.

Spinal Anesthesia in Infant Rats

Previous studies in infant rats and case-control studies of human infants undergoing surgery have raised concerns about potential neurodevelopmental toxicities of general anesthesia. Spinal anesthesia is an alternative to general anesthesia for some infant surgeries. To test for potential toxicity, a spinal anesthesia model in infant rats was developed. Rats of postnatal ages 7, 14, and 21 days were assigned to no treatment, 1% isoflurane for either 1 h or 6 h, or lumbar spinal injection of saline or bupivacaine at doses of 3.75 mg/kg (low dose) or 7.5 mg/kg (high dose). Subgroups of animals underwent neurobehavioral testing and blood gas analysis. Brain and lumbar spinal cord sections were examined for apoptosis using cleaved caspase-3 immunostaining. The lumbar spinal cord was examined histologically.Rats exposed to spinal or general anesthesia as infants underwent Rotarod testing of motor performance as adults. Data were analyzed using ANOVA with general linear models, Friedman tests, and Mann-Whitney U tests, as appropriate. Bupivacaine 3.75 mg/kg was effective for spinal anesthesia in all age groups. Impairments in sensory and motor function recovered in 40-60 min. Blood gases were similar among groups. Brain and spinal cord apoptosis increased in rats receiving 6 h of 1% isoflurane, but not among the other treatments. All groups showed intact motor performance at adulthood. Spinal anesthesia is technically feasible in infant rats and appears benign in terms of neuroapoptotic and neuromotor sequelae.

O20. The Role of muscle stem cells and denervation in sarcopenia

With ageing, the progressive loss of muscle mass and function (sarcopenia) results in frailty, loss of independence and is a major cause of increased falls and fractures. The precise reasons for sarcopenia are unclear. While sarcopenia is widely studied in rats, it has barely been studied in geriatric mice. We describe the time course of sarcopenia in mice and show significant loss of muscle mass at 24 and 27/29 months. We also demonstrate an excellent capacity of myogenic precursor cells in geriatric muscle (of mice aged 27/29 months) to form new muscle in regenerating muscle grafts cross-transplanted between young and very old mice [Shavlakadze et al., 2010. Biogerontology 11, 363–376]. Muscle function also requires electrical stimulation by a nerve, yet impaired innervation as a cause for sarcopenia has barely been considered. The extent of muscle denervation in geriatric mice was examined using immunostaining of whole intact muscles to identify the contribution of both muscle and nerve to the maintenance neuromuscular junctions (NMJ), and sections of lumbar spinal cord to quantify numbers of alpha-motoneurons. These analyses show striking morphological changes of NMJs and innervation, with a significant loss (~20%) of innervation in NMJ of geriatric muscles and myofibre type changes, but no significant loss of alpha-motoneurons cell bodies in the spinal cord of geriatric mice. Establishing these baseline data for geriatric mice is essential in order to take full advantage of the wealth of genetically modified mice available to study many aspects of sarcopenia, especially the molecular basis for this pronounced denervation.