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Neuroinflammation is a major contributor to neuropathic pain. Receptor interacting serine/threonine kinase 3 (RIP3) senses cellular stress, promotes inflammatory responses and activates c-Jun N-terminal kinase (JNK) signaling. Here, we assessed the involvement of RIP3-induced JNK signaling in chronic constriction injury (CCI)-induced neuropathic pain. We found that RIP3 inhibitors (GSK’872) and JNK inhibitors (SP600125) not only alleviated the radiant heat response and mechanical allodynia in CCI rats, but also reduced inflammatory factor levels in the lumbar spinal cord. CCI surgery induced RIP3 mRNA and protein expression in the spinal cord. GSK’872 treatment after CCI surgery reduced RIP3 and phosphorylated (p)-JNK expression in the spinal cord, whereas SP600125 treatment after CCI surgery had almost no effect on RIP3. Sinomenine treatment reduced RIP3, p-JNK and c-Fos levels in the spinal cords of CCI rats. These data demonstrated that RIP3 inhibition (particularly via sinomenine treatment) alleviates neuropathic pain by suppressing JNK signaling. RIP3 could thus be a new treatment target in patients with neuropathic pain.

Background Burgeoning evidence highlights seminal roles for microglia in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The receptor for advanced glycation end products (RAGE) binds ligands relevant to ALS that accumulate in the diseased spinal cord and RAGE has been previously implicated in the progression of ALS pathology. Methods We generated a novel mouse model to temporally delete Ager from microglia in the murine SOD1G93A model of ALS. Microglia Ager deficient SOD1G93A mice and controls were examined for changes in survival, motor function, gliosis, motor neuron numbers, and transcriptomic analyses of lumbar spinal cord. Furthermore, we examined bulk-RNA-sequencing transcriptomic analyses of human ALS cervical spinal cord. Results Transcriptomic analysis of human cervical spinal cord reveals a range of AGER expression in ALS patients, which was negatively correlated with age at disease onset and death or tracheostomy. The degree of AGER expression related to differential expression of pathways involved in extracellular matrix, lipid metabolism, and intercellular communication. Microglia display increased RAGE immunoreactivity in the spinal cords of high AGER expressing patients and in the SOD1G93A murine model of ALS vs. respective controls. We demonstrate that microglia Ager deletion at the age of symptomatic onset, day 90, in SOD1G93A mice extends survival in male but not female mice. Critically, many of the pathways identified in human ALS patients that accompanied increased AGER expression were significantly ameliorated by microglia Ager deletion in male SOD1G93A mice. Conclusions Our results indicate that microglia RAGE disrupts communications with cell types including astrocytes and neurons, intercellular communication pathways that divert microglia from a homeostatic to an inflammatory and tissue-injurious program. In totality, microglia RAGE contributes to the progression of SOD1G93A murine pathology in male mice and may be relevant in human disease.

Spinal cord injury (SCI) can lead to increased phosphorylation of p38 in spinal cord microglia. This is one of the main causes for the development of persistent pain. Recently, we reported our study on the activation of p38 mitogen-activated protein kinases (MAPK) in spinal microglia, which has been considered the key molecule for the onset and maintenance of neuropathic pain after peripheral nerve injury, using a rat model. We also reported that the RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) pathway mediates p38 activation in spinal microglia in peripheral nerve injury. But the precise mechanisms of neuropathic pain induced by SCI are still unclear.This study aimed to examine the activation of microglia and the p38 MAPK expression in the lumbar spinal cord after thoracic SCI in rats, and the correlation to the therapeutic effect of ROCK inhibitor ripasudil in rats with SCI.Male Sprague-Dawley rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon impactor device. SCI rats received ROCK inhibitor ripasudil (24 nmol/day or 240 nmol/day) from just before SCI to 3 days after SCI.The mechanical threshold in the rat's hind paws was measured over four weeks. Morphology of microglia and phosphorylation of p38 (p-p38) in the lumbar spinal cord and were analyzed using immunohistochemistry.The p-p38 positive cell and Iba1 (a maker of microglia) positive area were significantly increased at the lumbar spinal dorsal horn (L4-5) 3 days and 7 days after SCI compared with the sham-control (p.05), whereas phosphorylated p38 was co-localized with microglia. Three days after SCI, the intensity of phosphorylated p38 and Iba1 immunoreactive cells in the dorsal horn was significantly lower in the ripasudil treated groups than in the saline group. However, administration of ROCK inhibitor did not affect the numbers of microglia. Moreover, the withdrawal threshold of the ripasudil-treated rats was significantly higher than that of the saline-injected rats on 14 days and 28 days after SCI.Our results suggest that activation of ROCK in spinal cord microglia is likely to have an important role in the activation of p38 MAPK, which has been considered as a key molecule that switches on neuropathic pain after SCI. Inhibition of ROCK signaling may offer a means in developing a novel neuropathic pain treatment after SCI. It may help patients with neuropathic pain after SCI.The findings in the present study regarding intracellular mechanisms suggest that modulation of ROCK signaling may be a focus for novel treatment for neuropathic pain after SCI.

Objectives The burden of pain after spinal cord injury (SCI), which may occur above, at, or below injury level, is high worldwide. Spinal cord stimulation (SCS) is an important neuromodulation pain therapy, but its efficacy in SCI pain remains unclear. In SCI rats, we tested whether conventional SCS (50 Hz, 80% motor threshold [MoT]) and 1200 Hz, low-intensity SCS (40% MoT) inhibit hind paw mechanical hypersensitivity, and whether conventional SCS attenuates evoked responses of wide-dynamic range (WDR) neurons in lumbar spinal cord. Materials and methods Male rats underwent a moderate contusive injury at the T9 vertebral level. Six to eight weeks later, SCS or sham stimulation (120 min, n = 10) was delivered through epidural miniature electrodes placed at upper-lumbar spinal cord, with using a crossover design. Mechanical hypersensitivity was examined in awake rats by measuring paw withdrawal threshold (PWT) to stimulation with von Frey filaments. WDR neurons were recorded with in vivo electrophysiologic methods in a separate study of anesthetized rats. Results Both conventional SCS and 1200 Hz SCS increased PWTs from prestimulation level in SCI rats, but the effects were modest and short-lived. Sham SCS was not effective. Conventional SCS (10 min) at an intensity that evokes the peak Aα/β waveform of sciatic compound action potential did not inhibit WDR neuronal responses (n = 19) to graded or repeated electrical stimulation that induces windup. Conclusions Conventional SCS and 1200 Hz, low-intensity SCS modestly attenuated below-level mechanical hypersensitivity after SCI. Inhibition of WDR neurons was not associated with pain inhibition from conventional SCS.

Mangosteen fruit has been used for various disorders, including pain. The effects of alpha-mangostin, the main component of mangosteen, on the neuropathic pain caused by chronic constriction injury (CCI) were evaluated in rats. In treatment groups, alpha-mangostin (10, 50, 100 mg/kg/day, i.p.) was administered from Day 0, the day of surgery, for 14 days. The degree of heat hyperalgesia, cold, and mechanical allodynia was assessed on Days 0, 3, 5, 7, 10, and 14. The lumbar spinal cord levels of MDA, GSH, inflammatory markers (TLR-4, TNF-α, MMP2, COX2, IL-1β, iNOS, and NO), apoptotic markers (Bcl-2, Bax, and caspase-3) were measured by western blot on Days 7 and 14. Rats in the CCI group showed thermal hyperalgesia, cold, and mechanical allodynia on Days 3-14. All concentrations of alpha-mangostin alleviated CCI-induced behavioral alterations. MDA level augmented and GSH level decreased in the CCI group and alpha-mangostin (50, 100 mg/kg) reversed the alterations. An enhancement in the levels of all inflammatory markers, Bax, and caspase-3 was shown on Days 7 and 14, which was controlled by alpha-mangostin (50 mg/kg). The detected antinociceptive effects of alpha-mangostin may be mediated through antioxidant, anti-inflammatory, and antiapoptotic properties.

Axonal dystrophy (AD) is a common age-related neurohistological finding in vertebrates that can be congenital or induced by xenobiotics, vitamin E deficiency, or trauma/compression. To understand the incidence and location of AD as a background finding in Beagle dogs used in routine toxicity studies, we examined central nervous system (CNS) and selected peripheral nervous system (PNS) tissues in twenty 18- to 24-month-old and ten 4- to 5-year-old control males and females. Both sexes were equally affected. The cuneate, gracile, and cochlear nuclei and the cerebellar white matter (rostral vermis) were the most common locations for AD. Incidence of AD increased with age in the cuneate nucleus, cerebellar white matter (rostral vermis), trigeminal nuclei/tracts, and lumbar spinal cord. Axonal dystrophy in the CNS was not accompanied by neuronal degeneration/necrosis, nerve fiber degeneration, and/or glial reaction. Axonal dystrophy was not observed in the PNS (sciatic nerve, vagus nerve branches, or gastrointestinal mural autonomic plexuses).

Wallerian degeneration is defined as axonal fiber and myelin sheath degeneration that affects myelinated axons within the peripheral or central nervous system. Wallerian degeneration or anterograde axonal degeneration before myelination is rarely reported. Involvement of both corticospinal tracts (CSTs) is rarely documented in the literature. We present the postmortem neuropathologic findings of a 1-week-old male neonate born at 23 weeks of gestation with bilateral CST degeneration extending from the posterior limb of the internal capsule through the brainstem into the lumbar spinal cord. Abundant CD68- and CD163-positive macrophages were the prominent histopathology in both CSTs. The cerebrum, brainstem, and spinal cord were unmyelinated, as expected. In contrast, the spinal nerve roots demonstrated early myelination. This case illustrates that Wallerian degeneration occurs in unmyelinated axis cylinders.

Painful diabetic neuropathy (PDN) is a diabetes mellitus complication. Unfortunately, the mechanisms underlying PDN are still poorly understood. Adenosine triphosphate (ATP)-gated P2X7 receptor (P2X7R) plays a pivotal role in non-diabetic neuropathic pain, but little is known about its effects on streptozotocin (STZ)-induced peripheral neuropathy. Here, we explored whether spinal cord P2X7R was correlated with the generation of mechanical allodynia (MA) in STZ-induced type 1 diabetic neuropathy in mice. MA was assessed by measuring paw withdrawal thresholds and western blotting. Immunohistochemistry was applied to analyze the protein expression levels and localization of P2X7R. STZ-induced mice expressed increased P2X7R in the dorsal horn of the lumbar spinal cord during MA. Mice injected intrathecally with a selective antagonist of P2X7R and P2X7R knockout (KO) mice both presented attenuated progression of MA. Double-immunofluorescent labeling demonstrated that P2X7R-positive cells were mostly co-expressed with Iba1 (a microglia marker). Our results suggest that P2X7R plays an important role in the development of MA and could be used as a cellular target for treating PDN.

This study aimed to reveal functional and morphological changes in the corticospinal tract, a pathway shown to be susceptible to diabetes. Type 1 diabetes was induced in 13-week-old male Wistar rats administered streptozotocin. Twenty-three weeks after streptozotocin injection, diabetic animals and age-matched control animals were used to demonstrate the conduction velocity of the corticospinal tract. Other animals were used for morphometric analyses of the base of the dorsal funiculus of the corticospinal tract in the spinal cord using both optical and electron microscopy. The conduction velocity of the corticospinal tract decreased in the lumbar spinal cord in the diabetic animal, although it did not decrease in the cervical spinal cord. Furthermore, atrophy of the fibers of the base of the dorsal funiculus was observed along their entire length, with an increase in the g-ratio in the lumbar spinal cord in the diabetic animal. This study indicates that the corticospinal tract fibers projecting to the lumbar spinal cord experience a decrease in conduction velocity at the lumbar spinal cord of these axons in diabetic animals, likely caused by a combination of axonal atrophy and an increased g-ratio due to thinning of the myelin sheath.

Male sexual function in mammals is controlled by the brain neural circuits and the spinal cord centers located in the lamina X of the lumbar spinal cord (L3–L4). Recently, we reported that hypothalamic oxytocin neurons project to the lumbar spinal cord to activate the neurons located in the dorsal lamina X of the lumbar spinal cord (dXL) via oxytocin receptors, thereby facilitating male sexual activity. Sexual experiences can influence male sexual activity in rats. However, how this experience affects the brain–spinal cord neural circuits underlying male sexual activity remains unknown. Focusing on dXL neurons that are innervated by hypothalamic oxytocinergic neurons controlling male sexual function, we examined whether sexual experience affects such neural circuits. We found that &gt, 50% of dXL neurons were activated in the first ejaculation group and ~30% in the control and intromission groups in sexually naïve males. In contrast, in sexually experienced males, ~50% of dXL neurons were activated in both the intromission and ejaculation groups, compared to ~30% in the control group. Furthermore, sexual experience induced expressions of gastrin-releasing peptide and oxytocin receptors in the lumbar spinal cord. This is the first demonstration of the effects of sexual experience on molecular expressions in the neural circuits controlling male sexual activity in the spinal cord.

Spinally-administered local anesthetics provide effective perioperative anesthesia and/or analgesia for children of all ages. New preparations and drugs require preclinical safety testing in developmental models. We evaluated age-dependent efficacy and safety following 1% preservative-free 2-chloroprocaine (2-CP) in juvenile Sprague-Dawley rats. Percutaneous lumbar intrathecal 2-CP was administered at postnatal day (P)7, 14 or 21. Mechanical withdrawal threshold pre- and post-injection evaluated the degree and duration of sensory block, compared to intrathecal saline and naive controls. Tissue analyses one- or seven-days following injection included histopathology of spinal cord, cauda equina and brain sections, and quantification of neuronal apoptosis and glial reactivity in lumbar spinal cord. Following intrathecal 2-CP or saline at P7, outcomes assessed between P30 and P72 included: spinal reflex sensitivity (hindlimb thermal latency, mechanical threshold); social approach (novel rat versus object); locomotor activity and anxiety (open field with brightly-lit center); exploratory behavior (rearings, holepoking); sensorimotor gating (acoustic startle, prepulse inhibition); and learning (Morris Water Maze). Maximum tolerated doses of intrathecal 2-CP varied with age (1.0 μL/g at P7, 0.75 μL/g at P14, 0.5 μL/g at P21) and produced motor and sensory block for 10-15 minutes. Tissue analyses found no significant differences across intrathecal 2-CP, saline or naive groups. Adult behavioral measures showed expected sex-dependent differences, that did not differ between 2-CP and saline groups. Single maximum tolerated in vivo doses of intrathecal 2-CP produced reversible spinal anesthesia in juvenile rodents without detectable evidence of developmental neurotoxicity. Current results cannot be extrapolated to repeated dosing or prolonged infusion.

The pancreatic peptide, Amylin (AMY), reportedly affects nociception in rodents. Here, we investigated the potential effect of AMY on the tolerance to morphine and on the expression of BDNF at both levels of protein and RNA in the lumbar spinal cord of morphine tolerant rats. Animals in both groups of control and test received a single daily dose of intrathecal (i.t.) morphine for 10 days. Rats in the test group received AMY (1, 10 and 60 pmoles) in addition to morphine from days 6 to10. Morphine tolerance was established at day 5. AMY alone showed enduring antinociceptive effects for 10 days. Real-Time PCR, western blotting and ELISA were used respectively to assess levels of BDNF transcripts and their encoded proteins. Rats tolerant to i.t. morphine showed increased expression of exons I, IV, and IX of the BDNF gene, and had elevated levels of pro-BDNF and BDNF protein in their lumbar spinal cord. AMY, when co-administered with morphine from days 6 to 10, reversed morphine tolerance and adversely affected the morphine-induced expression of the BDNF gene at both levels of protein and mRNAs containing exons I, IV and IX. AMY alone increased levels of exons I and IV transcripts. Levels of pro-BDNF and BDNF proteins remained unchanged in the lumbar spinal cord of rats treated by AMY alone. These results suggest that i.t. AMY not only abolished morphine tolerance, but also reduced the morphine induced increase in the expression of both BDNF transcripts and protein in the lumbar spinal cord.

Aims Neuroinflammatory changes in the central nervous system are widely involved in the initiation and maintenance of neuropathic pain after peripheral nerve injury. The present study investigated how losartan treatment may affect the development of neuropathic pain and neuroinflammation. Main methods The effect of losartan treatment on the development of peripheral neuropathy was studied in L5 spinal nerve ligation (SNL) model in rats with systemic (100 mg/kg) or intrathecal (10 μl/ 20 μM solution) application of losartan. Electronic von Frey filament and plantar test were used to determine pain thresholds to mechanical and thermal stimulations. At the 7th post-operative day, CD68-positive cells in DRG and dorsal roots were quantified by immunohistochemistry and western blot analyses were used to compare the expression levels of neuroinflammatory markers in lumbar spinal cord (SC). Key findings Our data confirmed the presence of SNL-evoked heat hyperalgesia and mechanical allodynia. Losartan application blocked the SNL-induced hypersensitivity to thermal stimuli but failed to prevent mechanical allodynia. No significant difference between systemic and i.t. administration of losartan was observed. Immunohistochemistry confirmed the presence of infiltrated macrophages in the ipsilateral DRG that was significantly attenuated with the losartan treatment. Western blot SC tissue analysis revealed that systemic treatment with losartan prevented SNL-induced upregulation of CCR2, TNFα, TNFR1, and OX42 while its effect on CCL2 and AT1R expression was not significant. Significance Our results show that losartan treatment attenuates neuroinflammation and neuropathic pain after SNL. These effects of losartan represent an interesting direction for the development of novel treatments of peripheral neuropathy.

Background Chemotherapy-induced peripheral neuropathy (CIPN) is a common and intractable complication in chemotherapy-receiving patients. Insulin-like growth factor-1 (IGF-1) is a popular neurotrophin with various functions, such as maintaining neuronal survival and synaptic functioning in the central nervous system. Therefore, we hypothesized that the IGF-1 signaling pathway could be a candidate target for treating CIPN. Methods We established the CIPN model by injecting mice intraperitoneally with oxaliplatin and assessed IGF-1 protein expression, its receptor IGF1R, phospho-IGF1R (p-IGF1R), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and calcitonin gene-related peptide (CGRP) in the lumbar spinal cord with Western blot and immunofluorescence. To examine the effect of IGF-1 signaling on CIPN, we injected mice intrathecally or intraperitoneally with mouse recombinant IGF-1 (rIGF-1). Results IGF-1 protein expression decreased significantly in the spinal cord on D3 and D10 (the 3rd and 10th days after beginning oxaliplatin chemotherapy) and was co-localized with astrocytes primarily in the lumbar spinal cord, whereas IGF1R was predominantly expressed on neurons. Both intrathecally- and intraperitoneally-administered rIGF-1 relieved the chemotherapy-induced pain-like behavior and reduced IL-17A, TNF-α, and CGRP protein expressions in the spinal cord. Conclusion Our results indicate a vital role for IGF-1 signaling in CIPN. Targeting IGF-1 signaling could be a potent therapeutic strategy for treating CIPN in clinical settings.

Loss of motor neurons (MNs) after spinal root injury is a drawback limiting the recovery after palliative surgery by nerve or muscle transfers. Research based on preventing MN death is a hallmark to improve the perspectives of recovery following severe nerve injuries. Sigma-1 receptor (Sig-1R) is a protein highly expressed in MNs, proposed as neuroprotective target for ameliorating MN degenerative conditions. Here, we used a model of L4–L5 rhizotomy in adult mice to induce MN degeneration and to evaluate the neuroprotective role of Sig-1R ligands (PRE-084, SA4503 and BD1063). Lumbar spinal cord was collected at 7, 14, 28 and 42 days post-injury (dpi) for immunohistochemistry, immunofluorescence and Western blot analyses. This proximal axotomy at the immediate postganglionic level resulted in significant death, up to 40% of spinal MNs at 42 days after injury and showed markedly increased glial reactivity. Sig-1R ligands PRE-084, SA4503 and BD1063 reduced MN loss by about 20%, associated to modulation of endoplasmic reticulum stress markers IRE1α and XBP1. These pathways are Sig-1R specific since they were not produced in Sig-1R knockout mice. These findings suggest that Sig-1R is a promising target for the treatment of MN cell death after neural injuries.

Neonatal anoxia is a well-known world health problem which results in neurodevelopmental deficits, such as sensory alterations that are observed in patients with cerebral palsy and autism disorder, for which oxygen deprivation is a risk factor. Nociceptive response, as part of the sensory system, has been reported as altered in these patients. To determine whether neonatal oxygen deprivation alters nociceptive sensitivity and promotes medium- and long-term inflammatory feedback in the central nervous system, Wistar rats of around 30 h old were submitted to anoxia (100% nitrogen flux for 25 min) and evaluated on PND23 (postpartum day) and PND90. The nociceptive response was assessed by mechanical, thermal and tactile tests in the early postnatal and adulthood periods. The lumbar spinal cord (SC -L4-L6) motor neurons (MNs) and the posterior insular cortex neurons were counted and compared with their respective controls after anoxia. In addition, we evaluated the possible effect of anoxia on the expression of astrocytes in the spinal cord at adulthood. The results showed increased nociceptive responses in both males and females submitted to anoxia, although these responses were different according to the nociceptive stimulus. A decrease in MNs in adult anoxiated females and an upregulation of GFAP expression in the SC were observed. In the insular cortex, a decrease in the number of cells of anoxiated males was observed in the neonatal period. Our findings suggest that oxygen-deprived nervous systems in rats may affect their response at the sensorimotor pathways and respective controlling centers with sex differences, which were related to the used stimulus.

BACKGROUND Burn injury (BI) pain consists of inflammatory and neuropathic components and activates microglia. Nicotinic alpha 7 acetylcholine receptors (α7AChRs) expressed in microglia exhibit immunomodulatory activity during agonist stimulation. Efficacy of selective α7AChR agonist GTS-21 to mitigate BI pain and spinal pain-mediators was tested. METHODS Anesthetized rats after hind-paw BI received intraperitoneal GTS-21 or saline daily. Allodynia and hyperalgesia were tested on BI and contralateral paw for 21 days. Another group after BI receiving GTS-21 or saline had lumbar spinal cord segments harvested (day 7 or 14) to quantify spinal inflammatory-pain transducers or microglia activation using fluorescent marker, ionized calcium-binding adaptor protein (Iba1). RESULTS BI significantly decreased allodynia withdrawal threshold from baseline of ~9-10 to ~0.5-1 g, and hyperalgesia latency from ~16-17 to ~5-6 seconds by day 1. Both doses of GTS-21 (4 or 8 mg/kg) mitigated burn-induced allodynia from ~0.5-1 to ~2-3 g threshold (P = .089 and P = .010), and hyperalgesia from ~5-6 to 8-9 seconds (P < .001 and P < .001) by day 1. The GTS-21 group recovered to baseline pain threshold by day 15-17 compared to saline-treated, where the exaggerated nociception persisted beyond 15-17 days. BI significantly (P < .01) increased spinal cord microgliosis (identified by fluorescent Iba1 staining), microglia activation (evidenced by the increased inflammatory cytokine), and pain-transducer (protein and/or messenger RNA [mRNA]) expression (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], nuclear factor-kappa B [NF-κB], interleukin-6 [IL-6], Janus-associated kinase signal transducer and activator of transcription 3 [JAK-STAT3], and/or N-methyl-D-aspartate receptor [NMDAR]). GTS-21 mitigated pain-transducer changes. The α7AChR antagonist methyllycaconitine nullified the beneficial effects of GTS-21 on both increased nociception and pain-biomarker expression. CONCLUSIONS Nonopioid, α7AChR agonist GTS-21 elicits antinociceptive effects at least in part by decreased activation spinal-cord pain-inducers. The α7AChR agonist GTS-21 holds promise as potential therapeutic adjunct to decrease BI pain by attenuating both microglia changes and expression of exaggerated pain transducers.

Clinical management of neuropathic pain is unsatisfactory, mainly due to its resistance to the effects of available analgesics, including opioids. Converging evidence indicates the functional interactions between chemokine and opioid receptors and their influence on nociceptive processes. Recent studies highlight that the CC chemokine receptors type 2 (CCR2) and 5 (CCR5) seem to be of particular interest. Therefore, in this study, we investigated the effects of the dual CCR2/CCR5 antagonist, cenicriviroc, on pain-related behaviors, neuroimmune processes, and the efficacy of opioids in rats after chronic constriction injury (CCI) of the sciatic nerve. To define the mechanisms of action of cenicriviroc, we studied changes in the activation/influx of glial and immune cells and, simultaneously, the expression level of CCR2, CCR5, and important pronociceptive cytokines in the spinal cord and dorsal root ganglia (DRG). We demonstrated that repeated intrathecal injections of cenicriviroc, in a dose-dependent manner, alleviated hypersensitivity to mechanical and thermal stimuli in rats after sciatic nerve injury, as measured by von Frey and cold plate tests. Behavioral effects were associated with the beneficial impact of cenicriviroc on the activation/influx level of C1q/IBA-1-positive cells in the spinal cord and/or DRG and GFAP-positive cells in DRG. In parallel, administration of cenicriviroc decreased the expression of CCR2 in the spinal cord and CCR5 in DRG. Concomitantly, we observed that the level of important pronociceptive factors (e.g., IL-1beta, IL-6, IL-18, and CCL3) were increased in the lumbar spinal cord and/or DRG 7 days following injury, and cenicriviroc was able to prevent these changes. Additionally, repeated administration of this dual CCR2/CCR5 antagonist enhanced the analgesic effects of morphine and buprenorphine in neuropathic rats, which can be associated with the ability of cenicriviroc to prevent nerve injury-induced downregulation of all opioid receptors at the DRG level. Overall, our results suggest that pharmacological modulation based on the simultaneous blockade of CCR2 and CCR5 may serve as an innovative strategy for the treatment of neuropathic pain, as well as in combination with opioids.

Clinically, pain has an uneven incidence throughout lifespan and impacts more on the elderly. In contrast, preclinical models of pathological pain have typically used juvenile or young adult animals to highlight the involvement of glial populations, proinflammatory cytokines, and chemokines in the onset and maintenance of pathological signalling in the spinal dorsal horn. The potential impact of this mismatch is also complicated by the growing appreciation that the aged central nervous system exists in a state of chronic inflammation because of enhanced proinflammatory cytokine/chemokine signalling and glial activation. To address this issue, we investigated the impact of aging on the expression of genes that have been associated with neuropathic pain, glial signalling, neurotransmission and neuroinflammation. We used qRT-PCR to quantify gene expression and focussed on the dorsal horn of the spinal cord as this is an important perturbation site in neuropathic pain. To control for global vs region-specific age-related changes in gene expression, the ventral half of the spinal cord was examined. Our results show that expression of proinflammatory chemokines, pattern recognition receptors, and neurotransmitter system components was significantly altered in aged (24–32 months) versus young mice (2–4 months). Notably, the magnitude and direction of these changes were spinal-cord region dependent. For example, expression of the chemokine, Cxcl13, increased 119-fold in dorsal spinal cord, but only 2-fold in the ventral spinal cord of old versus young mice. Therefore, we propose the dorsal spinal cord of old animals is subject to region-specific alterations that prime circuits for the development of pathological pain, potentially in the absence of the peripheral triggers normally associated with these conditions.

Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the “spinal ejaculation generator (SEG).” We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion—a localized volume transmission—to reach oxytocin receptors on GRP neurons and facilitate male sexual function.

Background: Postoperative pain (PO) is a common form of acute pain. Inadequate PO treatment is an important health problem, as it leads to worse outcomes, such as chronic post-surgical pain. Therefore, it is necessary to acquire new knowledge on PO mechanisms to develop therapeutic options with greater efficacy than those available today and to lower the risk of adverse effects. For this reason, we evaluated the ability of micronized palmitoylethanolamide (PEA-m) to resolve the pain and inflammatory processes activated after incision of the hind paw in an animal model of PO. Methods: The animals were subjected to surgical paw incision and randomized into different groups. PEA-m was administered orally at 10 mg/kg at different time points before or after incision. Results: Our research demonstrated that the pre- and post-treatment with PEA-m reduced the activation of mast cells at the incision site and the expression of its algogenic mediator nerve growth factor (NGF) in the lumbar spinal cord. Furthermore, again at the spinal level, it was able to decrease the activation of phospho-extracellular signal-regulated kinases (p-ERK), ionized calcium binding adaptor molecule 1 (Iba1), glial fibrillary acidic protein (GFAP), and the expression of brain-derived neurotrophic factor (BDNF). PEA-m also reduced the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&kappa, B) spinal pathway, showing a protective effect in a rat model of PO. Conclusion: The results obtained reinforce the idea that PEA-m may be a potential treatment for the control of pain and inflammatory processes associated with PO. In addition, pre- and post-treatment with PEA-m is more effective than treatment alone after the surgery and this limits the time of taking the compound and the abuse of analgesics.

The endocannabinoid system (ECS) is known to modulate not only food intake but also pain, especially via the cannabinoid type 1 receptor (CB1R) expressed throughout the central nervous system and the peripheral tissues. Our previous study demonstrated that fasting produces an analgesic effect in adult male mice, which is reversed by intraperitoneal (i.p.) administration of CB1R antagonist (SR 141716). In the present study, we further examined the effect of CB1R expressed in the peripheral tissues. In the formalin-induced inflammatory pain model, i.p. administration of peripherally restricted CB1R antagonist (AM 6545) reversed fasting-induced analgesia. However, intraplantar administration of SR 141716 did not affect fasting-induced analgesia. Furthermore, mRNA expression of CB1R did not change in the formalin model by fasting in the dorsal root ganglia. The formalin-induced c-Fos expression at the spinal cord level was not affected by fasting, and in vivo recording from the superficial dorsal horn of the lumbar spinal cord revealed that fasting did not affect formalin-induced neural activity, which indicates minimal involvement of the spinal cord in fasting-induced analgesia. Finally, when we performed subdiaphragmatic vagotomy to block the hunger signal from the gastrointestinal (GI) system, AM 6545 did not affect fasting-induced analgesia, but SR 141716 still reversed fasting-induced analgesia. Taken together, our results suggest that both peripheral and central CB1Rs contribute to fasting-induced analgesic effects and the CB1Rs in the GI system which transmit fasting signals to the brain, rather than those in the peripheral sensory neurons, may contribute to fasting-induced analgesic effects.

Objective To explore the potential analgesic effect of melatonin and its underlying molecular mechanisms in a neuropathic pain model induced by spinal nerve ligation (SNL). Methods The experimental animals were divided into different groups including sham, vehicle, melatonin (MT) treatment, caspase-1 inhibitor (VX-765) treatment and MT2 antagonist (4P-PDOT) treatment. On the first three successive postoperative days, rats were intraperitoneally administered with MT, VX-765 or combination of MT and 4P-PDOT. Hyperalgesic behavior after SNL was evaluated using the paw withdrawal threshold (PWT). We then assessed expression of tumor necrosis factor-α (TNF-α), IL-18, interleukin-1β (IL-1β), NLRP3 inflammasome components, and nuclear factor-κB (NF-κB) activation using enzyme-linked immunosorbent assay kits (ELISA), real-time PCR, immunohistochemistry, and western blot, respectively, in spinal cord horn tissues extracted on postoperative day 7. Results The results showed that melatonin treatment alleviated SNL-induced allodynia. We observed an SNL-induced upregulation of TNF-α, IL-18, IL-1β, NLRP3, ASC, cleaved caspase-1, and NF-κB in the lumbar spinal cord horn of rats, which was significantly attenuated by intraperitoneal injection of melatonin or VX-765. Additionally, co-treatment of melatonin and 4P-PDOT abrogated the analgesic and anti-inflammatory effect of melatonin. Conclusion Melatonin had potent analgesic and anti-inflammatory effects in SNL-induced neuropathic pain via NF-κB/NLRP3 inflammasome signaling pathway. Our results therefore suggested that this pathway could represent a novel therapeutic target for the management of neuropathic pain.

Accumulating evidence shows that agents targeting gut dysbiosis are effective for improving symptoms of irritable bowel syndrome (IBS). However, the potential mechanisms remain unclear. In this study we investigated the effects of berberine on the microbiota-gut-brain axis in two rat models of visceral hypersensitivity, i.e., specific pathogen-free SD rats subjected to chronic water avoidance stress (WAS) and treated with berberine (200 mg· kg−1 ·d−1, ig, for 10 days) as well as germ-free (GF) rats subjected to fecal microbiota transplantation (FMT) from a patient with IBS (designated IBS-FMT) and treated with berberine (200 mg· kg−1 ·d−1, ig, for 2 weeks). Before the rats were sacrificed, visceral sensation and depressive behaviors were evaluated. Then colonic tryptase was measured and microglial activation in the dorsal lumbar spinal cord was assessed. The fecal microbiota was profiled using 16S rRNA sequencing, and short chain fatty acids (SCFAs) were measured. We showed that berberine treatment significantly alleviated chronic WAS-induced visceral hypersensitivity and activation of colonic mast cells and microglia in the dorsal lumbar spinal cord. Transfer of fecal samples from berberine-treated stressed donors to GF rats protected against acute WAS. FMT from a patient with IBS induced visceral hypersensitivity and pro-inflammatory phenotype in microglia, while berberine treatment reversed the microglial activation and altered microbial composition and function and SCFA profiles in stools of IBS-FMT rats. We demonstrated that berberine did not directly influence LPS-induced microglial activation in vitro. In both models, several SCFA-producing genera were enriched by berberine treatment, and positively correlated to the morphological parameters of microglia. In conclusion, activation of microglia in the dorsal lumbar spinal cord was involved in the pathogenesis of IBS caused by dysregulation of the microbiota–gut–brain axis, and the berberine-altered gut microbiome mediated the modulatory effects of the agent on microglial activation and visceral hypersensitivity, providing a potential option for the treatment of IBS.

The data obtained by transcriptome analysis of lumbar spinal cord segments, sciatic nerve, and the respiratory diaphragm of the mice performed after a space flight on board Bion-M1 biosatellite were processed by bioinformatic methods aimed at elucidation of the regularities in hypogravity-induced transcriptome changes in various compartments of motor neurons. The study revealed abnormalities of axonal transport in spinal motor neurons provoked by weightlessness. These data agree with the results of electron microscopy examination of the spinal cord in experimental animals. In space group mice sacrificed on the landing day, the content of perinuclear ribosomes in lumbar motoneurons surpassed that in control mice or in the recovery group examined 1 week after the flight. The data corroborate our hypothesis on contribution of axonal transport disturbances into pathogenesis of hypogravity motor syndrome. They can be employed as a launching pad for further study of hypogravity-triggered motor disorder mechanisms in order to elaborate the preventive therapy against the development of hypogravity motor syndrome in space flights.

The use of vaccines containing aluminum (Al) adjuvants is widespread in ovine production. Al adjuvants induce an effective immune-response but lead to the formation of post-vaccination granulomas from which Al can disseminate. This work aims to study the accumulation of Al in the central nervous system of sheep subcutaneously inoculated with Al-hydroxide containing products. Lumbar spinal cord and parietal lobe from 21 animals inoculated with 19 doses of Vaccine (n = 7), Adjuvant-only (n = 7) or phosphate-buffered saline as Control (n = 7) were analyzed with transversely heated graphite furnace atomic absorption spectroscopy and lumogallion staining for Al analytical measurements and Al tisular localization respectively. In the lumbar spinal cord, Al median content was higher in both the Adjuvant-only and Vaccine group (p = .001) compared with the Control group. Animals of the Adjuvant-only group showed the higher individual measurements in the lumbar spinal cord (14.36 μg/g and 7.83 μg/g). In the parietal lobe, Al median content tended to be higher in the Adjuvant-only group compared with Control group (p = .074). Except for three replicates of the Adjuvant-only group, Al content was always below 1 μg/g. In the lumbar spinal cord, lumogallion-reactive Al deposits were more abundant in the gray matter than in the white matter in both Vaccine (p = .034) and Adjuvant-only groups (p = .017) and Al deposits were mostly associated with glial-like cells (p = .042). In the parietal lobe, few Al deposits, which were sometimes related to blood vessels, were found. In sheep, Al-hydroxide adjuvants inoculated in the subcutaneous tissue selectively accumulate in the lumbar spinal cord., RM is a PhD student funded by the Department of Innovation, Research and University of Aragon, Spain. JA and ARL are PhD students funded by the Spanish Ministry of Science, Innovation and Universities (formerly Spanish Ministry of Education). This work was funded by grants from the Spanish Ministry of Economy, Industry and Competitiveness (AGL2013-49137-C3-1-R and RTI2018-096172-B-C33), the Ministry of Science, Innovation and Universities (RTI2018-096172-B-C31 and RTI2018-096172-B-C33) and the Government of Aragón (A17_17R, Animal Health and Reproduction).

Background. Treatment of diabetic neuropathic pain (DNP) continues to be a major challenge, and underlying mechanisms of DNP remain elusive. We investigated treatment effects of B vitamins on DPN- and DNP-associated alterations of neurochemical signaling in the nociceptive dorsal root ganglion (DRG) neurons and the spinal cord in rats. Methods. DNP was produced in male, adult, Sprague Dawley rats by single i.p. streptozotocin (STZ). Western blot analysis and immunohistochemistry were used to analyze protein expressions in DRG and ELISA to measure the proinflammatory cytokines in the spinal cord. Behaviorally expressed DNP was determined by measuring the sensitivity of hindpaw skin to mechanical and thermal stimulation. Results. There were 87.5% (77/88) rats which developed high blood glucose within 1-2 weeks following STZ injection. Of which, 70.13% (n = 54/77) animals exhibited DNP manifested as mechanical allodynia and/or thermal hyperalgesia. Intraperitoneal administration of vitamins B1/B6/B12 (100/100/2 mg/kg, one or multiple doses) significantly attenuated DNP without affecting the blood glucose. Expressions of P2X3 and TRPV1 in CGRP-positive and IB4-positive DRG neurons as well as the interleukin-1β, tumor necrosis factor-α, and nerve growth factor in the lumbar spinal cord were greatly increased in DNP rats. Such DNP-associated neurochemical alterations were also greatly suppressed by the B-vitamin treatment. Conclusions. B-vitamin treatment can greatly suppress chronic DNP and DNP-associated increased activities of P2X3 and TRPV1 in DRG and the spinal proinflammatory cytokines, which may contribute to the pathogenesis of DNP. Systematic administration of B vitamins can be a strategy for DNP management in clinic.

The organization of the mouse spinal dorsal horn has been delineated in 2D for the six Rexed laminae in our publication Atlas of the Spinal Cord: Mouse, Rat, Rhesus, Marmoset, and Human. in the present study, the tissue clearing technique CLARITY was used to observe the cyto- and chemoarchitecture of the mouse spinal cord in 3D, using a variety of immunohistochemical markers. We confirm prior observations regarding the location of glycine and serotonin immunoreactivities. Novel observations include the demonstration of numerous calcitonin gene-related peptide (CGRP) perikarya, as well as CGRP fibers and terminals in all laminae of the dorsal horn. We also observed sparse choline acetyltransferase (ChAT) immunoreactivity in small perikarya and fibers and terminals in all dorsal horn laminae, while gamma aminobutyric acid (GABA) and glutamate decarboxylase-67 (GAD67) immunoreactivities were found only in small perikarya and fibers. Finally, numerous serotonergic fibers were observed in all laminae of the dorsal horn. in conclusion, CLARITY confirmed the 2D immunohistochemical properties of the spinal cord. Furthermore, we observed novel anatomical characteristics of the spinal cord and demonstrated that CLARITY can be used on spinal cord tissue to examine many proteins of interest., National Health and Medical Research CouncilNational Health and Medical Research Council of Australia [APP1140295, APP1188744]; Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-114S405]; Rebecca L. Cooper Medical Research Foundation; Centre of Excellence for Integrative Brain Function, Australian Research Council [CE140100007], We thank Dr. Emma Schofield for her technical support. This research was funded by the National Health and Medical Research Council (APP1140295 and APP1188744, awarded to George Paxinos), Scientific and Technological Research Council of Turkey (awarded to Gulgun Sengul, SBAG-114S405), Rebecca L. Cooper Medical Research Foundation (no grant number available, awarded to Huazheng Liang), and Centre of Excellence for Integrative Brain Function, Australian Research Council (CE140100007, awarded to George Paxinos), and the APC was funded by the Rebecca L. Cooper Medical Research Foundation.

The neurotoxin 1-methyl,4-phenyl-1,2,3,6-tetrahydropiridine (MPTP) is widely used to produce experimental parkinsonism in rodents and primates. Among different administration protocols, continuous or chronic exposure to small amounts of MPTP is reported to better mimic cell pathology reminiscent of Parkinson's disease (PD). Catecholamine neurons are the most sensitive to MPTP neurotoxicity; however, recent studies have found that MPTP alters the fine anatomy of the spinal cord including motor neurons, thus overlapping again with the spinal cord involvement documented in PD. In the present study, we demonstrate that chronic exposure to low amounts of MPTP (10 mg/kg daily, × 21 days) significantly reduces motor neurons in the ventral lumbar spinal cord while increasing α-synuclein immune-staining within the ventral horn. Spinal cord involvement in MPTP-treated mice extends to Calbindin D28 KDa immune-reactive neurons other than motor neurons within lamina VII. These results were obtained in the absence of significant reduction of dopaminergic cell bodies in the Substantia Nigra pars compacta, while a slight decrease was documented in striatal tyrosine hydroxylase immune-staining. Thus, the present study highlights neuropathological similarities between dopaminergic neurons and spinal motor neurons and supports the pathological involvement of spinal cord in PD and experimental MPTP-induced parkinsonism. Remarkably, the toxic threshold for motor neurons appears to be lower compared with nigral dopaminergic neurons following a chronic pattern of MPTP intoxication. This sharply contrasts with previous studies showing that MPTP intoxication produces comparable neuronal loss within spinal cord and Substantia Nigra.

Clinical studies indicate that psychosocial stress contributes to adverse chronic pain outcomes in patients, but it is unclear how this is initiated or amplified by stress. Repeated social defeat (RSD) is a mouse model of psychosocial stress that activates microglia, increases neuroinflammatory signaling, and augments pain and anxiety-like behaviors. We hypothesized that activated microglia within the spinal cord facilitate increased pain sensitivity following RSD. Here we show that mechanical allodynia in male mice was increased with exposure to RSD. This stress-induced behavior corresponded with increased mRNA expression of several inflammatory genes, including IL-1β, TNF-α, CCL2, and TLR4 in the lumbar spinal cord. While there were several adhesion and chemokine-related genes increased in the lumbar spinal cord after RSD, there was no accumulation of monocytes or neutrophils. Notably, there was evidence of microglial activation selectively within the nociceptive neurocircuitry of the dorsal horn of the lumbar cord. Elimination of microglia using the colony stimulating factor 1 receptor antagonist PLX5622 from the brain and spinal cord prevented the development of mechanical allodynia in RSD-exposed mice. Microglial elimination also attenuated RSD-induced IL-1β, CCR2, and TLR4 mRNA expression in the lumbar spinal cord. Together, RSD-induced allodynia was associated with microglia-mediated inflammation within the dorsal horn of the lumbar spinal cord.SIGNIFICANCE STATEMENTMounting evidence indicates that psychological stress contributes to the onset and progression of adverse nociceptive conditions. We show here that repeated social defeat stress causes increased pain sensitivity due to inflammatory signaling within the nociceptive circuits of the spinal cord. Studies here mechanistically tested the role of microglia in the development of pain by stress. Pharmacological ablation of microglia prevented stress-induced pain sensitivity. These findings demonstrate that microglia are critical mediators in the induction of pain conditions by stress. Moreover, these studies provide a proof of principle that microglia can be targeted as a therapeutic strategy to mitigate adverse pain conditions.

Etidronate is widely used as a therapeutic agent for osteoporosis. We have recently shown that intrathecal administration of etidronate into mice produces an analgesic effect against the capsaicin-induced nociceptive behavior. However, the effect of etidronate on neuropathic pain at the spinal level remains unknown. Therefore, we examined whether etidronate attenuates pain after partial sciatic nerve ligation (PSNL). We evaluated tactile allodynia 7 days after PSNL by measuring paw withdrawal with the von Frey filament test. The mRNA and protein levels of SLC17A9 in the ipsilateral lumbar dorsal spinal cord of PSNL-operated mice were determined using real-time PCR and western blotting, respectively. PSNL-induced tactile allodynia was attenuated by oral and intrathecal administration of etidronate, with maximum efficiency at 90 and 60 min after injection, respectively. The anti-allodynic effect of intrathecally administered etidronate was completely inhibited by an intrathecal administration of adenosine triphosphate (ATP). The solute carrier family, SLC17, mediates the transport of pain transmitters, like ATP and glutamate. Indeed, we detected several members of the SLC17 family in the mouse dorsal lumbar spinal cord. Among the detected mRNAs, only Slc17a9, encoding for neuronal vesicular ATP transporter, was significantly increased upon PSNL. SLC17A9 protein levels were also significantly increased. In mice subjected to PSNL, SLC17A9 was present in neurons and microglia, but not in astrocytes of the lumbar superficial dorsal horn. Collectively, our results suggest that etidronate produces its anti-allodynic effects by inhibiting SLC17A9-dependent exocytotic ATP release from the dorsal horn in mice subjected to PSNL.

BACKGROUND The aim of this study was to assess the clinical and radiological outcomes of surgical treatment for primary spinal ependymoma in children. MATERIAL AND METHODS Medical records of 46 primary spinal ependymoma patients who underwent surgery in BRSHH hospital during a 12-year period from 2004 to 2015 were retrospectively reviewed. All pediatric patients (patient age

A 2-year-old male neutered mixed breed dog with a body weight of 30 kg was presented for evaluation of a soft subcutaneous mass on the dorsal midline at the level of the caudal thoracic spine. A further clinical sign was intermittent pain on palpation of the area of the subcutaneous mass. The owner also described a prolonged phase of urination with repeated interruption and re-initiation of voiding. The findings of the neurological examination were consistent with a lesion localization between the 3rd thoracic and 3rd lumbar spinal cord segments. Magnetic resonance imaging revealed a spina bifida with a lipomeningocele and diplomyelia (split cord malformation type I) at the level of thoracic vertebra 11 and 12 and secondary syringomyelia above the aforementioned defects in the caudal thoracic spinal cord. Surgical resection of the lipomeningocele via a hemilaminectomy was performed. After initial deterioration of the neurological status postsurgery with paraplegia and absent deep pain sensation the dog improved within 2 weeks to non-ambulatory paraparesis with voluntary urination. Six weeks postoperatively the dog was ambulatory, according to the owner. Two years after surgery the owner recorded that the dog showed a normal gait, a normal urination and no pain. Histopathological diagnosis of the biopsied material revealed a lipomeningocele which confirmed the radiological diagnosis.Ein 2-jähriger Mischlingsrüde mit einem Körpergewicht von 30 kg wurde aufgrund einer dorsalen subkutanen Masse im Bereich der kaudalen Brustwirbelsäule vorgestellt. Ein weiteres klinisches Symptom war eine intermittierende Schmerzhaftigkeit bei Palpation im Bereich dieser Masse. Der Besitzer beschrieb zudem eine verlängerte Phase des Urinabsatzes mit wiederholter Unterbrechung und Wiederaufnahme der Miktion. Die Befunde der neurologischen Untersuchung entsprachen der Lokalisation der Rückenmarksabschnitte zwischen dem 3. Brust- und 3. Lendenwirbel. Die magnetresonanztomographische Untersuchung zeigte eine Spina bifida mit einer Lipomeningozele und Diplomyelie (Split-Cord-Malformation Typ I) auf Höhe des 11. und 12. Brustwirbels und eine sekundäre Syringomyelie kranial des besagten Defekts im Bereich der kaudalen Brustwirbelsäule. Es wurde eine chirurgische Resektion der Lipomeningozele mittels Hemilaminektomie vorgenommen. Nach anfänglicher postoperativer Verschlechterung des neurologischen Zustands mit Paraplegie und fehlender Tiefenschmerzwahrnehmung kam es innerhalb von 2 Wochen zu einer Verbesserung. Der Hund wies eine nicht gehfähige Paraparese auf und konnte selbstständig Urin absetzen. Nach Aussage des Besitzers war der Hund 6 Wochen postoperativ gehfähig. Zwei Jahre nach der Operation berichtete der Besitzer, der Hund zeige einen normalen Gang, einen unauffälligen Urinabsatz und keine Anzeichen für Schmerzen. Die histopathologische Untersuchung bestätigte die radiologische Diagnose einer Lipomeningozele.

Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia. The TLR4-induced hepoxilin production was also observed in primary spinal microglia, but not in astrocytes, and was accompanied by increased microglial expression of the 12/15-lipoxygenase enzyme 15-LOX-1. Intrathecal KLA-induced tactile allodynia was completely prevented by spinal pretreatment with the 12/15-lipoxygenase inhibitor CDC or a selective antibody targeting rat 15-LOX-1. Similarly, pretreatment with the selective inhibitors ML127 or ML351 both reduced activity of the rat homolog of 15-LOX-1 heterologously expressed in HEK-293T cells and completely abrogated nonsteroidal anti-inflammatory drug-unresponsive allodynia in vivo after IT KLA. Finally, spinal 12/15-lipoxygenase inhibition by nordihydroguaiaretic acid (NDGA) both prevents phase II formalin flinching and reverses formalin-induced persistent tactile allodynia. Taken together, these findings suggest that spinal TLR4-mediated hyperpathic states are mediated at least in part through activation of microglial 15-LOX-1.

This study tested the hypothesis that selective ablation of transient receptor potential vanilloid type 1 (TRPV1)-positive nerve fibers by intrathecal injection of resiniferatoxin (RTX) enhances renal sympathoexcitatory responses and salt sensitivity. Intrathecal injection of RTX (1.8 μg/kg) to the levels of lower thoracic and upper lumbar spinal cord (T8–L3) increased mean arterial pressure (MAP) in rats fed a normal (NS, 1% NaCl) or high-sodium (HS, 8% NaCl) diet for 4 weeks compared to vehicle-treated rats (NS: 121 ± 2 vs. 111 ± 2; HS: 154 ± 2 vs. 134 ± 2 mm Hg, both P

Previous work from our laboratory showed that motor nerve injury by lumbar 5 ventral root transection (L5-VRT) led to interleukin-6 (IL-6) over-expression in bilateral spinal cord, and that intrathecal administration of IL-6 neutralizing antibody delayed the induction of mechanical allodynia in bilateral hind paws. However, early events and upstream mechanisms underlying spinal IL-6 expression following L5-VRT require elucidation. The model of L5-VRT was used to induce neuropathic pain, which was assessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Calpain-2 (CALP2, a calcium-dependent protease) knockdown or over-expression and microglia depletion were conducted intrathecally. Western blots and immunohistochemistry were performed to explore the possible mechanisms. Here, we provide the first evidence that both IL-6 and CALP2 levels are increased in lumbar spinal cord within 30 min following L5-VRT. IL-6 and CALP2 co-localized in both spinal dorsal horn (SDH) and spinal ventral horn. Post-operative (PO) increase in CALP2 in ipsilateral SDH was evident at 10 min PO, preceding increased IL-6 at 20 min PO. Knockdown of spinal CALP2 by intrathecal CALP2-shRNA administration prevented VRT-induced IL-6 overproduction in ipsilateral spinal cord and alleviated bilateral mechanical allodynia. Spinal microglia activation also played a role in early IL-6 up-regulation. Macrophage/microglia markers ED1/Iba1 were increased at 30 min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20 min PO and peaked at 3 days. Morphology changed from a small soma with fine processes in resting cells to an activated ameboid shape. Depletion of microglia using Mac-1-saporin partially prevented IL-6 up-regulation and attenuated VRT-induced bilateral mechanical allodynia. Taken together, our findings provide evidence that increased spinal cord CALP2 and microglia cell activation may have early causative roles in IL-6 over-expression following motor nerve injury. Agents that inhibit CALP2 and/or microglia activation may therefore prove valuable for treating neuropathic pain.

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by progressive muscular dystrophy and paralysis; most ALS patients die from respiratory failure within 3 to 5 years, and there is currently no effective treatment. Some studies have indicated sex differences in the incidence of ALS, and evidence suggests a neuroprotective role for estrogen. MATERIAL AND METHODS We used human Cu/Zn superoxide dismutase (hSOD1-G93A) transgenic mice to determine the effects of ovariotomy on the onset of disease and behavior; we also used Western blotting to measure the expression of aromatase and estrogen receptors, as well as the inflammatory cytokines and apoptosis markers, in the lumbar spinal cord to determine the mechanism of estrogen-mediated neuroprotection. RESULTS Ovariectomy advanced the onset of disease, down-regulated aromatase and estrogen receptor alpha (ER-a) expression, and inhibited expression of the anti-inflammatory factors arginase-1 and the anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) in the lumbar spinal cord of hSOD1-G93A transgenic mice. CONCLUSIONS Ovariectomy resulted in earlier disease onset and attenuated the anti-inflammatory and anti-apoptotic actions of estrogen in hSOD1-G93A transgenic mice. Therefore, estrogen may play an important role in protecting spinal cord motor neurons.

SUMMARY To understand the cellular basis of behavior, it is necessary to know the cell types that exist in the nervous system and their contributions to function. Spinal networks are essential for sensory processing and motor behavior and provide a powerful system for identifying the cellular correlates of behavior. Here, we used massively parallel single nucleus RNA sequencing (snRNA-seq) to create an atlas of the adult mouse lumbar spinal cord. We identified and molecularly characterized 43 neuronal populations. Next, we leveraged the snRNA-seq approach to provide unbiased identification of neuronal populations that were active following a sensory and a motor behavior, using a transcriptional signature of neuronal activity. This approach can be used in the future to link single nucleus gene expression data with dynamic biological responses to behavior, injury, and disease., In Brief Sathyamurthy et al. use massively parallel single nucleus RNA-seq to probe spinal cord cell types and present an atlas of 43 neuronal populations. By using this approach after a sensory and a motor behavior, they were able to detect and molecularly identify activated neurons associated with each function.

Background Cumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown. Methods A total of 120 male Wistar rats were used in the present study. The neuropathic pain model was established by chronic constrictive injury (CCI) of the sciatic nerve. The rats were randomly divided into sham group, CCI group, and sham CCI + EA group, and CCI + EA group. EA was applied to bilateral Zusanli (ST36)-Yanlingquan (GB34). The mechanical (both time and force responses) and thermal pain thresholds (PTs) of the bilateral hind-paws were measured. The number of microgliacytes and activity of astrocytes in the dorsal horns (DHs) of lumbar spinal cord (L4–5) were examined by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) protein was detected by western blot. Results Following CCI, both mechanical and thermal PTs of the ipsilateral hind-paw were significantly decreased beginning from the 3rd day after surgery (P

Monoamine oxidase-B (MAOB), a flavin adenine dinucleotide (FAD), is an enzyme which catalyzes the oxidation of amines. MAOB is proposed to play a major role in the pathogenesis of neurodegeneration through the production of reactive oxygen species (ROS) and neurotoxins. The present study was designed to outline the effects of the MAOB inhibitor (MAOB-I) on neuroprotection of spinal neurons, regeneration of sciatic nerve fibers, and recovery of sensory-motor functions in the sciatic nerve crush injury model. Male Wistar rats (4-months-old) were assigned to i) Naive (N), ii) Sham (S), iii) Sciatic nerve crush and treated with saline (CRUSH + SALINE) and iv) Sciatic nerve crush and treated with MAOB inhibitor (CRUSH + MAOB-I) groups (n = 10/group). In groups iii and iv, the crush injury was produced by crushing the sciatic nerve followed by treatment with saline or MAOB-I (Selegiline® 2.5 mg/kg) intraperitoneally for 10 days. Behavioral tests were conducted from week 1 to week 6. At the end of the study, sciatic nerve and lumbar spinal cord were examined by immunohistochemistry, light and electron microscopy. MAOB-I treatment showed significant improvement in sensory and motor functions compared to saline treatment (p

OBJECTIVEThere has been no established animal model of syringomyelia associated with lumbosacral spinal lipoma. The research on the pathophysiology of syringomyelia has been focused on Chiari malformation, trauma, and inflammation. To understand the pathophysiology of syringomyelia associated with occult spinal dysraphism, a novel animal model of syringomyelia induced by chronic mechanical compression of the lumbar spinal cord was created.METHODSThe model was made by epidural injection of highly concentrated paste-like kaolin solution through windows created by partial laminectomy of L-1 and L-5 vertebrae. Behavioral outcome in terms of motor (Basso-Beattie-Bresnahan score) and urinary function was assessed serially for 12 weeks. Magnetic resonance images were obtained in some animals to confirm the formation of a syrinx and to monitor changes in its size. Immunohistochemical studies, including analysis for glial fibrillary acidic protein, NeuN, CC1, ED-1, and caspase-3, were done.RESULTSBy 12 weeks after the epidural compression procedure, syringomyelia formation was confirmed in 85% of the rats (34 of 40) on histology and/or MRI. The syrinx cavities were found rostral to the epidural compression. Motor deficit of varying degrees was seen immediately after the procedure in 28% of the rats (11 of 40). In 13 rats (33%), lower urinary tract dysfunction was seen. Motor deficit improved by 5 weeks after the procedure, whereas urinary dysfunction mostly improved by 2 weeks. Five rats (13%, 5 of 40) died 1 month postoperatively or later, and 3 of the 5 had developed urinary tract infection. At 12 weeks after the operation, IHC showed no inflammatory process, demyelination, or accelerated apoptosis in the spinal cords surrounding the syrinx cavities, similar to sham-operated animals.CONCLUSIONSA novel experimental model for syringomyelia by epidural compression of the lumbar spinal cord has been created. The authors hope that it will serve as an important research tool to elucidate the pathogenesis of this type of syringomyelia, as well as the CSF hydrodynamics of the lumbar spinal cord.

Estrogen exerts protective roles in amyotrophic lateral sclerosis (ALS). However, the expression of aromatase (ARO) and estrogen receptors (ERs) in the motoneurons of spinal cord, has not yet been elucidated. By immunohistochemistry, we found that ARO and ERs were present in the ventral horn of adult mice lumbar spinal cord, and colocalized with SMI-32, a motoneuron specific marker. Within motoneurons, we observed that ARO is detected primarily in the cytoplasm, with fewer ARO in the nucleus; ERα and ERβ mainly localized in the nucleus with less in the cytoplasm; while GPR30 is located in soma and processes. In conclusion, we found that ERs and ARO are expressed in the motoneurons of lumbar spinal cord in adult mice. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in ALS.

A non-invasive, auricular percutaneous electrical nerve field stimulation (PENFS) has been suggested to modulate central pain pathways. We investigated the effects of BRIDGE® device on the responses of amygdala and lumbar spinal neurons and the development of post-colitis hyperalgesia. Male Sprague-Dawley rats received intracolonic trinitrobenzene sulfonic acid (TNBS) and PENFS on the same day. Control rats had sham devices. The visceromotor response (VMR) to colon distension and paw withdrawal threshold (PWT) was recorded after 7days. A different group of rats had VMR and PWT at baseline, after TNBS and following PENFS. Extracellular recordings were made from neurons in central nucleus of the amygdala (CeA) or lumbar spinal cord. Baseline firing and responses to compression of the paw were recorded before and after PENFS. Sham-treated rats exhibited a much higher VMR (>30mmHg) and lower PWT compared to PENFS-treated rats (p

Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide (NO). NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95 kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein-protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95-nNOS protein-protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within lumbar spinal dorsal horn of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95-nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95-nNOS protein-protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. © 2016 Wiley Periodicals, Inc.

Impaired signaling via CX3CR1, the fractalkine receptor, promotes recovery after traumatic spinal contusion injury in mice, a benefit achieved in part by reducing macrophage-mediated injury at the lesion epicenter. Here, we tested the hypothesis that CX3CR1-dependent changes in microglia and macrophage functions also will enhance neuroplasticity, at and several segments below the injury epicenter. New data show that in the presence of inflammatory stimuli, CX3CR1-deficient (CX3CR1−/−) microglia and macrophages adopt a reparative phenotype and increase expression of genes that encode neurotrophic and gliogenic proteins. At the lesion epicenter (mid-thoracic spinal cord), the microenvironment created by CX3CR1−/−microglia/macrophages enhances NG2 cell responses, axon sparing, and sprouting of serotonergic axons. In lumbar spinal cord, inflammatory signaling is reduced in CX3CR1−/−microglia. This is associated with reduced dendritic pathology and improved axonal and synaptic plasticity on ventral horn motor neurons. Together, these data indicate that CX3CR1, a microglia-specific chemokine receptor, is a novel therapeutic target for enhancing neuroplasticity and recovery after SCI. Interventions that specifically target CX3CR1 could reduce the adverse effects of inflammation and augment activity-dependent plasticity and restoration of function. Indeed, limiting CX3CR1-dependent signaling could improve rehabilitation and spinal learning.SIGNIFICANCE STATEMENTPublished data show that genetic deletion of CX3CR1, a microglia-specific chemokine receptor, promotes recovery after traumatic spinal cord injury in mice, a benefit achieved in part by reducing macrophage-mediated injury at the lesion epicenter. Data in the current manuscript indicate that CX3CR1 deletion changes microglia and macrophage function, creating a tissue microenvironment that enhances endogenous repair and indices of neuroplasticity, at and several segments below the injury epicenter. Interventions that specifically target CX3CR1 might be used in the future to reduce the adverse effects of intraspinal inflammation and augment activity-dependent plasticity (e.g., rehabilitation) and restoration of function.

The transcription factor Pax2 is required for the differentiation of GABAergic neurons in the mouse dorsal horn. Pax2 continues to be expressed in the adult murine spinal cord and has been used as a presumed marker of GABAergic neurons in the superficial dorsal horn of the adult mouse, although a strict association between adult Pax2 expression and presence of GABA throughout the dorsal horn has not been firmly established. Moreover, whether Pax2 is selectively expressed in GABAergic dorsal horn neurons also in the rat is unknown. Here, immunofluorescent labeling of Pax2 and GABA in the lumbar spinal cord of adult rats was used to investigate this issue. Indeed, essentially all GABA immunoreactive neurons in laminae I-V were immunolabeled for Pax2. Conversely, essentially all Pax2 immunopositive neurons in these laminae exhibited somatic GABA immunolabeling. These results indicate persistent Pax2 expression in GABAergic neurons in the adult rat dorsal horn, supporting the hypothesis that Pax2 may be required for the maintenance of a GABAergic phenotype in mature inhibitory dorsal horn neurons in the rat. Furthermore, Pax2 may be used as a selective and specific general somatic marker of such neurons.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers. Injection of AAV–shRNA below the pial surface of the spinal cord prevents onset or ameliorates progression in a mouse model of ALS, and achieves widespread delivery to the spinal cord and brain motor centers in adult pigs and non-human primates.

Neuropathic pain, or pain after nerve injury, is a disorder with a significant reliance on the signalling of cytokines such as IL-1β. However, quantifying the cytokine release repeatedly over time in vivo is technically challenging.To evaluate if changes in IL-1β are correlated with the presentation of mechanical allodynia over time, by repeatedly quantifying intrathecal IL-1β concentrations following chronic constriction injury of the sciatic nerve in rats. Also, to establish any possible correlation between biochemical spinal marker expression and the in vivo quantification of IL-1β. Finally, to assess the expression of the mature IL-1β in lumbar spinal cord samples.The Chronic Constriction Injury model (CCI) was used to initiate nerve injury in male Sprague Dawley rats and the generation of behavioural mechanical allodynia was quantified. Using an indwelling intrathecal catheter, a stainless steel (SS) wire biosensing device was repeatedly introduced to quantify intrathecal IL-1β concentrations at three timepoints of 0, 7, and 14 days post CCI. Fixed spinal cord samples (L4-L5), collected on day 14, were imaged for the expression of glial fibrillary acidic protein (GFAP, astrocytes) and ionized calcium binding adaptor molecule 1 (IBA1, microglia). Snap frozen spinal cord tissues (L4-L5) were also processed for western blot analysis.Using the novel SS based biosensing device we established that CCI caused a significant increase in intrathecal IL-1β concentrations from day 0 to day 7 (p = 0.001) and to day 14 (p 0.0001), while the sham group did not show any significant increase. We also further showed that the degree of mechanical allodynia correlated positively with the increase in the intrathecal concentration of IL-1β in the active CCI animals (p = 0.0007). While there was a significant increase in the ipsilateral GFAP expression in injured animals compared to sham animals (p = 0.03), we did not find any significant correlation between in vivo IL-1β concentration on days 7 and 14 and the area of dorsal horn GFAP or IBA1 positive structures on day 14. The result of western blot analysis of whole lumbar spinal cord revealed that there was no significant change (p = 0.7579) in IL-1β expression on day 14 in the CCI group compared to the sham group.For the first time we have established that the SS based immunosensing platform technology can repeatedly sample the intrathecal space for bioactive peptides, such as IL-1β. Using this novel approach, we have been able to establish the correlation of the intrathecal concentration of IL-1β with the extent of mechanical allodynia, providing a molecular biomarker of the degree of the exaggerated pain state.

BackgroundDown syndrome (DS), caused by the triplication of chromosome 21, results in a constellation of clinical features including changes in intellectual and motor function. Although altered neural development and function have been well described in people with DS, few studies have investigated the etiology underlying the observed motor phenotypes. Here, we examine the development, patterning, and organization of the spinal cord throughout life in the Ts65Dn mouse, a model that recapitulates many of the motor changes observed in people with DS.MethodsSpinal cords from embryonic to adult animals were processed for gene and protein expression (immunofluorescence) to track the spatiotemporal development of excitatory and inhibitory neurons and oligodendroglia. Postnatal analyses were focused on the lumbar region due to the reflex and gait abnormalities found in Ts65Dn mice and locomotive alterations seen in people with DS.ResultsBetween embryonic days E10.5 and E14.5, we found a larger motor neuron progenitor domain in Ts65Dn animals containing more OLIG2-expressing progenitor cells. These disturbed progenitors are delayed in motor neuron production but eventually generate a large number of ISL1+ migrating motor neurons. We found that higher numbers of PAX6+ and NKX2.2+ interneurons (INs) are also produced during this time frame. In the adult lumbar spinal cord, we found an increased level ofHb9and a decreased level ofIrx3gene expression in trisomic animals. This was accompanied by an increase in Calretinin+ INs, but no changes in other neuronal populations. In aged Ts65Dn animals, both Calbindin+ and ChAT+ neurons were decreased compared to euploid controls. Additionally, in the dorsal corticospinal white matter tract, there were significantly fewer CC1+ mature OLs in 30- and 60-day old trisomic animals and this normalized to euploid levels at 10–11 months. In contrast, the mature OL population was increased in the lateral funiculus, an ascending white matter tract carrying sensory information. In 30-day old animals, we also found a decrease in the number of nodes of Ranvier in both tracts. This decrease normalized both in 60-day old and aged animals.ConclusionsWe show marked changes in both spinal white matter and neuronal composition that change regionally over the life span. In the embryonic Ts65Dn spinal cord, we observe alterations in motor neuron production and migration. In the adult spinal cord, we observe changes in oligodendrocyte maturation and motor neuron loss, the latter of which has also been observed in human spinal cord tissue samples. This work uncovers multiple cellular perturbations during Ts65Dn development and aging, many of which may underlie the motor deficits found in DS.