Spinal Cord Sections Research Articles

Injury-Specific Promoters Enhance Herpes Simplex Virus–Mediated Gene Therapy for Treating Neuropathic Pain in Rodents

Chronic neuropathic pain is often difficult to treat with current pain medications. Gene therapy is presently being explored as a therapeutic approach for the treatment of neuropathic and cancer pain. In this study, we sought to use an injury-specific promoter to deliver the mu-opioid receptor (MOR) transgene such that expression would occur during the injured state only in response to release of injury-specific galanin. To determine whether an injury-specific promoter can produce neuron-specific MOR expression and enhanced antinociception, we compared animals infected with a galanin promoter virus (galMOR) or a human cytomegalovirus promoter virus (cmvMOR). In behavioral assays, we found an earlier onset and a larger magnitude of antinociception in animals infected with galMOR compared with cmvMOR. Immunohistochemical analysis of dorsal root ganglion neurons revealed a significant increase in MOR-positive staining in cmvMOR- and galMOR-treated mice. Spinal cord sections from galMOR-treated mice showed a greater increase in density but not area of MOR-positive staining. These results suggest that using injury-specific promoters to drive gene expression in primary afferent neurons can influence the onset and magnitude of antinociception in a rodent model of neuropathic pain and can be used to upregulate MOR expression in populations of neurons that are potentially injury specific. PerspectiveAn injury-specific promoter (galMOR) was used to drive MOR expression in a population- and injury-specific manner. GalMOR increased antinociception and density of MOR staining in the spinal cord. This article presents evidence that promoter selection is an important component in successful gene expression in an injury- and population-specific manner.

Dynamic expression of ATF3 as a novel tool to study activation and migration of endogenous spinal stem cells and their role in neural repair.

One of the major problems of modern neurobiology is how to replace dead or damaged neurons in the human brain or spinal cord after injury or as a consequence of neurodegenerative diseases. In fact, because adult mammalian neurons are post-mitotic cells that cannot divide to replace dead cells, loss due to lesion or disease is permanent. Furthermore, surviving neurons have modest capacity to regenerate their damaged axons and re-establish functional connections. Thus, a gradual neurodegenerative scenario with certain similarities in stroke, brain or spinal cord injuries and neurological diseases like Alzheimer's disease is produced. These conditions represent the major disease burden of the modern world in terms of mortality, disability, productivity loss and health-care costs (World Health Organization, 2008). While much effort has been directed to understand the molecular and cellular mechanisms involved in the pathology of these diseases to set new effective treatments, many neuroprotective and regenerative approaches, although showing positive results in preclinical studies, have so far failed to provide strong benefit to patients.

Local expression and role of BMP-2/4 in injured spinal cord.

We investigated local changes in BMP-2/4 expression in rat spinal cords 1 week following injury to study the damage effects of BMP-2/4 in spinal cord injury (SCI). Sprague Dawley rats (45, 4 months old) were randomized into three groups comprising 15 rats each: a SHAM group, an SCI without noggin group (SCIO), and an SCI with noggin group (SCID). The SCIO and SCID groups were subjected to spinal cord hemisection, and motor activity was assessed using the BBB score. Expression of BMP-2/4 in each injured spinal cord section was examined by hematoxylin and eosin staining, immunohistochemistry, and western blot. There were no significant differences in BBB scores among the three groups (P > 0.05). Following hemisection, the BBB score in the SHAM group was significantly higher than in the other two groups on the 1st day after modeling (P < 0.05), and the BBB scores in the SCIO and SCID groups were not significantly different (P > 0.05). Seven days after modeling, the BBB score in the SHAM group was significantly higher than in the other two groups (P < 0.05), and the BBB score in the SCID group was obviously higher than in the SCIO group (P < 0.05). The expression of BMP-2/4 was highest in the SCIO group and lowest in the SHAM group (P < 0.05). SCI can cause severe impairment of motor activity in rats. Seven days after SCI, the local expression of BMP-2/4 had obviously increased; noggin can effectively inhibit the expression of BMP-2/4 and reduce impairment.

Establishment of minimally acute spinal cord injury in mice model by the self-made device

Objective Try to construct the self-made spinal impactor and the simple interspinous process distraction stabilizing device.Discussion on the feasibility,stability and reliability of method establishing the acute spinal cord injury in mice model retaining the lamina.Methods Using a electric push fool,a pressure sensor and so on,to manufacture the spinal inpactor and the interspinous process distraction stabilizing device.32 adult female rats were randomly divided into four groups,using the self-made spinal impactor device to make varying degrees on the spinal cord of the mice model retaining the lamina,non-combat (0 g),mild (5 g),medium (10 g),and severe (20 g).Taking two mice from every group randomly and making pathological section of damage zone spinal cord after 12 h,testing the behavior BBB grade post operation 1,3 d,7,14 w,in order to evaluate its correlation with the strike force.Results The spinal impactor device and the interspinous process distraction stabilizing device was producted successfully,and the minimally invasive spinal cord injury model retaining the lamina was built.Pathological examination and behavioral score was related with the strike force closely and the grade differences was obvious (P ＜ 0.05).Conclusion A a stable and reliable spinal cord injury model retaining the lamina can be made by the self-made device,and this conducive to the further research on spinal cord injury. Key words: Self-made device; Minimally invasive; Spinal cord injury; Mice model

(-)-Epigallocatechin-3-gallate modulates spinal cord neuronal degeneration by enhancing growth-associated protein 43, B-cell lymphoma 2, and decreasing B-cell lymphoma 2-associated x protein expression after sciatic nerve crush injury.

Our previous studies have established that (-)-epigallocatechin-3-gallate (EGCG) has both neuroprotective and -regenerative capacity after sciatic nerve injury. Moreover, this improvement was evident on the behavioral level. The aim of this study was to investigate the central effects of ECGC on spinal cord motor neurons after sciatic nerve injury. Our study showed that administering 50 mg/kg intraperitoneally i.p. of EGCG to sciatic nerve-injured rats improved their performance on different motor functions and mechanical hyperesthesia neurobehavioral tests. Histological analysis of spinal cords of EGCG-treated sciatic nerve-injured (CRUSH+ECGC) animals showed an increase in the number of neurons in the anterior horn, when compared to the naïve, sham, and saline-treated sciatic nerve-injured (CRUSH) control groups. Additionally, immunohistochemical study of spinal cord sections revealed that EGCG reduced the expression of glial fibrillary acidic protein and increased the expression of growth-associated protein 43, a marker of regenerating axons. Finally, EGCG reduced the ratio of B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 and increased the expression of survivin gene. This study may shed some light on the future clinical use of EGCG and its constituents in the treatment of peripheral nerve injury.

Efectos neurotóxicos de la levobupivacaína y el fentanilo sobre la médula espinal de ratones

BackgroundThe purpose of the study was to compare the neurotoxic effects of intrathecally administered levobupivacaine, fentanyl and their mixture on rat spinal cord. MethodsIn experiment, there were 4 groups with medication and a control group. Rats were injected 15μL saline or fentanyl 0.0005μg/15μL, levobupivacaine 0.25%/15μL and fentanyl 0.0005μg+levobupivacaine 0.25%/15μL intrathecally for four days. Hot plate test was performed to assess neurologic function after each injection at 5th, 30th and 60thmin. Five days after last lumbal injection, spinal cord sections between the T5 and T6 vertebral levels were obtained for histologic analysis. A score based on subjective assessment of number of eosinophilic neurons –red neuron– which means irreversible neuronal degeneration. They reflect the approximate number of degenerating neurons present in the affected neuroanatomic areas as follows: 1, none; 2, 1–20%; 3, 21–40%; 4, 41–60%; and 5, 61–100% dead neurons. An overall neuropathologic score was calculated for each rat by summating the pathologic scores for all spinal cord areas examined. ResultsIn the results of hot plate test, comparing the control group, analgesic latency statistically prolonged for all 4 groups.In neuropathologic investment, the fentanyl and fentanyl+levobupivacaine groups have statistically significant high degenerative neuron counts than control and saline groups. ConclusionsThese results suggest that, when administered intrathecally in rats, fentanyl and levobupivacaine behave similar for analgesic action, but fentanyl may be neurotoxic for spinal cord. There was no significant degeneration with levobupivacaine, but fentanyl group has had significant degeneration.

Mannan-conjugated myelin peptides prime non-pathogenic Th1 and Th17 cells and ameliorate experimental autoimmune encephalomyelitis

Antigen presenting cells (APC) are critical for regulating immune responses. We tested mannan-peptide conjugates for targeting myelin peptides to APC to induce T cell tolerance and resistance to experimental autoimmune encephalomyelitis (EAE). Myelin peptides conjugated to mannan in oxidized (OM) or reduced (RM) forms protected mice against EAE in prophylactic and therapeutic protocols, with OM-conjugated peptides giving best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation, but not alterations in Th1, Th17 and Treg cell differentiation or T cell apoptosis compared to EAE controls. Bone marrow-derived dendritic cells (DC) loaded with OM-MOG showed up-regulated expression of co-stimulatory molecules, reduced PD-L1 expression and enhanced CD40-inducible IL-12 and IL-23 production compared to MOG DC, features consistent with immunogenic DC. OM-MOG induced active T cell tolerance because i.d. administration or passive transfer of OM-MOG DC suppressed ongoing EAE, while OM-MOG-vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. As in vivo, MOG-specific T cells cultured with OM-MOG DC showed reduced proliferation and equal Th1 and Th17 cell differentiation compared to those with MOG DC, but surprisingly cytokine production was unresponsive to CD40 engagement. Impaired effector T cell function was further evidenced in spinal cord sections from OM-MOG-vaccinated EAE mice, where markedly reduced numbers of CD3+ T cells were present, restricted to leptomeninges and exceptional parenchymal lesions. Our results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and APC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells.

Efeitos neurotóxicos de levobupivacaína e fentanil sobre a medula espinhal de ratos

BackgroundThe purpose of the study was to compare the neurotoxic effects of intrathecally administered levobupivacaine, fentanyl and their mixture on rat spinal cord. MethodsIn experiment, there were four groups with medication and a control group. Rats were injected 15μL saline or fentanyl 0.0005μg/15μL, levobupivacaine 0.25%/15μL and fentanyl 0.0005μg+levobupivacaine 0.25%/15μL intrathecally for four days. Hot plate test was performed to assess neurologic function after each injection at 5th, 30th and 60th min. Five days after last lumbal injection, spinal cord sections between the T5 and T6 vertebral levels were obtained for histologic analysis. A score based on subjective assessment of number of eosinophilic neurons – Red neuron – which means irreversible neuronal degeneration. They reflect the approximate number of degenerating neurons present in the affected neuroanatomic areas as follows: 1, none; 2, 1–20%; 3, 21–40%; 4, 41–60%; and 5, 61–100% dead neurons. An overall neuropathologic score was calculated for each rat by summating the pathologic scores for all spinal cord areas examined. ResultsIn the results of HPT, comparing the control group, analgesic latency statistically prolonged for all four groups.In neuropathologic investment, the fentanyl and fentanyl+levobupivacaine groups have statistically significant high degenerative neuron counts than control and saline groups. ConclusionsThese results suggest that, when administered intrathecally in rats, fentanyl and levobupivacaine behave similar for analgesic action, but fentanyl may be neurotoxic for spinal cord. There was no significant degeneration with levobupivacaine, but fentanyl group has had significant degeneration.

Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice

Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.

Enhanced in Vivo Efficacy of a Type I Interferon Superagonist with Extended Plasma Half-life in a Mouse Model of Multiple Sclerosis

IFNβ is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNβ. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35-55-induced experimental autoimmune encephalomyelitis compared with IFNβ, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.

Prevalencia de contraindicaciones a succinilcolina en unidades de cuidados intensivos

ObjectiveTo determine the prevalence of contraindications to succinylcholine in patients admitted to the ICU. DesignAn observational study was carried out, describing the frequency of succinylcholine contraindication by stays, and associating the contraindications to population variables. SettingThe adult general ICU of a reference hospital. PatientsClinical-surgical critically ill patients. Thirty consecutive days were analyzed. InterventionsNone. VariablesPopulation variables: age, sex, APACHE II and SOFA scores, illness, days of hospitalization, mechanical ventilation and renal replacement therapy. Contraindications: hyperkalemia, bedridden patients, rhabdomyolysis, muscle trauma, burns, infusion of neuromuscular blocking agents, acute renal failure, chronic renal failure, intraocular hypertension, intracranial hypertension, statin use, malignant hyperthermia, neuromuscular disease, spinal cord sectioning, bradycardia < 50 bpm, succinylcholine allergy, and pseudo-cholinesterase deficiency. ResultsA total of 102 patients were admitted, aged 60±16 years (56.9% males), with the generation of 657 stays. Main diseases: elective heart surgery (33%), respiratory failure (22%) and sepsis (14%). We documented 466 stays (70.9%) with contraindications, particularly bedridden patients (n=140), neuromuscular blocking agents (n=101), acute renal failure (n=126), statin use (n=167), neuromuscular disease (n=159) and bradycardia (n=76). We recorded 74 stays (11.3%) at high risk of complications, secondary to hyperkalemic risk associated to receptor dysregulation. A high risk of complications was associated to advanced age, prolonged hospitalization, prolonged length of stay in the ICU, higher APACHE II and SOFA scores, mechanical ventilation and renal replacement therapy. ConclusionGiven the high prevalence of contraindications to succinylcholine observed, we discourage its use in patients admitted to the ICU.

Spinal cord dopamine D2/D3 receptors: in vivo and ex vivo imaging in the rat using 18F/11C-fallypride

ObjectivesThe spinal cord is known to be innervated with dopaminergic cells with catecholaminergic projections arising from the medulla and pons and dopaminergic transmission in the spinal cord is vital for sensory and motor function. Our goal was to evaluate and compare the imaging capability of dopamine D2/D3 receptors in the rat spinal cord using PET ligands 18F-fallypride and 11C-fallypride. MethodsMale Sprague–Dawley rats were used in all in vitro and in vivo studies. Spinal cord and brain sections were used for in vitro autoradiography and ex vivo autoradiography. For in vivo studies animals received a 18F-fallypride scan or a 11C-fallypride PET scan. The spinal cord and the brain were then harvested, flash-frozen and imaged ex vivo. For in vivo analysis Logan plots with cerebellum as a reference was used to evaluate binding potentials (BP). Tissue ratios were used for ex vivo analysis. Drug effects were evaluated using clozapine, haloperidol and dopamine were evaluated on spinal cord sections in vitro. ResultsIn vitro studies showed 18F-fallypride binding to superficial dorsal horn (SDH), dorsal horn (DH), ventral horn (VH) and the pars centralis (PC). In the cervical section, the greatest amount of binding appeared to be in the SDH. Ex vivo studies showed approximately 6% of 18F-fallypride in SDH compared to that observed in the striatum. In vivo analysis of both 18F-fallypride and 11C-fallypride in the spinal cord were comparable to that in the extrastriatal regions. Haloperidol and clozapine displaced more than 75% of the 18F-fallypride in spinal cord sections. ConclusionsOur studies showed 18F-fallypride and 11C-fallypride binding in the spinal cord in vitro and in vivo. The binding pattern correlates well with the known distribution of dopamine D2/D3 receptors in the spinal cord.

Detection of Neuronal and Glial Antigens After Decalcification in Formic Acid Solution and Fixation in Zinc-Ethanol-Formaldehyde

The aim of the present work was to identify the possibility of immunocytochemical detection of various tissue antigens on paraffin sections of rat spinal cord surrounded by spinal bony tissue and adjacent muscles fixed in zinc-ethanol-formaldehyde after decalcification with formic acid in adult (n = 5) and neonatal (n = 2) Wistar rats. These studies used antibodies to glial fibrillary acidic protein, neurofilaments, neuronal nuclear protein, tyrosine hydroxylase, the synaptic vesicle protein synaptophysin, and the intermediate filament protein vimentin. The combination of fixation in zinc-ethanol-formaldehyde and decalcification in 21% formic acid solution provided optimal preservation of the antigens of interest.

Coronavirus-induced demyelination of neural pathways triggers neurogenic bladder overactivity in a mouse model of multiple sclerosis.

In the present study, we aimed to determine whether mice with coronavirus-induced encephalomyelitis (CIE) develop neurogenic bladder dysfunction that is comparable with the neurogenic detrusor overactivity observed in patients with multiple sclerosis. Adult mice (C57BL/6J, 8 wk of age, n = 146) were inoculated with a neurotropic strain of mouse hepatitis virus (A59 strain) and followed for 4 wk. Inoculation with the virus caused a significant neural deficit in mice with an average clinical symptom score of 2.6 ± 0.5 at 2 wk. These changes were accompanied by 25 ± 5% weight loss at 1 and 2 wk postinoculation (P ≤ 0.001 vs. baseline) followed by a recovery phase. Histological analysis of spinal cord sections revealed multifocal sites of demyelinated lesions. Assessment of micturition patterns by filter paper assay determined an increase in the number of small and large urine spots in CIE mice starting from the second week after inoculation. Cystometric recordings in unrestrained awake animals confirmed neurogenic bladder overactivity at 4 wk postinoculation. One week after inoculation with the A59 strain of mouse hepatitis virus, mice became increasingly sensitive to von Frey filament testing with responses enhanced by 45% (n = 8, P ≤ 0.05 vs. baseline at 4 g); however, this initial increase in sensitivity was followed by gradual and significant diminution of abdominal sensitivity to mechanical stimulation by 4 wk postinoculation. Our results provide direct evidence showing that coronavirus-induced demyelination of the central nervous system causes the development of a neurogenic bladder that is comparable with neurogenic detrusor overactivity observed in patients with multiple sclerosis.

Bifurcation analysis of anti-phase oscillations and synchrony in the tadpole central pattern generator

Recent experimental findings in the laboratory of Wen-Chang Li (St. Andrews University, UK) demonstrate that the same neural Central Pattern Generator (CPG) within the hindbrain/spinal cord of the Xenopus tadpole can produce both reliable anti-phase oscillations between the left and right sides of the body (swimming) and long bouts of in-phase synchronous activity. The key element of the CPG circuit includes a pair of neurons on each side of the body at about the same position: an excitatory descending interneuron (dIN) and an inhibitory commissural interneuron (cIN). A simplified description of the CPG activity in swimming regime follows from experimental facts: dIN spiking on one side leads to excitation of the cIN on the same side, which inhibits the dIN on the opposite side causing a spike due to post-inhibitory rebound (PIR). Similarly, experiments suggest that synchrony appears as a result of simultaneous firing of dINs on both sides which leads to simultaneous firing of cINs on both sides, followed by inhibition of dINs (on opposite sides) and a new cycle of simultaneous dIN firing due to PIR. To study these exciting experimental findings we use biologically realistic anatomical and functional computational models [1-3]. The tadpole swimming model represents a 1.5 mm section of tadpole spinal cord with ~ 1,500 neurons, interconnected by ~ 85,000 synapses. Neuron dynamics are simulated according to a conductance based model of the Hodgkin-Huxley type. Additionally, gap junctions between dINs within 100µm of each other in the rostro-caudal direction are included. From studying the dynamics of this model and bifurcation analysis of the key element of the CPG circuit, we conclude: 1. There are two limit cycles in the phase space of the model: an anti-phase limit cycle with period T which corresponds to swimming activity, and an in-phase limit cycle with period T/2 which corresponds to synchrony (see Figure ​Figure11). Figure 1 Phase portraits of the system during anti-phase (green) and in-phase (blue) activity. Horizontal and vertical axes correspond to membrane potentials of dINs at similar positions on the left and right body side respectively. 2. The anti-phase (swimming) cycle is stable and robust with a large basin of attraction. The in-phase (synchrony) cycle can be initiated from swimming regime by mid-cycle stimulation of dIN neurons, but is generally unstable or has a very small basin of attraction. 3. The in-phase cycle is near a fold of limit cycles bifurcation. For a minority of generated connections the stable in-phase limit cycle exists. However, for most connections this stable cycle does not exist, although its “ghost” is still visible: after several synchronous cycles the system returns to swimming oscillations. 4. The stability of synchrony is extremely sensitive to the synaptic/conductance delays between dINs and cINs, with longer delays stabilizing synchrony. 5. It is unclear whether synchrony has a functional purpose. If it does, we hypothesize that in this early developmental stage the system may be near to the critical bifurcation point in order to provide flexibility in locomotion control. As the tadpole develops, longer synaptic delays may act to stabilize the synchronous firing pattern.

Spatial elucidation of spinal cord lipid- and metabolite- regulations in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressing disease of the central nervous system that is characterized by motor neuron degeneration in the brain stem and the spinal cord. We employed time of flight secondary ion mass spectrometry (ToF-SIMS) to profile spatial lipid- and metabolite- regulations in post mortem human spinal cord tissue from ALS patients to investigate chemical markers of ALS pathogenesis. ToF-SIMS scans and multivariate analysis of image and spectral data were performed on thoracic human spinal cord sections. Multivariate statistics of the image data allowed delineation of anatomical regions of interest based on their chemical identity. Spectral data extracted from these regions were compared using two different approaches for multivariate statistics, for investigating ALS related lipid and metabolite changes. The results show a significant decrease for cholesterol, triglycerides, and vitamin E in the ventral horn of ALS samples, which is presumably a consequence of motor neuron degeneration. Conversely, the biogenic mediator lipid lysophosphatidylcholine and its fragments were increased in ALS ventral spinal cord, pointing towards neuroinflammatory mechanisms associated with neuronal cell death. ToF-SIMS imaging is a promising approach for chemical histology and pathology for investigating the subcellular mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis.

Acute estrogen surge enhances inflammatory nociception without altering spinal Fos expression

Chronic pain is a major neurological disorder that can manifest differently between genders or sexes. The complex actions of sex hormones may underlie these differences; previous studies have suggested that elevated estrogen levels can enhance pain perception. The purpose of this study was to investigate the hypothesis that acute, activational effects of estradiol (E2) increase persistent inflammatory nociception, and anatomically where this modulation occurs. Spinal expression of Fos is widely used as a marker of nociceptive activation. This study used formalin-evoked nociception in ovariectomized (OVX) adult female rats and measured late-phase hindlimb flinching and Fos expression in the spinal cord, and their modification by acute estrogen supplementation similar to a proestrus surge. Six days after ovariectomy, female rats were injected subcutaneously (s.c.) with 10μg/kg E2 or vehicle. Twenty-four hours later, 50μL of 1.25% or 100μL of 5% formalin was injected into the right hindpaw; hindlimb flinches were counted, and spinal cords removed 2h after formalin injection. The numbers of Fos-expressing neurons in sections of the lumbar spinal cord were analyzed using immunohistochemistry. Formalin-induced inflammation produced a dose-dependent increase in late-phase hindlimb flinching, and E2 pretreatment increased flinching following 5%, but not 1.25% formalin injection. Despite the modification of behavior by E2, the number of spinal Fos-positive neurons was not altered by E2 pretreatment. These findings demonstrate that an acute proestrus-like surge in serum estrogen can produce a stimulus-intensity-dependent increase in inflammation-evoked nociceptive behavior. However, the lack of effect on spinal Fos expression suggests that this enhancement of nociceptive signaling by estrogen is independent of changes in peripheral activation of, expression of the immediate early gene Fos by, or signal throughput of spinal nociceptive neurons.

Programmed death 1 deficiency induces the polarization of macrophages/microglia to the M1 phenotype after spinal cord injury in mice.

The inflammatory response following spinal cord injury (SCI) involves the activation of resident microglia and the infiltration of macrophages. Macrophages and microglia can be polarized into the classically activated proinflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype. Programmed cell death 1 (PD-1) is a critical immune inhibitory receptor involved in innate and adaptive immune responses. However, whether PD-1 is involved in the modulation of macrophage/microglial polarization is unknown. In this study, the mRNA levels of pd1 gradually increased after SCI, and PD-1 protein was found in macrophages/microglia in injured spinal cord sections. PD-1 knockout (KO) mice showed poor locomotor recovery after spinal cord crushing compared with wild-type mice. M1-type macrophages/microglia accumulated in greater numbers in the injured spinal cord of PD-1-KO mice. Under polarized stimulation, induced expression of PD-1 occurred in cultured macrophages and microglia. PD-1 suppressed M1 polarization by reducing the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and promoted M2 polarization by increasing STAT6 phosphorylation. In PD-1-KO mice, the M1 response was enhanced via the activation of STAT1 and nuclear factor-kappa B. Furthermore, PD-1 played various roles in phagocytosis in macrophages and microglia. Therefore, our results suggest that PD-1 signaling plays an important role in the regulation of macrophage/microglial polarization. Thus, deregulated PD-1 signaling may induce the polarization of macrophages/microglia toward the M1 phenotype. Overall, our results provide new insights into the modulatory mechanisms of macrophage/microglial polarization, thereby possibly facilitating the development of new therapies for SCI via the regulation of macrophage/microglial polarization through PD-1 signaling.Electronic supplementary materialThe online version of this article (doi:10.1007/s13311-013-0254-x) contains supplementary material, which is available to authorized users.

Spinal cord development in marsupials in relation to birthing strategies and in comparison with monotremes and the laboratory rat

Marsupials are born in an immature state, followed by prolonged nurturing of pouch young by maternal lactation. Spinal cord sections held in the collections at the Museum für Naturkunde, Berlin were used to test the relationship between structural maturity of the spinal cord and the locomotor challenges that face young marsupials and monotremes. Analysis of variance indicated that body length is a much stronger determinant of variation in anatomical measures of spinal cord maturation than mammal type.

Effect of tacolimus postconditioning on serine-threonine protein kinase signaling pathway via regulating generation of reactive oxygen species after spinal cord ischemia-reperfusion injury

Objective To explore the influence of tacrolimus postconditioning (TP) on serine-threonine protein kinase (Akt) signaling pathway afier spinal cord ischemia-reperfusion injury (SCIRI)and the role of reactive oxygen species (ROS) in the mechanism.Methods The rat model of SCIRI was prepared on the basis of Taira' s method.Eighty male SD rats were randomly divided into 4 groups:sham operation (SO) group,ischemia-reperfusion (IR) group,TP group,and TP plus N-Acetylcysteine (TP +N) group.IR group underwent reperfusion for 20 min after spinal cord ischemia.TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery right at the onset of reperfusion,with remaining procedures the same as IR group.TP + N group was injected through the left common carotid artery with N-Acetylcysteine (150 mg/kg) 5 min before spinal cord ischemia,with remai-ning procedures the same as TP group.SO group received exclusively femoral artery catheterization.Fluo-rospectrophotometry was employed to detect the level of ROS in injured spinal cord segments,and immuno-blotting was used to determine the expression of phosphorylated Akt (p-Akt) at 15 min after reperfusion.Flow cytometry was applied to assess apoptosis of neural cells at 24 h after reperfusion.Nissl's staining was utilized to observe pathological changes of injured spinal cord segments at 7th day after reperfusion.Re-sults The ROS levels in spinal cord tissue were 49.0 ± 3.5,145.3±11.7,79.0±6.3 and12.8 ± 0.9in SO group,IR group,TP group,and TP + N group at 15 rain after reperfusion,respectively.The ROSlevel in TP group was significantly lower than that in IR group at 15 min after reperfusion (P ＜0.01),and that in TP + N group was significantly lower than the other three groups (P ＜ 0.01).The p-Akt expression in spinal cord tissue was 0.54 ± 0.06,0.34 ± 0.03,0.42 ± 0.07,and 0.28 ± 0.05 in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The p-Akt expression was sig-nificantly lower in IR group than in SO group (P ＜ 0.01),higher in TP group than in IR group (P ＜0.05),and lower in TP + N group than in SO group (P ＜0.01) or in TP group (P ＜0.01).The percentages of apoptotic cells in spinal cord tissue were (7.5 ± 1.8) ％,(31.4 ± 5.5) ％,(22.3-± 4.0) ％,and (33.3 ± 3.1) ％ in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The percentage of apoptotic cells was significantly higher in IR or TP + N group than in TP group (P ＜0.01),while there was no significant difference between IR group and TP + N group (P ＞0.05).The spinal cord sections revealed that pathological changes could hardly be observed in SO group,and mild in TP group but serious in IR and TP + N groups.Conclusion TP produces protective effect on ischemic spinal cord via positive regulation of Akt signaling pathway,and ROS,acting as a signaling molecule,plays an indispensable role in initiating the mechanism. Key words: Spinal cord ischemia; Tacrolimus ; Reactive oxygen species ; Serine-threonine protein kinase