Spinal Sections Research Articles

337 Cellular senescence contributes to inflammation and disease progression in an animal model of multiple sclerosis

OBJECTIVES/GOALS: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. MS affects more than two million people worldwide, resulting in physical disability, cognitive impairment, and decreased quality of life. We are investigating the role of senescent cells, a hallmark of aging, in inflammation and disease progression in MS. METHODS/STUDY POPULATION: Female mice on a C57BL/6 background were subjected to the Hooke Laboratory EK-2110 experimental autoimmune encephalomyelitis protocol (EAE; n=10) to induce hind limb paralysis, and matching control mice received no injections (naīve; n=10). Immunofluorescent staining was used to visualize senescence cells and their localization in spinal cord tissue sections from naīve and EAE mice based on antibody detection of cell surface or intracellular proteins. Tissue sections from each group were analyzed in duplicates for each antibody (n=3-4/group). Flow cytometry was performed to immunophenotype the senescence cells using conjugated fluorescent antibodies to cell surface or intracellular proteins (n=5/group). RESULTS/ANTICIPATED RESULTS: Immunostaining demonstrated an increase in the cell senescence markers p16 (15-fold; unpaired t-test, p=0.01; n=3) and p21 (15-fold; unpaired t-test, p=0.003; n=3) in the spinal cord of EAE mice compared to naīve mice. Next, we showed that p16+ and p21+ cells increase with disease progression in the meninges adjacent to the ventral demyelinating lesions (one-way ANOVA, p=0.0009; n=3/group). We further found that 30±9.7% of M2 macrophages, a subset of myeloid cells, express p21 in the spinal cord of EAE mice by using flow cytometry analysis compared to only 5±1.6% in naīve mice (unpaired t-test, p=0.002; n=5/group). DISCUSSION/SIGNIFICANCE: Our findings demonstrate that senescent cells accumulate in the meningeal compartment following EAE induction, suggesting that decreasing senescent cell burden is a promising avenue in delaying disease progression and preventing neuroinflammation in MS.

The Effectiveness of Parecoxib on Inflammatory Cytokines, Prostaglandin Levels, and Visual Analogue Scale Value among Preeclamptic Patients that Undergo Caesarean Section

Introduction: Preeclampsia is produced by the maternal immune system and trophoblast proinflammatory cytokines. Effective perioperative analgesics reduce surgery-related pain and inflammation. It explores how parecoxib affects IL-1, IL-6, prostaglandin and VAS in preeclampsia patients who had a spinal anesthesia cesarean. Materials and methods: This experiment uses a 2-group pretest and posttest. Samples were taken consecutively. The study group (n = 18) received parecoxib 40 mg IV bolus every 12 h 4 times and paracetamol 1,000 mg IV infusion every 8 h until 48 h postoperatively, while the control group (n = 18) received placebo NaCl 0.9 % IV bolus every 12 h 4 times and paracetamol 1,000 mg IV drip every 8 to 48 h. Participants’ postoperative pain was assessed by VAS before, 12, 24 and 36 h after surgery. Statistical analysis was conducted on biomolecular examination data, including IL-1β, IL-6, PGE2 and VAS values. Results and discussion: The study group had lower IL-1β levels than the control group, although the difference was not significant (p > 0.05). In the study group, IL-6 levels were 4,782 ± 5,673 lower than in the control group (6,147 ± 5,957). Study group IL-6 levels were higher than control group values, although not significantly (p > 0.05). The study group had lower PGE2 than the control group, but not significantly (p > 0.05). The study group’s mean VAS value was 1.722 ± 1.994, lower than the control group’s 1.833 ± 2.007 and not significant (p > 0.05). The study group had considerably lower VAS values than the control group (p < 0.05). Conclusions: In preeclampsia patients who underwent caesarean delivery under spinal anesthesia, parecoxib administration influences IL-1β levels after 24 h and VAS values after 12 h. It did not affect IL-6 or PGE2 levels in preeclamptic individuals who had spinal anesthesia caesarean sections. HIGHLIGHTS Parecoxib, a powerful analgesic, anti-inflammatory drug and moderately toxic maternal and newborn drug Parecoxib has the ability to reduce IL-1β levels after 24 h in preeclampsia patients undergoing cesarean section with spinal anesthesia After 12 h, parecoxib has an effect on the pain scale (VAS) values of preeclampsia patients who underwent a caesarean section under spinal anesthesia GRAPHICAL ABSTRACT

Impact of Melatonin as a Premedication Agent in Caesarean Section on Blood Loss and Postoperative Pain Level.

Postpartum hemorrhage (PPH) is a serious postdelivery condition with a high incidence of morbidity and mortality for women who undergo childbirth with or without a caesarean section. Melatonin has been suggested to increase the contractility of myometrium and reduce the pain score postoperatively, therefore it is believed that the use of melatonin before surgery may decrease blood loss, reduce pain score, and decrease the need for postoperative opioids. The main objectives of this study are focused on the investigation of melatonin as a premedication agent to reduce blood loss and decrease pain score postoperatively in patients undergoing cesarean section under spinal anesthesia. 80 patients were scheduled for spinal anesthesia-based cesarean sections and randomly assigned to two groups, melatonin group (M) 40 patients and placebo group (P) 40 patients to receive either 10 mg of sublingual melatonin or a placebo of 90 minutes preoperatively. Hemoglobin levels were been measured preoperative and 12 hrs. Postoperatively, blood loss volume was calculated by measuring both the weight of used materials before and after the surgery and the volume sucked in the suction bottle after placental delivery. Postoperative visual pain score and analgesic requirements were used to evaluate pain levels. Analyzed collected data showed a significant decrease in blood loss in the melatonin group in comparison with the placebo group as measured by the hemoglobin level. On the other hand, there is a significant decrease in pain score and analgesia requirement with the melatonin group compared to the placebo group. Melatonin is a promising premedication drug that has a significant impact on postpartum hemorrhage by reducing blood loss and pain levels of mothers who have undergone C-sections.

Nogo-A antibody delivery through the olfactory mucosa mitigates experimental autoimmune encephalomyelitis in the mouse CNS

Systemic administration of Nogo-A-neutralizing antibody ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the blood-brain barrier (BBB) is a major obstacle limiting the passage of systemically applied antibody to the CNS. To bypass the BBB, in the present study we tested the intranasal route of administration by targeting the olfactory mucosa with the Nogo-A-blocking antibody 11C7 mAb in myelin oligodendrocyte glycoprotein-induced EAE. Antibodies were specifically administered onto the olfactory mucosa using a microcatheter. Antibody distribution was examined in the CNS by ELISA and light-sheet microscopy. The effects of 11C7 mAb on Nogo-A signaling were assessed by Western blotting. EAE-induced deficits were monitored daily. Demyelination was observed on spinal cord histological sections. Gene expression changes were followed by trancriptomic analyses. A sensitive capture ELISA revealed a rapid and widespread distribution of 11C7 mAb in the CNS, including the olfactory bulb, the cerebellum and the lumbar spinal cord, but not in the CSF. Light-sheet microscopy allowed to observe antibody accumulation in the parenchyma, thus demonstrating nose-to-brain transfer of IgG. At the functional level, the widespread penetration of 11C7 mAb in the CNS, including the thoracolumbar spinal cord, resulted in the improvement of motor symptoms and in the preservation of myelin in the spinal cord of EAE mice. This was accompanied by Nogo-A signaling downregulation, as reflected by the decreased level of phosphorylated cofilin observed by Western blotting in the cerebellum. In the brain of EAE score-matched animals, 11C7 modified the expression of genes that can influence neurotransmission and cognitive functions, independently of the demyelination phenotype in the spinal cord. In conclusion, our data show the feasibility of olfactory mucosa-directed administration for the delivery of therapeutic antibodies targeting CNS antigens in EAE mice.

Motor neuron loss and neuromuscular transmission failure following chemogenetic inhibition of BDNF/TrkB signaling in adult rats

Brain derived neurotrophic factor (BDNF) signaling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. However, the specific effects of reduced BDNF/TrkB signaling on neuromuscular structure and function in adult mammals are not as well known. In this study, we employed a novel 1NMPP1 sensitive TrkBF616 rat model to evaluate the effect of 14 days inhibition of BDNF/TrkB signaling on the number and morphology of phrenic motor neurons (PhMNs) and diaphragm muscle (DIAm) neurotransmission in adult animals. We hypothesized that reduced BDNF/TrkB signaling results in PhMN loss and impaired DIAm neuromuscular transmission. Adult female and male TrkBF616 rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were labeled via intrapleural injection of Alexa-Fluor647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor488 CTB. After 14 days of treatment, the spinal cord was excised and 100 μm thick spinal sections containing PhMNs were imaged in 3D using two-channel confocal microscopy. Phrenic nerve and DIAm was excised en bloc, and the extent of neuromuscular transmission failure (NMTF) was assessed by comparing DIAm isometric forces evoked by nerve (40 Hz in 330 ms duration trains repeated each s) compared to direct muscle stimulation (40 Hz 330 ms duration train) superimposed every 15 s. Presynaptic (synaptophysin immunoreactivity) and postsynaptic (α-Bungarotoxin) compartments of neuromuscular junctions (NMJs) were labeled and imaged in 3D by confocal microscopy and the extent of pre- and postsynaptic apposition assessed. 1NMPP1-induced BDNF/TrkB inhibition reduced the total number of PhMNs by ~16%, reduced the mean PhMN somal surface areas by ~25%, reduced PhMN dendritic surface area by ~38%, impaired secondary CTB uptake by 2.5-fold and impaired neuromuscular transmission by ~33%, with no overt evidence of NMJ derangement. We conclude that inhibition of BDNF/TrkB signaling in adult TrkBF616 rats is sufficient to lead to PhMN loss, morphological degeneration, deficits in retrograde axonal transport and impaired DIAm neurotransmission. Supported by NIH: R01-AG44615 and R01-HL146114 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chemogenetic inhibition of TrkB signalling reduces phrenic motor neuron survival and size

Brain derived neurotrophic factor (BDNF) signalling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. Here, we employed a novel 1NMPP1 sensitive TrkBF616 rat model to evaluate the effect of 14 days inhibition of TrkB signalling on phrenic motor neurons (PhMNs). Adult female and male TrkBF616 rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were initially labeled via intrapleural injection of Alexa-Fluor-647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal uptake/transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor-488 CTB. After 14 days of treatment, the spinal cord was excised 100 μm thick spinal sections containing PhMNs were imaged using two-channel confocal microscopy. TrkB inhibition reduced the total number of PhMNs by ∼16 %, reduced the mean PhMN somal surface areas by ∼25 %, impaired CTB uptake 2.5-fold and reduced the estimated PhMN dendritic surface area by ∼38 %. We conclude that inhibition of TrkB signalling alone in adult TrkBF616 rats is sufficient to lead to PhMN loss, morphological degeneration and deficits in retrograde axonal uptake/transport.

Composite Fibrin and Carbon Microfibre Implant to Modulate Postraumatic Inflammation after Spinal Cord Injury.

Poor functional recovery after spinal cord injury (SCI) drives the development of novel strategies to manage this devastating condition. We recently showed promising immunomodulatory and pro-regenerative actions of bio-functionalized carbon microfibres (MFs) implanted in a rodent model of SCI. In order to maximize tissue repair while easing MF implantation, we produced a composite implant based on the embedding of several MFs within a fibrin hydrogel. We used intravital imaging of fluorescent reporter mice at the early stages and spinal sections of the same animals 3 months later to characterize the neuroinflammatory response to the implant and its impact on axonal regeneration. Whereas fibrin alone was inert in the first week, its enzymatic degradation drove the chronic activation of microglial cells and axonal degeneration within 3 months. However, the presence of MFs inside the fibrin hydrogel slowed down fibrin degradation and boosted the early recruitment of immune cells. Noteworthy, there was an enhanced contribution of monocyte-derived dendritic cells (moDCs), preceding a faster transition toward an anti-inflammatory environment with increased axonal regeneration over 3 months. The inclusion of MF here ensured the long-term biocompatibility of fibrin hydrogels, which would otherwise preclude successful spinal cord regeneration.

Role of diffusion kurtosis imaging in evaluating microstructural changes in spinal cord of patients with cervical spondylosis.

Analytical cross-sectional study. To study the role of diffusion kurtosis imaging (DKI) in evaluating microstructural changes in patients with cervical spondylosis. Cervical spondylosis is a common progressive degenerative disorder of the spine. Conventional magnetic resonance imaging (MRI) can only detect the changes in the spinal cord once there are visual signal changes; hence, it underestimates the extent of the injury. Newer imaging techniques like Diffusion Tensor and Kurtosis Imaging can evaluate the microstructural changes in cervical spinal cord before the obvious signal changes appear. Conventional MRI, diffusion tensor imaging (DTI), and DKI scans were performed for 90 cervical spondylosis patients on 1.5-T MR Siemens Magnetom aera after obtaining informed consent. Eight patients were excluded due to poor image quality. Fractional anisotropy (FA) colour maps and diffusion kurtosis (DK) maps corresponding to spinal cord cross sections at C2-C3 intervertebral disc level (control) and at the most stenotic levels were obtained. Modified Japanese Orthopaedic Association (mJOA) scoring was used for clinical assessment of the spinal cord function. The changes in DTI and DKI parameters and their correlation with mJOA scores were analysed by SPSS 23 software. In our study, mean FA and mean kurtosis (MK) values at the stenotic level (0.54, 1.02) were significantly lower than values at the non-stenotic segment (0.70, 1.27). The mean diffusivity (MD) value at the stenotic segment (1.25) was significantly higher than in the non-stenotic segment (1.09). We also observed a strong positive correlation between mJOA score and FA and MK values and a negative correlation between mJOA score and MD values, suggesting a correlation of FA, MK, and MD with the clinical severity of the disease. Addition of DTI and DKI sequences helps in early identification of the disease without any additional cost incurred by the patient.

Tocilizumab promotes repair of spinal cord injury by facilitating the restoration of tight junctions between vascular endothelial cells

BackgroundOur previous study demonstrated that M1 macrophages could impair tight junctions (TJs) between vascular endothelial cells by secreting interleukin-6 (IL-6) after spinal cord injury (SCI). Tocilizumab, as a humanized IL-6 receptor (IL-6R) monoclonal antibody approved for the clinic, has been applied in the treatment of neurological diseases in recent years, but the treatment effect of Tocilizumab on the TJs restoration of the blood-spinal cord barrier (BSCB) after SCI remains unclear. This study aimed to explore the effect of Tocilizumab on the restoration of TJs between vascular endothelial cells and axon regeneration after SCI.MethodsIn this study, the mouse complete spinal cord crush injury model was used, and Tocilizumab was continuously injected intrathecally until the day of sample collection. A PBS injection in the same location was included as a control. At 14 days postinjury (dpi) and 28 dpi, spinal cord tissue sections were examined via tissue immunofluorescence. The Basso Mouse Scale (BMS) scores and footprint analysis were used to verify the effect of Tocilizumab on the recovery of motor function in mice after SCI.ResultsWe demonstrated that depletion of macrophages has no effect on axon regeneration and motor functional recovery after SCI, but mice subjected to Tocilizumab showed a significant increase in axon regeneration and a better recovery in motor function during the chronic phase after SCI. Moreover, our study demonstrated that at 14 and 28 dpi, the expression of claudin-5 (CLDN5) and zonula occludens-1 (ZO-1) between vascular endothelial cells was significantly increased and the leakage of BSCB was significantly reduced in the injured core after daily intrathecal injection of Tocilizumab. Notably, the infiltration of CD68+ macrophages/microglia and the formation of fibrotic scar were decreased in the injured core after Tocilizumab treatment. Tocilizumab treatment could effectively reduce the IL-6 expression in macrophages in the injured core.ConclusionThe application of Tocilizumab to antagonize IL-6R can effectively reduce the expression of IL-6 in macrophages and facilitate TJs restoration of the BSCB, which is beneficial for axon regeneration and motor functional recovery after SCI. Hence, Tocilizumab treatment is a potential therapeutic strategy for SCI.

MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue.

Gangliosides are acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the analysis of isomeric lipid structures in situ. Here, we demonstrate the gas-phase separation and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a standard mixture of both isomers, a biological extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were determined in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.

Ral GTPases are critical regulators of spinal cord myelination and homeostasis.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

Effect of Intravenous Ketamine on Hypocranial Pressure Symptoms in Patients with Spinal Anesthetic Cesarean Sections: A Systematic Review and Meta-Analysis.

Background: Pregnant women are more likely to suffer post-puncture symptoms such as headaches and nausea due to the outflow of cerebrospinal fluid after spinal anesthesia. Because ketamine has the effect of raising intracranial pressure, it may be able to improve the symptoms of perioperative hypocranial pressure and effectively prevent the occurrence of hypocranial pressure-related side effects. Method: Keywords such as ketamine, cesarean section, and spinal anesthesia were searched in databases including Medline, Embase, Web of Science, and Cochrane from 1976 to 2021. Thirteen randomized controlled trials were selected for the meta-analysis. Results: A total of 12 randomized trials involving 2099 participants fulfilled the inclusion criteria. There was no significant association between ketamine and the risk of headaches compared to the placebo (RR = 1.12; 95% CI: 0.53, 2.35; p = 0.77; I² = 62%). There was no significant association between ketamine and nausea compared to the placebo (RR = 0.66; 95% CI: 0.40, 1.09; p = 0.10; I² = 57%). No significant associations between ketamine or the placebo and vomiting were found (RR = 0.94; 95% CI: 0.53, 1.67; p = 0.83; I² = 72%). Conclusion: Intravenous ketamine does not improve the symptoms caused by low intracranial pressure after spinal anesthesia in patients undergoing cesarean section.

Effects of C5a and Receptor CD88 on Glutamate and N-Methyl-D-Aspartic Acid Receptor Expression in the Mouse Model of Optic Neuromyelitis

Objective To analyze the role of C5a, C5a receptor (CD88), glutamic acid, and N-methyl-D-aspartic acid receptors (NMDAR1 and NMDAR2B) in the onset of neuromyelitis optica (NMO) disease in mice. Method To select C57BL/6 wild-type (WT) mice and C5a receptor gene knockout (C5aR-/-) mice, use NMO-IgG and hemolytic complement to intervene in spinal cord tissue sections and optic nerves to establish an NMO model in vitro. The experiment was carried out with five groups (control group, WT group, C5aR-/- group, C5a group, and C5a+C5aRA group), with six mice in each group. The differences of American spinal cord injury (ASIA) motor scores were compared among all groups. The expressions of aquaporin (AQP4), glial fibrillary acidic protein (GFAP), NMDAR1, and NMDAR2B in spinal cord and optic nerve tissues were detected. The difference of glutamic acid (Glu) concentrations in culture solutions of the spinal cord and optic nerves was compared. Result The ASIA motor score of the control group was significantly lower than that of the other four groups. The C5a-/- group was significantly higher than the WT group. The C5a+C5aRA group was significantly higher than the C5a group in terms of ASIA motor score. In the control group, AQP4 and GFAP showed expression loss. The C5aR-/- group's loss rate was significantly higher than that of the WT group. The loss rate of the C5a+C5aRA group was significantly higher than that of the C5a group. In the control group, the protein expressions of NMDAR1 and NMDAR2B were significantly lower than that of the other four groups. The C5aR-/- group was significantly higher than the WT group. The C5a+C5aRA group was significantly higher than the C5a group in protein expression. In the control group, the concentration of Glu in the C5aR-/- group was significantly higher than that in the WT group, and the C5a group was significantly lower than the C5a+C5aRA group. Conclusion The deletion of the C5a receptor promotes NMDAR activity, which affects the toxic excitatory effect of NMDAR in NMO and regulates the neurotoxic effect of glutamic acid, thus participating in the pathogenesis of NMO.

Three-Dimensional Modeling and In Silico Kinematic Evaluation of Interspinous Ligament Desmotomy in Horses.

Background: Interspinous ligament desmotomy (ISLD) has been shown to improve the comfort of horses diagnosed with overriding dorsal spinous processes (DSP), but its effects on spine mobility are unknown. Objective: To objectively quantify the change in mobility of thoracic vertebrae following ISLD using CT and medical modeling software. Study design: Prospective cadaveric manipulation of seven equine thoracolumbar spines collected from T11-L1. Methods: Spines were collected from T11-L1 with the musculature intact. Flexion and extension phases were achieved with a ratchet device calibrated to 2000N. Bone volume CT scans were performed in resting, flexion, and extension phase preoperatively. Interspinous ligament desmotomy was performed at each intervertebral space (n = 8), and bone volume CT imaging was repeated for each phase. The spinal sections were individually segmented and imported into medical software for kinematic evaluation. T11 of each phase were superimposed, the distance between each dorsal spinous process, the total length of the spine, and the maximal excursion of the first lumbar vertebra along with angular rotational information were recorded. Results: The mean distance between each dorsal spinous process increased by 5.6 ± 4.9 mm, representing a 24 ± 21% increase in mobility following ISLD. L1 dorsoventral excursion increased by 15.3 ± 11.9 mm, craniocaudal motion increased by 6.9 ± 6.5 mm representing a 47 ± 36.5% and 14.5 ± 13.7% increase, respectively. The rotation of L1 about the mediolateral axis increased by 6.5° post-ISLD. Conclusion and Clinical Relevance: ISLD increases dorsoventral, craniocaudal, and rotational motion of the equine spine. The computer modeling methodology used here could be used to evaluate multiplanar spinal kinematics between treatments.

Massive Sequential Spatial Transcriptional RNA Sequencing by Capturing Mouse Spinal Cord Tissue Sections

Massive Sequential Spatial Transcriptional RNA Sequencing by Capturing Mouse Spinal Cord Tissue Sections

Selected Thiadiazine-Thione Derivatives Attenuate Neuroinflammation in Chronic Constriction Injury Induced Neuropathy.

Neuropathic pain refers to a lesion or disease of peripheral and/or central somatosensory neurons and is an important body response to actual or potential nerve damage. We investigated the therapeutic potential of two thiadiazine-thione [TDT] derivatives, 2-(5-propyl-6-thioxo-1, 3, 5-thiadiazinan-3-yl) acetic acid [TDT1] and 2-(5-propyl-2-thioxo-1, 3, 5-thiadiazinan-3-yl) acetic acid [TDT2] against CCI (chronic constriction injury)-induced neuroinflammation and neuropathic pain. Mice were used for assessment of acute toxicity of TDT derivatives and no major toxic/bizarre responses were observed. Anti-inflammatory activity was assessed using the carrageenan test, and both TDT1 and TDT2 significantly reduced carrageenan-induced inflammation. We also used rats for the induction of CCI and performed allodynia and hyperalgesia-related behavioral tests followed by biochemical and morphological analysis using RT-qPCR, immunoblotting, immunohistochemistry and immunofluorescence. Our findings revealed that CCI induced clear-cut allodynia and hyperalgesia which was reversed by TDT1 and TDT2. To determine the function of TDT1 and TDT2 in glia-mediated neuroinflammation, Iba1 mRNA and protein levels were measured in spinal cord tissue sections from various experimental groups. Interestingly, TDT1 and TDT2 substantially reduced the mRNA expression and protein level of Iba1, implying that TDT1 and TDT2 may mitigate CCI-induced astrogliosis. In silico molecular docking studies predicted that both compounds had an effective binding affinity for TNF-α and COX-2. The compounds interactions with the proteins were dominated by both hydrogen bonding and van der Waals interactions. Overall, these results suggest that TDT1 and TDT2 exert their neuroprotective and analgesic potentials by ameliorating CCI-induced allodynia, hyperalgesia, neuroinflammation and neuronal degeneration in a dose-dependent manner.

Spinal Cord Stimulation Reduces Ventricular Arrhythmias by Attenuating Reactive Gliosis and Activation of Spinal Interneurons.

This study investigated spinal cord neuronal and glial cell activation during cardiac ischemia-reperfusion (IR)-triggered ventricular arrhythmias and neuromodulation therapy by spinal cord stimulation (SCS). Myocardial ischemia induces changes in cardiospinal neural networks leading to sudden cardiac death. Neuromodulation with SCS decreases cardiac sympathoexcitation; however, the molecular mechanisms remain unknown. Yorkshire pigs (n=16) were randomized to Control, IR, or IR+SCS groups. A 4-pole SCS lead was placed in the T1-T4 epidural space with stimulation for 30minutes before IR (50Hz, 0.4-ms duration, 90% motor threshold). Cardiac electrophysiological mapping and Ventricular Arrhythmia Score (VAS) were recorded. Immunohistochemistry of thoracic spinal sections was used to map and identify Fos-positive neuronal and glial cell types during IR with and without SCS. IR increased cardiac sympathoexcitation and arrhythmias (VAS=6.2 ± 0.9) that were attenuated in IR+ SCS (VAS=2.8 ± 0.5; P=0.017). IR increased spinal cellular Fos expression (#Fos+ cells Control=23 ± 2 vs IR=88 ± 5; P< 0.0001) in T1-T4, with the greatest increase localized to T3, and the greatest %Fos+ cells being microglia and astrocytes. Fos expression was attenuated by IR+ SCS (62 ± 4; P< 0.01), primarily though a reduction in Fos+ microglia and astrocytes, as SCS also led to increase in Fos+ neurons in deep dorsal laminae. In a porcine model, cardiac IR was associated with astrocyte and microglial cell activation. Our results suggest that preemptive thoracic SCS decreased IR-induced cardiac sympathoexcitation and ventricular arrhythmias through attenuation of reactive gliosis and activation of inhibitory interneurons in the dorsal horn of spinal cord.

Model of spinal cord lateral hemi-excision at the lower thoracic level for the tasks of reconstructive and experimental neurosurgery

Purpose. To test the model of spinal cord lateral hemiexcision in young rats. Materials and methods. Animals ‒ male rats (age about 1 month, body weight about 50 g, inbred derivatives of the Wistar line); the number of experimental groups is: 1) lateral spinal cord hemisection at the level of segments about T12–T13 (Sect; n=11); 2) lateral spinal cord hemiexcision about 1 mm long at the similar level (Exc; n=8). Assessment of motor Function Index (FI) and the Spasticity Index (SI) of the paretic hindlimb was carried out using the Basso–Beattie–Bresnahan (BBB) scale and Ashworth scale, respectively, in our technical modifications. The non-inclusion criteria: the BBB score above 9 points of FI for the ipsilateral hindlimb in a week after injury ‒and / or BBB score less than or equal to 14 points of FI of the contralateral hindlimb during a long follow-up period (in general, 2 animals in the Sect group, 3 animals ‒ in the Exc group). Asymptotic differences in the timing of testing between subgroups and groups were revealed during the first three weeks of follow-up. Interpolation reproduction of individual values of FI and SI was used in exceptional cases. The total follow-up period was 5 months. Statistical analysis was performed using the Mann-Whitney U Test, Wilcoxon Matched Pairs Test, Spearman’s Rank Order Correlation. For pathomorphological study, the method of silver impregnation of the spinal cord longitudinal sections of the Exc group animals obtained in 5 months after the simulation of injury was used. Results. One week after injury, the FI in the Sect group was 5.9±1.1 according to BBB points, a statistically significant increase in the FI lasted for the first 3 weeks (p&lt;0.05; Wilcoxon Matched Pairs Test), the FI maximum in the group was 10.1±1.1 BBB points, and the FI value at the end of the study was 9.5±1.0 BBB points. In the Exc group, 1 week after injury, the FI was 0.9±0.5 BBB points, during the next week it reached the actual maximum (1.9±0.7 BBB points), by the end of the 5th month it significantly decreased to 0.8±0.3 BBB points (p&lt;0.05; Wilcoxon Matched Pairs Test). One week after injury, the SI value in the Sect group was 0.3±0.1 points according to Ashworth scale, in the Exc group ‒ 0.7±0.1 Ashworth points, a significant increase (p&lt;0.05; Wilcoxon Matched Pairs Test) in SI in the Sect group was noted during the 2nd week and the 2nd month, in the Exc group ‒ during the 2nd and 6th week, as well as the 3rd and 5th month after injury. The SI final and maximal score for the Sect group was 0.8±0.2 Ashworth points, and for the Exc group ‒ 3.6±0.3 Ashworth points. For both groups, there was no correlation between the mean FI value and a significant positive correlation of the mean SI value with the value of the follow-up period (p&lt;0.05; Spearman’s Rank Order Correlation), as well as the absence of correlation between the mean FI and SI values during the total follow-up period. A significant negative correlation (p&lt;0.05; Spearman’s Rank Order Correlation) between individual FI and SI values was found after 1 and 4 weeks, 3 and 5 months after the injury for the Sect group, as well as after 5, 7, 8 weeks and after 3 and 4 months for the Exc group. At all periods of follow-up, the difference in both FI and SI mean values of both groups was significant (p&lt;0.05; the Mann-Whitney U Test). Conclusions. The studied model of spinal cord injury in young rats is the means of choice for testing solid neural transplantation means for the spinal cord injury restorative treatment. The interpretation of data obtained using the BBB scale on models of lateral half spinal cord injury should be carried out with caution, and the methodology for verifying spasticity requires significant improvement. We recommend that the optimal timing for the FI and SI monitoring after lateral half spinal cord injury is 7 days, 14 days and in 1, 2, 3, 4, 5, 6, and 7 months.

Piperine Improves Experimental Autoimmune Encephalomyelitis (EAE) in Lewis Rats Through its Neuroprotective, Anti-inflammatory, and Antioxidant Effects.

Inflammation, demyelination, glial activation, and oxidative damage are the most pathological hallmarks of multiple sclerosis (MS). Piperine, a main bioactive alkaloid of black pepper, possesses antioxidant, anti-inflammatory, and neuroprotective properties whose therapeutic potential has been less studied in the experimental autoimmune encephalomyelitis (EAE) models. In this study, the efficiency of piperine on progression of EAE model and myelin repair mechanisms was investigated. EAE was induced in female Lewis rats and piperine and its vehicle were daily administrated intraperitoneally from day 8 to 29 post immunization. We found that piperine alleviated neurological deficits and EAE disease progression. Luxol fast blue and H&E staining and immunostaining of lumbar spinal cord cross sections confirmed that piperine significantly reduced the extent of demyelination, inflammation, immune cell infiltration, microglia, and astrocyte activation. Gene expression analysis in lumbar spinal cord showed that piperine treatment decreased the level of pro-inflammatory cytokines (TNF-α, IL-1β) and iNOS and enhanced IL-10, Nrf2, HO-1, and MBP expressions. Piperine supplementation also enhanced the total antioxidant capacity (FRAP) and reduced the level of oxidative stress marker (MDA) in the CNS of EAE rats. Finally, we found that piperine has anti-apoptotic and neuroprotective effect in EAE through reducing caspase-3 (apoptosis marker) and enhancing BDNF and NeuN expressing cells. This study strongly indicates that piperine has a beneficial effect on the EAE progression and could be considered as a potential therapeutic target for MS treatment. Upcoming clinical trials will provide a deeper understanding of piperine's role for the treatment of the MS.

A comparison, using X-ray micro-computed tomography, of the architecture of cancellous bone from the cervical, thoracic and lumbar spine using 240 vertebral bodies from 10 body donors.

The vertebral trabecular bone has a complex three-dimensional microstructure with an inhomogeneous morphology. Correct identification and assessment of the weakest segments of the cancellous bone may lead to better prediction of fracture risk. The aim of this study was to compare cancellous bone from 240 vertebrae of the cervical, thoracic and lumbar spine of ten body donors with osteoporosis in regard to bone volume fraction (BVF), trabecular thickness, separation, trabecular number and degree of anisotropy, to ascertain why cervical vertebrae rarely fracture, even with severe osteoporosis. Samples were obtained from all vertebrae with a Jamshidi needle (8 Gauge). The investigations were performed with a micro-computed tomography (micro-CT) device (SKYSCAN 1172, RJL Micro & Analytic GmbH, Karlsdorf-Neuthard, Germany). Existing vertebral fractures and the bone mineral density of the lumbar spine were assessed with quantitative CT. Regarding the micro-CT parameters, statistically significant differences were observed between the various sections of the spine. We found a higher BVF, trabecular number and trabecular thickness, as well as a lower trabecular separation of the cervical vertebrae compared to other vertebrae. In addition, the degree of anisotropy in the cervical spine is lower than in the other spinal column sections. These results are age and sex dependent. Thus, the cervical spine has special structural features, whose causes must be determined in further investigations.