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[Display omitted] • Non-canonical NF-κB pathways are activated in the liver, spinal cord, and intestine in animal models of ALS.2. • The administration of the NIK inhibitor B022 significantly prolonged lifespan and grip strength and improved neuroinflammation in an animal model of ALS. • SOD1G93A in conjunction with TBK1-UKO produces intestinal inflammation possibly due to activation of non-canonical NF-κB pathway. Amyotrophic lateral sclerosis (ALS) is a typical neurodegenerative disorder typically characterized by inflammation activation. However, the relationship between non-canonical NF-κB (ncNF-κB) pathway activation and ALS progression is not clear. We tested the ncNF-κB pathway in the ALS animal model including hSOD1-G93A transgenic mice and TBK1 deletion mice.We treated age-matched SOD1-G93A mice with B022 (a NIK inhibitor) to investigate the role of NIK in the ALS animal model. We also established a new mice model by crossing SOD1-G93A mice with NIK+/- mice to further evaluate the interrelationship between the NIK and the disease progression in ALS animal model. In this study, we found the ncNF-κB pathway was activated in SOD1-G93A animal model and TBK1 deletion model. Inhibition of NIK activity by small molecule B022 significantly improved the motor performance of the ALS animal model. However, NIK deletion enhanced the mutant SOD1 toxicity by inflammatory infiltration. TBK1 deletion and mutant SOD1 shared the common pathological feature possibly via effects on NIK activation and inhibitor of NIK could be a novel strategy for treating ALS. [ABSTRACT FROM AUTHOR]

• Sig-1R activation reduced the iron accumulation and inhibited ferroptosis in spinal cord tissues after SCI in mice. • Sigma-1 receptor agonists were able to ameliorate ferroptosis and IRE1α were significantly upregulated after SCI. • Sigma-1 receptor activation could improve functional recovery after SCI. Spinal Cord Injury (SCI) is a debilitating disease associated with a significant economic burden owing to its high level of disability; however, current treatment options have only limited efficacy. Past research has shown that iron-dependent programmed cell death, also known as ferroptosis, plays a critical role in the pathogenesis of SCI. The sigma-1 receptor (Sig-1R) is widely distributed in the central nervous system, and has been implicated in the pathophysiology of several neurological and psychiatric disorders. Several in vivo and ex vivo studies have shown that Sig-1R activation exerts unique neuroprotective effects. However, the underlying mechanisms remain unclear. To date, no study has yet demonstrated the association between Sig-1R activation and ferroptosis in patients with SCI. However, the present study found that Sig-1R activation effectively promoted the recovery of motor function in mice after spinal cord injury, attenuated neuronal apoptosis, reduced the production of pro-inflammatory cytokines and iron accumulation, and inhibited ferroptosis in spinal cord tissues following SCI in mice. Ferroptosis and IRE1α were significantly upregulated after spinal cord injury, while sigma-1 receptor agonists were able to facilitate this result through the elimination of inositol-requiring enzyme-1 alpha (IRE1α)-mediated neuronal ferroptosis. Therefore, sigma-1 receptor activation could attenuate ferroptosis after SCI by reducing IRE1α and improving functional recovery after SCI, potentially representing a new therapeutic strategy for treating SCI. [ABSTRACT FROM AUTHOR]

[Display omitted] • Central poststroke pain (CPSP) is a type of central neuropathic pain whose mechanisms remain unknown. • We hypothesized that spinal glial cell-derived molecules promoted the development of CPSP. • We used a bilateral common carotid occlusion (BCAO)-induced CPSP mouse model. • BCAO mice showed hypersensitivity to mechanical stimuli and astrocyte activation in the spinal cord. • Lipocalin 2 (LCN2) in the spinal cord of BCAO mice were identified as a responsible factor for CPSP. Central poststroke pain (CPSP) is a type of central neuropathic pain whose mechanisms remain unknown. Recently, we showed that activated astrocytes and microglial cells are present in the spinal cord of CPSP model mice. Activated glial cells exacerbate cerebral ischemic pathology by increasing the expression of inflammatory factors. However, the involvement of spinal glial cells in CPSP remains unknown. We hypothesized that spinal glial cell-derived molecules cause hyperexcitability or promoted the development of CPSP. In this study, we identified glial cell-derived factors involved in the development of CPSP using a bilateral common carotid occlusion (BCAO)-induced CPSP mouse model. Male ddY mice were subjected to BCAO for 30 min. The von Frey test assessed mechanical hypersensitivity in the right hind paw of mice. BCAO mice showed hypersensitivity to mechanical stimuli and astrocyte activation in the spinal cord 3 days after treatment. DNA microarray analysis revealed a significant increase in lipocalin 2 (LCN2), is known as neutrophil gelatinase-associated lipocalin, in the superficial dorsal horns of BCAO-induced CPSP model mice. LCN2 colocalized with GFAP, an astrocyte marker. Spinal GFAP-positive cells in BCAO mice co-expressed signal transducer and activator of transcription 3 (STAT3). The increase in the fluorescence intensity of LCN2 and GFAP in BCAO mice was suppressed by intrathecal injection of AG490, an inhibitor of JAK2 and downstream STAT3 activation, or anti-LCN2 antibody. Our findings indicated that LCN2 in spinal astrocytes may be a key molecule and may be partly involved in the development of CPSP. [ABSTRACT FROM AUTHOR]

[Display omitted] • BMS-345541 avoided the development of REMSD-induced allodynia in female rats. • BMS-345541 did not avoid the development of REMSD-induced allodynia in male rats. • ERα antagonism or run-down of ERα in female rats abolished BMS-345541′s effect. • REMSD increased ERα protein expression in DRG/DSC in females but not in male rats. • ERα activation or ERα overexpression allowed the effect of BMS-345541 in male rats. Several signaling pathways that converge in NF-kB activation have been linked to developing and maintaining different types of pathological pain. In addition, some mechanisms implied in the establishment of chronic pain have been demonstrated to have a sex-dependent correlation. This study aimed to determine if the IKKs/NF-kB signaling pathway is involved in establishing REM sleep deprivation (REMSD) induced mechanical allodynia in rats and its possible regulation depending on estradiol and estrogen receptors. Intrathecal administration of BMS-345541 or minocycline, two drugs that reduce the IKKs/NF-kB activity, avoided the development of mechanical allodynia in female but not in male rats subjected to 48 h of REMSD. Ovariectomy in female rats abolished the effect of BMS-345541 and minocycline. Meanwhile, the 17-β-estradiol restitution restored it. Intrathecal administration of MPP, a selective ERα antagonist, but not PHTPP, a selective ERβ antagonist, avoided the effect of BMS-345541 in female rats without hormonal manipulation. In addition, the transient run-down of ERα in female rats abolished the effect of BMS-345541. All data suggest an important role of ERα as a regulator of the IKKs/NF-kB activity. REMSD increased the ERα protein expression in the dorsal root ganglia and the dorsal spinal cord in females but not in male rats. Interestingly, ERα activation or ERα overexpression allowed the effect of BMS-345541 in male rats. Data suggest an important regulatory role of ERα in the IKKs/NF-kB activity on establishing mechanical allodynia induced by REMSD in female rats. [ABSTRACT FROM AUTHOR]

Study of ADAM10 expression in the murine CNS by immunohistochemistry (IHC) and immunocytochemistry (ICC). Created with BioRender.com. [Display omitted] • ADAM10 protein shows a widespread distribution in the adult CNS. • ADAM10 is mainly detected in vivo in neurons, in oligodendrocytes with a lower extent. • ADAM10 is expressed in neurons, oligodendrocytes, astrocytes and microglia in vitro. • Subcellular localisation of ADAM10 reveals a membrane, cytoplasmic and nuclear expression. A Disintegrin And Metalloprotease 10 (ADAM10), is able to control several important physiopathological processes through the shedding of a large number of protein substrates. Although ADAM10 plays a crucial role in the central nervous system (CNS) development and function, its protein distribution in the CNS has not been fully addressed. Here, we described the regional and cellular ADAM10 protein expression in C57BL/6 mice examined by immunofluorescence 1) throughout the adult mouse brain, cerebellum and spinal cord in vivo and 2) in different cell types as neurons, astrocytes, oligodendrocytes and microglia in vitro. We observed ADAM10 expression through the whole CNS, with a strong expression in the hippocampus, in the hypothalamus and in the cerebral and piriform cortex in the brain, in the Purkinje and in granular cell layers in the cerebellum and in the spinal cord to a lower extent. In vivo , ADAM10 protein expression was mainly found in neurons and in some oligodendroglial cell populations. However, in primary cultures we observed ADAM10 expression in neurons, oligodendrocytes, astrocytes and microglia. Interestingly, ADAM10 was not only found in the membrane but also in cytoplasmic vesicles and in the nucleus of primary cultured cells. Overall, this work highlights a wide distribution of ADAM10 throughout the CNS. The nuclear localization of ADAM10, probably due to its intracellular domain, emphasizes its role in cell signalling in physiological and pathological conditions. Further investigations are required to better elucidate the role of ADAM10 in glial cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Dorsal horn compression at the upper cervical cord causes facial allodynia. • Ultrastructural changes were observed in trigeminal ganglion and infraorbital nerve. • Neuroinflammation and apoptosis are main supportive factors of this model. Idiopathic trigeminal neuralgia (TN) cases encountered frequently in daily practice indicate significant gaps that still need to be illuminated in the etiopathogenesis. In this study, a novel TN animal model was developed by compressing the dorsal horn (DH) of the upper cervical spinal cord. Eighteen rabbits were equally divided into three groups, namely control (CG), sham (SG), and spinal cord compression (SCC) groups. External pressure was applied to the left side at the C3 level in the SCC group. Dorsal hemilaminectomy was performed in the SG, and the operative side was closed without compression. No procedure was implemented in the control group. Samples from the SC, TG, and ION were taken after seven days. For the histochemical staining, damage and axons with myelin were scored using Hematoxylin and Eosin and Toluidine Blue, respectively. Immunohistochemistry, nuclei, apoptotic index, astrocyte activity, microglial labeling, and CD11b were evaluated. Mechanical allodynia was observed on the ipsilateral side in the SCC group. In addition, both the TG and ION were partially damaged from SC compression, which resulted in significant histopathological changes and increased the expression of all markers in both the SG and SCC groups compared to that in the CG. There was a notable increase in tissue damage, an increase in the number of apoptotic nuclei, an increase in the apoptotic index, an indication of astrocytic gliosis, and an upsurge in microglial cells. Significant increases were noted in the SG group, whereas more pronounced significant increases were observed in the SCC group. Transmission electron microscopy revealed myelin damage, mitochondrial disruption, and increased anchoring particles. Similar changes were observed to a lesser extent in the contralateral spinal cord. Ipsilateral trigeminal neuropathic pain was developed due to upper cervical SCC. The clinical finding is supported by immunohistochemical and ultrastructural changes. Thus, alterations in the DH due to compression of the upper cervical region should be considered as a potential cause of idiopathic TN. [ABSTRACT FROM AUTHOR]

[Display omitted] • ON causes occult cervical cord damage, as demonstrated by DTI abnormalities. • The DTI abnormalities are positively correlated with cognitive impairment in ON patients. • Quantitative spinal MRI may hold potential for contributing to clinical evaluations in patients with ON. Previous studies have demonstrated that optic neuritis (ON) affects brain plasticity. However, whether ON affects the spinal cord remains unclear. We aimed to investigate the spinal cord changes in ON and their associations with disability. A total of 101 ON patients, and 41 healthy controls (HC) were retrospectively recruited. High-resolution imaging was conducted using a Magnetization Prepared Rapid Acquisition Gradient-Echo (MP-RAGE) sequence for T1-weighted images and an echo planar imaging (EPI) sequence for Diffusion Tensor Imaging (DTI) data collection. Additionally, patients' disability and cognitive impairment were evaluated using the Expanded Disability Status Scale (EDSS) and the Paced Auditory Serial Addition Test (PASAT), respectively. The quantitative spinal MRI was employed to examine the cross-sectional area (CSA) and diffusion indicators, with a specific focus on calculating the average values across the C2-C7 cervical spinal cord segments. CSA, fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were compared between groups. Correlation analyses were performed between CSA, diffusion indicators, and clinical variables. No significant differences were found in CSA between ON patients and HCs. MD (p = 0.007) and RD (p = 0.018) were increased in ON patients compared with HCs, and AD was decreased in ON (p = 0.013). The AD values of the ON patients were significantly positively correlated with PASAT scores (r = 0.37, p < 0.001). This study provided imaging evidence for DTI abnormalities in patients with ON. Spinal cord DTI can improve our knowledge of the path physiology of ON, and clinical progression. [ABSTRACT FROM AUTHOR]

We verified the neuroprotection and molecular mechanism of wnt3a following spinal cord injury (SCI). wnt-3a significantly inhibited the apoptosis and inflammatory response of neuronal cells, reduced the loss of motor neurons in the anterior horn of the spinal cord, promoted the repair of injured tissues, and built a good microenvironment for recovery of motor nerve function, ultimately promotes the recovery of motor nerve function after SCI. This neuroprotective effect of wnt-3a is exerted through activating the β-catenin signaling pathway. [Display omitted] • Wnt3a promotes the recovery of neurological function after SCI. • Wnt3a inhibits inflammation and apoptosis via wnt/β-catenin signaling pathway after SCI. • Wnt3a exerts neuroprotection via wnt/β-catenin signaling pathway after SCI. The specific molecular mechanism of neuroprotective effects of wnt-3a on spinal cord injury (SCI) has not been elucidated. In our study, we evaluated the recovery of motor function after SCI by BBB, observed neuronal apoptosis by western blot and TUNEL, observed the changes of neuronal inflammation by western blot and immunofluorescence staining, and observed the changes of motoneurons and spinal cord area in the anterior horn of the spinal cord via Nissl and HE staining. We found that wnt-3a could significantly promote the recovery of motor function, reduce the loss of motor neurons in the anterior horn of the spinal cord, promote the recovery of injured spinal cord tissue, inhibit neuronal apoptosis and inflammatory response, and ultimately promote neuronal function after SCI. However, when XAV939 inhibits the wnt/β-catenin signaling pathway, the neuroprotective effects of wnt-3a are also significantly inhibited. The above results together indicated that wnt-3a exerts its neuroprotective effect on after SCI via activating the wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

• We focused on the aspartoacylase (ASPA) and mitochondrion-related metabolites to clarify the mechanism of myelin pathology in dmy rats. • Aspa mRNA and ASPA protein was significantly decreased in the dmy rats. • It is intriguing that prior to the myelin destruction disrupted expression of Aspa mRNA and ASPA protein undergoes from early stage of myelinogenesis. The dmy rat is an autosomal recessive mutant that exhibits severe rapid myelin breakdown throughout the central nervous system at 7–8 weeks of age. The dmy rat has a point mutation in Mrs2 gene, which encodes an essential component of the major electrophoretic Mg2+ influx system in the mitochondria. However, it remains unknown how mitochondrial dysfunction leads to the myelin breakdown. We focused on the aspartoacylase (ASPA) and mitochondrion-related metabolites to clarify the mechanism of myelin pathology in dmy rats. Aspa mRNA was significantly decreased in both the gray matter and the ventral white matter of spinal cord in the dmy rats from 4 to 8 weeks of age. Very faint immunohistochemical expression for ASPA was noted in the gray and white matter of the affected dmy rats at 8 weeks. Liquid chromatography mass spectrometry revealed no different amount of N -acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A (CoA) in neurons, in the brain and spinal cord between the dmy and control rats. Our results indicated that the pyruvate dehydrogenase activity might be reduced due to the loss of Mg2+ transport activity in the mitochondria of the dmy rats, suggesting acetyl CoA production might be reduced. The number of oligodendrocytes was well preserved until 7 weeks. It is intriguing that prior to the myelin destruction at 7–8 weeks, disrupted expression of Aspa mRNA and ASPA protein undergoes from early stage of myelinogenesis. These data indicate that ASPA expression would be a useful index to evaluate a function of oligodendrocyte in the dmy rat. [ABSTRACT FROM AUTHOR]

• Plantar incision resulted in increase of MMP-9/2 in the wounded tissue, ipsilateral DRG, and spinal dorsal horn. • The upregulated MMP-9 via p38/IL-1β, and MMP-2 via Erk/IL-1β contribute to postoperative pain. • MMP-9/2 might be therapeutic target to the treatment of postoperative pain. Emerging evidence implicates the upregulation of matrix metalloproteinase (MMP)-9/2 in the dorsal root ganglion (DRG) and spinal cord as a contributor to the pathogenesis of chronic pain. In the current study, the expression of MMP-9/2 in wounded tissue, ipsilateral DRG, and the spinal dorsal horn as well as its role in the development of postoperative pain were examined following plantar incision in rats. Our results showed that plantar incision resulted in increased expression of MMP-9/2 in wounded tissue and ipsilateral L4/5 DRGs. Although gelatin zymography detected an increased activity of MMP-9, only MMP-2 protein was increased in the spinal cord. Results of double immunofluorescence staining showed MMP-2 expression in DRG neurons and satellite glial cells, but MMP-9 was found only in neurons. In the spinal cord, MMP-2 was expressed in neurons and astrocytes, and MMP-9 was expressed in neurons and somewhat in microglial cells. Planter incision also elicited increased expression of p-Erk, p-p38, and IL-1β in wounded tissue, ipsilateral L4/5 DRGs, and dorsal horn. Prior intraplantar or intrathecal injection of MMP-9- and MMP-2-specific inhibitors partially prevented reductions of paw withdrawal threshold and paw withdrawal latency following plantar incision. The maturation of IL-1β was also inhibited by the treatment. Moreover, MMP-9 inhibition suppressed p38, and MMP-2 inhibitor reduced the Erk phosphorylation in wounded tissue, DRGs, and dorsal horn. Immunofluorescence staining detected colocalization of MMP-9 with p38 and MMP-2 with Erk in DRG and spinal cord. Together, the above results reveal that upregulated MMP-9 via p38/IL-1β and MMP-2 via Erk/IL-1β signaling in the wounded tissue, ipsilateral DRG, and dorsal horn contribute to the development of postoperative pain. [ABSTRACT FROM AUTHOR]

• A transition zone (TZ) lies in rat dysgranular sensorimotor cortex. • TZ neurons are highly responsive to thermonoxious skin stimuli. • TZ is a likely rat homolog of nociresponsive region area 3a in primates. • TZ inactivation increases the latency of the nociceptive withdrawal reflex. • TZ likely exerts tonic facilitation over spinal cord neurons producing this reflex. The primary somatosensory cortex (S1) comprises a number of functionally distinct regions, reflecting the diversity of somatosensory receptor submodalities innervating the body. In particular, two spatially and functionally distinct nociceptive regions have been described in primate S1 (Vierck et al., 2013; Whitsel et al., 2019). One region is located mostly in Brodmann cytoarchitectonic area 1, where a subset of neurons exhibit functional characteristics associated with myelinated Aδ nociceptors and perception of 1st/sharp, discriminative pain. The second region is located at the transition between S1 and primary motor cortex (M1) in area 3a, where neurons exhibit functional characteristics associated with unmyelinated C nociceptors and perception of 2nd/slow, burning pain. To test the hypothesis that in rats the transitional zone (TZ) – which is a dysgranular region at the transition between M1 and S1 – is the functional equivalent of the nociresponsive region of area 3a in primates, extracellular spike discharge activity was recorded from TZ neurons in rats under general isoflurane anesthesia. Thermonoxious stimuli were applied by lowering the contralateral forepaw or hindpaw into a 48–51 °C heated water bath for 5–10 s. Neurons in TZ were found to be minimally affected by non-noxious somatosensory stimuli, but highly responsive to thermonoxious skin stimuli in a slow temporal summation manner closely resembling that of nociresponsive neurons in primate area 3a. Selective inactivation of TZ by topical lidocaine application suppressed or delayed the nociceptive withdrawal reflex, suggesting that TZ exerts a tonic facilitatory influence over spinal cord neurons producing this reflex. In conclusion, TZ appears to be a rat homolog of the nociresponsive part of monkey area 3a. A possibility is considered that this region might be primarily engaged in autonomic aspects of nociception. [ABSTRACT FROM AUTHOR]

• We tested effects of µ-, δ- and κ-opioid receptor agonists on spinal reflexes. • All these agonists suppressed stronger contra- than ipsilateral nociceptive reflexes. • Opioid system regulates sensorimotor processing in the spinal commissural pathways. Nociceptive discharges caused by the unilateral tissue damage are processed in the spinal cord by both ipsi- and contralateral neuronal circuits. The mechanisms of the neurotransmitter control of this bilateral excitation spread is poorly understood. Spinally administered opiates are known to suppress nociceptive transmission and nociceptive withdrawal reflexes. Here we investigated whether three major types of opioid receptors are involved in the bilateral control of the spinal nociceptive sensorimotor processing. Effects of the µ-, δ- and κ-opioid receptor agonists on the ipsi- and contralateral nociceptive reflexes were studied by recording slow ventral root potentials in an isolated spinal cord preparation of the new-born rat. Absolute levels of expression of the opioid genes were analyzed by the droplet digital PCR. Ipsi- and contralateral slow ventral root potentials were most strongly suppressed by the µ-opioid receptor agonist DAMGO, by 63% and 85%, followed by the κ-opioid receptor agonist U-50488H, by 44% and 73%, and δ-opioid receptor agonist leucine-enkephalin, by 27% and 49%, respectively. All these agonists suppressed stronger contra- than ipsilateral responses. Naloxone prevented effects of the agonists indicating that they act through opioid receptors, which, as we show, are expressed in the neonatal spinal cord at the levels similar to those in adults. Thus, opioid receptor agonists suppress the segmental nociceptive reflexes. Stronger contralateral effects suggest that the endogenous opioid system regulates sensorimotor processing in the spinal commissural pathways. These effects of opioids may be relevant for treatment of symmetric clinical pain symptoms caused by unilateral tissue injury. [ABSTRACT FROM AUTHOR]

• Dynorphin A increment contributed to the initiation and maintenance of neuropathic pain after SNL surgery. • Dynorphin A stimulated IL-1β and TNF-α release in the acute phase of neuropathic pain. • Activation of alpha7 n acetylcholine receptor improved neuropathic pain behaviors by decreasing dynorphin A release. Dynorphin A is increased in neuropathic pain models. Activation of α7 n acetylcholine receptor (nAchR) reduces inflammation and pain. Whether activation of α7 nAchR affects dynorphin A release is unknown. The experiments evaluated the proinflammatory effect of dynorphin A in the spinal nerve ligation-induced neuropathic pain models and the effect of α7 nAchR activation on the dynorphin A content. α7 nAchR agonist, PHA-543613 and its antagonist, methyllycaconitine citrate were used and dynorphin A content was measured after spinal nerve ligation and in microglia cultures to test the analgesic mechanisms of α7 nAchR activation. The results showed that dynorphin A content peaked 3 to 7 days after nerve injury, and dynorphin A anti-serum intrathecal injection decreased IL-β and TNF-α content a week after nerve injury. Activation of α7 nAchR by PHA-543613 alleviated neuropathic pain behaviors and decreased dynorphin A concentration in the ipsilateral spinal cords. Also, PHA-543613 decreased dynorphin A release from the microglia cultures to LPS stimulation by activation of α7 nAchR. Our results suggest that dynorphin A contribute to the development and maintenance of neuropathic pain and that decreasing dynorphin A content by activation of α7 AchR of microglia is a potential therapeutic target for treating neuropathic pain. [ABSTRACT FROM AUTHOR]

Highlights • 3 Days culturing of the rat isolated spinal cord reveals an inhibitory synaptopathy. • Loss of inhibitory synapses disrupts cyclic oscillations of locomotor networks. • Loss of inhibitory synapses does not occur in opossum spinal cord in vitro. Abstract The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity. [ABSTRACT FROM AUTHOR]

Highlights • Adult Sema6A knockout mice have corticospinal tract (CST) defects. • The aberrant trajectories were evaluated by 3D reconstruction of PKC γ -stained fibers. • The CST defects in Sema6A knockout mice were highly complex and variable. • 3D reconstruction is a powerful method to visualize these CST axon guidance defects. Abstract The corticospinal tract (CST) has a complex and long trajectory that originates in the cerebral cortex and ends in the spinal cord. Semaphorin 6A (Sema6A), a member of the semaphorin family, is an important regulator of CST axon guidance. Previous studies have shown that postnatal Sema6A mutant mice have CST defects at the midbrain – hindbrain boundary and medulla. However, the routes the aberrant fibers take throughout the Sema6A mutant brain remain unknown. In this study, we performed 3D reconstruction of immunostained CST fibers to reevaluate the details of the abnormal CST trajectories in the brains of adult Sema6A mutant mice. Our results showed that the axon guidance defects reported in early postnatal mutants were consistently observed in adulthood. Those abnormal trajectories revealed by 3D analysis of brain sections were, however, more complex and variable than previously thought. In addition, 3D analysis allowed us to identify a few new patterns of aberrant projections. First, a subset of fibers that separated from and descended in parallel to the main bundle projected laterally at the caudal pons, subsequently changed direction by turning caudally, and extended to the medulla. Second, some abnormal fibers returned to the correct trajectory after deviating substantially from the original tract. Third, some fibers reached the pyramidal decussation normally but did not enter the dorsal funiculus. Section immunostaining combined with 3D reconstruction is a powerful method to track long projection fibers and to examine the entire nerve tracts of both normal and abnormal animals. [ABSTRACT FROM AUTHOR]

Highlights • Oligodendrocyte differentiation program fails after spinal cord injury. • Pg upregulates the oligodendrocyte differentiation program. • Pg increases the number of TGFβ1 + astrocytes and microglial cells. • TGFβ1 emerges as a possible mediator of Pg differentiating effects. Abstract Spinal cord lesions result in chronic demyelination as a consequence of secondary injury. Although oligodendrocyte precursor cells proliferate the differentiation program fails. Successful differentiation implies progressive decrease of transcriptional inhibitors followed by upregulation of activators. Progesterone emerges as an anti-inflammatory and pro-myelinating agent which improves locomotor outcome after spinal cord injury. In this study, we have demonstrated that spinal cord injury enhanced oligodendrocyte precursor cell number and decreased mRNA expression of transcriptional inhibitors (Id2, Id4, hes5). However, mRNA expression of transcriptional activators (Olig2, Nkx2.2, Sox10 and Mash1) was down-regulated 3 days post injury. Interestingly, a differentiation factor such as progesterone increased transcriptional activator mRNA levels and the density of Olig2- expressing oligodendrocyte precursor cells. The differentiation program is regulated by extracellular signals which modify transcriptional factors and epigenetic players. As TGFβ1 is a known oligodendrocyte differentiation factor which is regulated by progesterone in reproductive tissues, we assessed whether TGFβ1 could mediate progesterone remyelinating actions after the lesion. Notwithstanding that astrocyte, oligodendrocyte precursor and microglial cell density increased after spinal cord injury, the number of these cells which expressed TGFβ1 remained unchanged regarding sham operated rats. However, progesterone treatment increased TGFβ1 mRNA expression and the number of astrocytes and microglial TGFβ1 expressing cells which would indirectly enhance oligodendrocyte differentiation. Therefore, TGFβ1 arises as a potential mediator of progesterone differentiating effects on oligodendrocyte linage. [ABSTRACT FROM AUTHOR]

Highlights • In neonatal mice the restoration of CPG output occurs few days after high lumbar SCI. • After injury multi-rhythmic state recovers with few noticeable differences. • After recovery, the CPG shows a higher sensitivity to 5-HT. • Expression for 5-HT receptor subtypes is altered while DA receptors remain unchanged. Abstract The spinal locomotor central pattern generator (CPG) in neonatal mice exhibits diverse output patterns, ranging from sub-rhythmic to multi-rhythmic to fictive locomotion, depending on its general level of excitation and neuromodulatory status. We have recently reported that the locomotor CPG in neonatal mice rapidly recovers the ability to produce neurochemically induced fictive locomotion following an upper lumbar spinal cord compression injury. Here we address the question of recovery of multi-rhythmic activity and the serotonin-sensitivity of the CPG. In isolated spinal cords from control and 3 days post-injury mice, application of dopamine and NMDA elicited multi-rhythmic activity with slow and fast components. The slow component comprised 10–20 s episodes of activity that were synchronous in ipsilateral or all lumbar ventral roots, and the fast components involved bursts within these episodes that displayed coordinated patterns of alternation between ipsilateral roots. Rhythm strength was the same in control and injured spinal cords. However, power spectral analysis of signal within episodes showed a reduced peak frequency after recovery. In control spinal cords, serotonin triggered fictive locomotion only when applied at high concentration (30 µM, constant NMDA). By contrast, in about 50% of injured preparations fictive locomotion was evoked by 2–3 times lower serotonin concentrations (10–15 µM). This increased serotonin sensitivity was correlated with post-injury changes in the expression of specific serotonin receptor transcripts, but not of dopamine receptor transcripts. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Ferroptosis, an iron dependent cell death pathway, contributed to secondary injury of SCI. • Ferroptosis inhibitor SRS 16-86 inhibits ferroptosis in SCI by up-regulating GPX4, GSH and xCT, and downregulation of 4HNE. • Inhibiting ferroptosis is a novel approach to repair traumatic spinal cord injury. Abstract Cell death is a key issue in spinal cord secondary injury. Ferroptosis is recently discovered as an iron-dependent type of cell death that is distinct from other forms of cell death pathways such as apoptosis and necrosis. This research is aimed to investigate the role of ferroptosis in spinal cord injury (SCI) pathophysiology, and to explore the effectiveness of ferroptosis inhibitor on SCI. We examined the ferroptosis markers and the factors in a rat contusion SCI model. Seen from transmission electron microscopy (TEM) following SCI, mitochondria showed ferroptotic characteristic changes. Treatment with a ferroptosis inhibitor SRS 16-86 enhanced functional recovery after SCI through the upregulation of anti-ferroptosis factor GPX4, GSH and xCT, and the downregulation of the lipid peroxidation marker 4HNE. SRS 16-86 treatment alleviated astrogliosis and enhanced neuronal survival after SCI. The inflammatory cytokine levels (IL-1β, TNF-α and ICAM-1) were decreased significantly post SRS 16-86 treatment after SCI. These findings suggest strong correlation between ferroptosis and the secondary injury of SCI. The effectiveness of ferroptosis inhibitor SRS-16-86 on SCI repair leads to the identification of a novel therapeutic target for SCI. [ABSTRACT FROM AUTHOR]

The effects of substance P on the morphine-evoked release of adenosine were examined. Substance P alone produced a multiphasic effect on release of adenosine, with release occurring at low nanomolar concentrations and at a micromolar concentration, but not at intermediate concentrations. An inactive dose of substance P augmented the morphine-evoked release of adenosine at a nanomolar concentration of morphine. Release of adenosine by substance P alone (1 nM) or substance P/morphine (100 nM/10 nM) was Ca2(+)-dependent and originated from capsaicin-sensitive nerve terminals.

The present study determined the Ca(2+)-dependence of the release of adenosine 5'-triphosphate (ATP) from dorsal spinal cord synaptosomes evoked by depolarization with K+ and capsaicin, and the effect of intrathecal capsaicin pretreatment, dorsal rhizotomy and intrathecal pretreatment with 6-hydroxydopamine on such release. Release of ATP evoked by K+ was Ca(2+)-dependent, while that evoked by capsaicin was Ca(2+)-independent. Capsaicin pretreatment (60 micrograms, 7 days), which lesions small diameter afferents, did not alter release of ATP evoked by either K+ or capsaicin. Dorsal rhizotomy, which lesions all afferents, produced a significant reduction in the amount of ATP released from the rhizotomized side compared to the intact side. Pretreatment with 6-hydroxydopamine (100 micrograms, 7 days) to destroy adrenergic nerve terminals, markedly reduced spinal cord noradrenaline levels, but did not alter the K(+)-evoked release of ATP. These results suggest that some K(+)-evoked release of ATP could originate from large but not small diameter afferent nerve terminals in the spinal cord. ATP does not appear to originate from small diameter afferents as, although ATP is released by in vitro exposure to capsaicin, such release occurs only at high concentrations, release is Ca(2+)-independent and it is unaltered by pretreatment with capsaicin. The bulk of the ATP released from the spinal cord does not originate from descending noradrenergic nerve terminals.

Highlights • HBO 2 -induced antinociception is mediated by spinal GABA A but not GABA B receptors. • Acetic acid reduced expression of nNOS and GABA A β3pSER 408/409. • HBO 2 treatment restored nNOS and GABA A β3pSER 408/409 expression to control levels. • Protein expression was altered in lumbar but not thoracic spinal cord. Abstract New pain treatments are in demand due to the pervasive nature of pain conditions. Hyperbaric oxygen (HBO 2) has shown potential in treating pain in both clinical and preclinical settings, although the mechanism of this effect is still unknown. The aim of this study was to investigate whether the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) is involved in HBO 2 -induced antinociception in the central nervous system (CNS). To accomplish this goal, pharmacological interactions between GABA drugs and HBO 2 were investigated using the behavioral acetic acid abdominal constriction test. Western blotting was used to quantify protein changes that might occur as a result of the interactions. GABA A but not GABA B receptor antagonists dose-dependently reduced HBO 2 antinociception, while antagonism of the GABA reuptake transporter enhanced this effect. Western blot results showed an interaction between the pain stimulus and HBO 2 on expression of the phosphorylated β3 subunit of the GABA A receptor at S408/409 in homogenates of the lumbar but not thoracic spinal cord. A significant interaction was also found in neuronal nitric oxide synthase (nNOS) expression in the lumbar but not thoracic spinal cord. These findings support the notion that GABA may be involved in HBO 2 -induced antinociception at the GABA A receptor but indicate that more study will be needed to understand the intricacies of this interaction. [ABSTRACT FROM AUTHOR]

Highlights • Spinal glutamatergic and AT1 receptors lead to sympathoexcitation in 2K1C rats. • Spinal Glutamate and Ang II control the sympathetic reflex responses to the kidneys. • AT1 receptors are selectively upregulated in lower thoracic levels in 2K1C rats. Abstract The role of spinal cord neurons in renal sympathoexcitation remains unclear in renovascular hypertension, represented by the 2-kidney, 1-clip (2K1C) model. Thus, we aimed to assess the influence of spinal glutamatergic and AT1 angiotensin II receptors on renal sympathetic nerve activity (rSNA) in 2K1C Wistar rats. Hypertension was induced by clipping the renal artery with a silver clip. After six weeks, a catheter (PE-10) was inserted into the subarachnoid space and advanced to the T10-11 vertebral level in urethane-anaesthetized rats. The effects of intrathecally (i.t.) injected kynurenic acid (KYN) or losartan (Los) on blood pressure (BP) and rSNA were analysed over 2 consecutive hours. KYN induced a significantly larger drop in rSNA among 2K1C rats than among control (CTL) rats (CTL vs. 2K1C: −8 ± 3 vs. −52 ± 9 spikes/s after 120′). Los also evoked a significantly larger drop in rSNA among 2K1C rats than among CTL rats starting at 80′ after administration (CTL vs. 2K1C – 80 min: −10 ± 2 vs. –32 ± 6∗; 100 min: −15 ± 4 vs. −37 ± 9∗; 120 min: −12 ± 5 vs. −37 ± 8∗ spikes/s). KYN decreased BP similarly in the CTL and 2K1C groups; however, Los significantly decreased BP in the 2K1C group only. We found upregulation of AT1 gene expression in the T11-12 spinal segments in the 2K1C group but no change in gene expression for AT2 or ionotropic glutamate (NMDA, kainate and AMPA) receptors. Thus, our data show that spinal ionotropic glutamatergic and AT1 receptors contribute to increased rSNA in the 2K1C model, leading to the maintenance of hypertension; however, the participation of spinal AT1 receptors seems to be especially important in the establishment of sympathoexcitation in this model. The origins of those projections, i.e., the brain areas involved in establishing the activity of spinal glutamatergic and angiotensinergic pathways, remain unclear. [ABSTRACT FROM AUTHOR]

There is evidence that neuronal injury can affect uninjured neurons in the same neural circuit. The overall goal of this study was to understand the effects of peripheral nerve injury on uninjured neurons located in the central nervous system (CNS). As a model, we examined whether axotomy (transection of postganglionic axons) of the superior cervical ganglion (SCG) affected the uninjured, preganglionic neurons that innervate the SCG. At 7 days post-injury a reduction in choline acetyltransferase (ChAT) and synaptophysin immunoreactivity in the SCG, both markers for preganglionic axons, was observed, and this reduction persisted at 8 and 12 weeks post-injury. No changes were observed in the number or size of the parent cell bodies in the intermediolateral cell column (IML) of the spinal cord, yet synaptic input to the IML neurons was decreased at both 8 and 12 weeks post-injury. In order to understand the mechanisms underlying these changes, protein levels of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) were examined and reductions were observed at 7 days post-injury in both the SCG and spinal cord. Taken together these results suggest that axotomy of the SCG led to reduced BDNF in the SCG and spinal cord, which in turn influenced ChAT and synaptophysin expression in the SCG and also contributed to the altered synaptic input to the IML neurons. More generally these findings provide evidence that the effects of peripheral injury can cascade into the CNS and affect uninjured neurons. [ABSTRACT FROM AUTHOR]

NLX-112 (a.k.a. F13640 or befiradol) possesses marked activity in a variety of animal models of pain and of neuropsychiatric disorders; it exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine1A (5–HT 1A ) receptors. Although NLX-112 has been shown to activate 5-HT 1A postsynaptic heteroreceptors in the prefrontal cortex (PFC), a brain region involved in the control of depressive states, the influence of NLX-112 on spinal cord 5-HT 1A receptors (implicated in the control of pain) has not been described. Here we report on the ability, in rats, of NLX-112 to elicit analgesia in the intraplantar formalin model of nociceptive pain following intrathecal (i.t.) administration, and its ability to produce antidepressant-like activity in the forced swim test (FST) following in situ PFC microinjection. NLX-112, injected i.t. (L5-L6 region) induced analgesic effects in the formalin model of tonic nociceptive pain. At 20 µg, it almost abolished the effect of formalin on both the paw licking and paw elevation measures, and in both the early (0–5 min after formalin administration, reflecting acute pain) and the late (22.5–27.5 min, reflecting inflammatory pain) phases. The effects of NLX-112 (20 µg i.t.) were reversed by co-administration of 20 µg i.t. of the 5-HT 1A receptor antagonist, WAY100635. Furthermore, the analgesic effects of systemically administered NLX-112 (0.63 mg/kg i.p.) were reversed by i.t. administration of WAY100635 (20 µg), most notably on paw licking. Finally, microinjection of NLX-112, bilaterally in the PFC, dose-dependently (MED 4 µg) and markedly reduced immobility in the FST (circa 90% reduction at 32 µg). In conclusion, the present data demonstrate that activation of spinal cord-located 5-HT 1A receptors is sufficient for NLX-112 to mediate its analgesic effects in a rat model of tonic nociceptive pain. The data also highlight the involvement of PFC 5-HT 1A receptors in the antidepressant-like activity of NLX-112 in the FST. Overall, the study suggests that highly selective and high efficacy 5–HT 1A receptors agonists, such as NLX-112, could be useful to treat painful conditions associated with depressive states, through activation of different sub-populations of 5-HT 1A receptors. [ABSTRACT FROM AUTHOR]

Extensive pathological evidence indicates that axonal degeneration represents an early and critical event in amyotrophic lateral sclerosis (ALS). Unfortunately, few MRI studies have focused in the early detection of white matter (WM) alterations in the spinal cord region. To unveil these WM changes, we performed high resolution diffusion tensor imaging (DTI) and correlated the results with histological analysis of adjacent slices taken from the spinal cords of presymptomatic mice. The DTI studies demonstrated a significant reduction in fractional anisotropy (FA) as well as axial diffusivities (AD) and an increase in radial diffusivity (RD), predominantly at lower segments of the spinal cord. Increases in FA and a reduction in AD and RD were observed in spinal cord (SC) gray matter (GM). Diffusion changes are associated with early and progressive alterations in axonal connectivity following a distal to proximal progression. Histological data tagging neuronal, axonal and glial cell markers demonstrated presymptomatic alterations in spinal cord WM and GM. This study demonstrates that DTI methods are optimal preclinical imaging tools to detect structural anomalies in WM and GM spinal cord during early stages of the disease. [ABSTRACT FROM AUTHOR]

• Spinal astrocytes in Stress-induced gastric mucosal damage in rat. • Astrocytic toxin L-a-aminoadipate (L-AA) prevented the stress-induced gastric damage. • Astrocytes contributed to the stress-induced gastric damage via CX43. Rat restraint water-immersion stress (RWIS) is a compound stress of high intensity and is widely used to study the pathological mechanisms of stress gastric ulcers. The spinal cord, as a part of the central nervous system, plays a dominant role in the gastrointestinal tract, but whether the spinal cord is involved in rat restraint water-immersion stress (RWIS)-induced gastric mucosal damage has not been reported. In this study, we examined the expression of spinal astrocytic glial fibrillary acidic protein (GFAP), neuronal c-Fos, connexin 43 (Cx43), and p-ERK1/2 during RWIS by immunohistochemistry and Western blotting. In addition, we intrathecally injected the astrocytic toxin L-a-aminoadipate (L-AA), gap junction blocker carbenoxolone (CBX), and ERK1/2 signaling pathway inhibitor PD98059 to explore the role of astrocytes in the spinal cord in RWIS-induced gastric mucosal damage and its possible mechanism in rats. The results showed that the expression of GFAP, c-Fos, Cx43, and p-ERK1/2 was significantly elevated in the spinal cord after RWIS. Intrathecal injection of both the astrocyte toxin L-AA and the gap junction blocker CBX significantly attenuated RWIS-induced gastric mucosal damage and decreased the activation of astrocytes and neurons induced in the spinal cord. Meanwhile, the ERK1/2 signaling pathway inhibitor PD98059 significantly inhibited gastric mucosal damage, gastric motility and RWIS-induced activation of spinal cord neurons and astrocytes. These results suggest that spinal astrocytes may regulate the RWIS-induced activation of neurons via CX43 gap junctions and play a critical role in RWIS-induced gastric mucosa damage through the ERK1/2 signaling pathway. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate proteoglycans produced in glial scar tissue are a major inhibitory factor for axonal regeneration after central nervous system injury in mammals. The inhibition is largely due to chondroitin sulfates, whose effects differ according to the sulfation pattern. In contrast to mammals, fish nerves spontaneously regenerate beyond the scar tissue after spinal cord injury, although the mechanisms that allow for axons to pass through the scar are unclear. Here, we used immunohistochemistry to examine the expression of two chondroitin sulfates with different sulfation variants at the lesion site in goldfish spinal cord. The intact spinal cord was immunoreactive for both chondroitin sulfate-A (CS-A) and chondroitin sulfate-C (CS-C), and CS-A immunoreactivity overlapped extensively with glial processes positive for glial fibrillary acidic protein. At 1 week after inducing the spinal lesion, CS-A immunoreactivity was observed in the cell bodies and extracellular matrix, as well as in glial processes surrounding the lesion center. At 2 weeks after the spinal lesion, regenerating axons entering the lesion center overtook the CS-A abundant area. In contrast, at 1 week after lesion induction, CS-C immunoreactivity was significantly decreased, and at 2 weeks after lesion induction, CS-C immunoreactivity was observed along the regenerating axons entering the lesion center. The present findings suggest that after spinal cord injury in goldfish, chondroitin sulfate proteoglycans are deposited in the extracellular matrix at the lesion site but do not form an impenetrable barrier to the growth of regenerating axons. [ABSTRACT FROM AUTHOR]

Lumbar spinal cord stimulation (LSCS) is reportedly effective for the recovery of locomotive intraspinal neural network, motor cortex and basal ganglia in animals with complete spinal cord injury and parkinsonism. We evaluated the effect of LSCS in combination with gait training on the recovery of locomotive function and brain plasticity using a rat model of brain ischemia. Adult male Sprague Dawley rats with ischemia were randomly assigned into one of four groups: sham treatment (group 1), LSCS only (group 2), LSCS with gait training and 50% (group 3) and 80% (group 4) of body weight support. Evaluations before randomization and 4 weeks after intervention included motor scoring index, real-time PCR and Western blot. Motor scoring index was significantly improved after the intervention in groups 2 and 3. The ratio of phospho-protein kinase C (PKC) to PKC measured in the infarcted area tended to be higher in groups 3 and 4. Protein expression of mGluR2 and mRNA expression of mGluR1 measured in the contralateral cortex were lower in groups 3 and 4. The ratio of phospho-Akt to Akt and mRNA expression of vascular endothelial growth factor measured in the ischemic border zone were higher in group 2. The mRNA expression of MAP1b measured in the infarcted area was significantly higher in group 2. The findings suggest that LSCS and gait training with an adequate amount of body weight support may promote brain plasticity and facilitate the functional recovery. [ABSTRACT FROM AUTHOR]

Paraplegia caused by spinal cord ischemia is a severe complication following surgeries in the thoracic aneurysm. HMGB1 has been recognized as a key mediator in spinal inflammatory response after spinal cord injury. Electroacupuncture (EA) pretreatment could provide neuroprotection against cerebral ischemic injury through inhibition of HMGB1 release. Therefore, the present study aims to test the hypothesis that EA pretreatment protects against spinal cord ischemia-reperfusion (I/R) injury via inhibition of HMGB1 release. Animals were pre-treated with EA stimulations 30 min daily for 4 successive days, followed by 20-min spinal cord ischemia induced by using a balloon catheter placed into the aorta. We found that spinal I/R significantly increased mRNA and cytosolic protein levels of HMGB1 after reperfusion in the spinal cord. The EA-pretreated animals displayed better motor performance after reperfusion along with the decrease of apoptosis, HMGB1, TNF-α and IL-1β expressions in the spinal cord, whereas these effects by EA pretreatment was reversed by rHMGB1 administration. Furthermore, EA pretreatment attenuated the down-regulation of LXA 4 receptor (ALX) expression induced by I/R injury, while the decrease of HMGB1 release in EA-pretreated rats was reversed by the combined BOC-2 (an inhibitor of LXA 4 receptor) treatment. In conclusion, EA pretreatment may promote spinal I/R injury through the inhibition of HMGB1 release in a LXA 4 receptor-dependent manner. Our data may represent a new therapeutic technique for treating spinal cord ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

• CFA-induced inflammatory pain upregulated caspase-11 expression in the spinal cord of mice. • Caspase-11 deficient mice attenuated pain hypersensitivity in the later phase of CFA-induced pain of mice. • Caspase-11 deficient mice attenuated CFA-induced neuroinflammation. Chronic pain is a common disease that severely disrupts the quality of life. Persistent neuroinflammation and central sensitization play important roles in its pathogenesis. Caspase-11 is a critical modulator of inflammation of central nervous system. However, its role in chronic pain remains elusive. In this study, chronic pain and acute pain were induced via injecting complete Freund's adjuvant (CFA) and 5 % formalin into the plantar of the right hind paw of wild-type (WT) and Caspase-11 deficient (Caspase-11−/−) mice, respectively. In WT mice, CFA injection significantly decreased the hind paw mechanical pain threshold in Von Frey test on 1–7 days after injection and increased the caspase-11 level of ipsilateral dorsal horn of spinal cord on day 2 and day 5 after injection. Compared to the WT mice, Caspase-11−/− mice showed significantly higher mechanical pain threshold in the later phase of CFA-induced pain, but not in the early phase, and had no significant difference in 5 % formalin induced licking and flinching behavior. In addition, the microglial activation, and the mRNA levels of caspase-1 and IL-18 in the spinal cord of Caspase-11−/− mice restored to baseline on the day 5 after CFA injection, but not in WT mice. Our data indicated that Caspase-11 contributed to persistent inflammation in ipsilateral dorsal horn of spinal cord, and consequently pain hypersensitivity in the later phase of CFA-induced pain. [ABSTRACT FROM AUTHOR]

Spinal cord injuries result in severe neurological deficits and neuronal loss, with poor functional recovery. Mesenchymal stem cells have shown promising results; therefore the present objective of this work was to compare motor recovery after treatment with human dental pulp stem cells (hDPSC) cultivated in monolayer (2D) or as spheroids (3D), following avulsion and reimplantation of spinal motor roots in adult rats. Thus, 72 adult female Lewis rats were divided into 4 groups: avulsion (AV); avulsion followed by reimplantation (AR); avulsion associated with reimplant and 2D cell therapy (AR + 2D), and avulsion associated with reimplant and 3D cell therapy (AR + 3D). The application of the cells in 2D and 3D was performed by microsurgery, with subsequent functional assessment using a walking track test (Catwalk system), immunohistochemistry, neuronal survival, and qRT-PCR in 1-, 4-, and 12-weeks post-injury. The animals in the AR + 2D and AR + 3D groups showed the highest neuronal survival rates, and immunofluorescence revealed downregulation of GFAP, and Iba-1, with preservation of synaptophysin, indicating a reduction in glial reactivity, combined with the maintenance of pre-synaptic inputs. There was an increase in anti-inflammatory (IL-4, TGFβ) and a reduction of pro-inflammatory factors (IL-6, TNFα) in animals treated with reimplantation and hDPSC. As for the functional recovery, in all analyzed parameters, the AR + 2D group performed better and was superior to the avulsion alone. Overall, our results indicate that the 2D and 3D cell therapy approaches provide successful immunomodulation and motor recovery, consistent with advanced therapies after spinal cord injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

• GlyT2 is upregulated in rats with neuropathic pain. • BoNT/A counters the increase of GlyT2 in CCI rats. • Overexpression of GlyT2 abolished the antinociceptive effect of BoNT/A. Botulinum toxin type A (BoNT/A) is a potent toxin, acts by cleaving synaptosome-associated-protein-25 (SNAP-25) to regulate the release of the neural transmitter and shows analgesic effect in neuropathic pain. However, the mechanisms of BoNT/A actions involved in nociceptions remain unclear. Glycine transporter 2 (GlyT2) is an isoform of glycine transporters, which plays an important role in the regulation of glycinergic neurotransmission. Inhibition of GlyTs could decrease pain sensation in neuropathic pain, the role of GlyT2 in the analgesic effect of BoNT/A has not been studied yet. In our present study, we demonstrated that the protein levels of GlyT2 and SNAP-25 were upregulated in the spinal cord after the development of chronic constriction injury (CCI)-induced neuropathic pain. Intraplantar application of BoNT/A (20 U/kg) attenuated mechanical allodynia induced by CCI and downregulated GlyT2 expression in the spinal cord. The application of BoNT/A s also decreased the expression of GlyT2 in pheochromocytoma (PC12) cells. Moreover, intrathecal application of lentivirus-mediated GlyT2 reversed the antinociceptive effect of BoNT/A in CCI rats. These findings indicate that GlyT2 contributes to the antinociceptive effect of BoNT/A and suggest a novel mechanism underlying BoNT/A's antinociception action. [ABSTRACT FROM AUTHOR]

In this study, we examined whether electroacupuncture (EA) represses pruritogen-induced microglial activation. Immunohistochemical studies revealed that a subcutaneous (s.c.) injection of the pruritogen 5′-guanidinonaltrindole (GNTI; 0.3 mg/kg) to the back of the neck in mice induced acute expression of the ionized calcium-binding adaptor molecule 1 (Iba1) in both gray and white matter of the spinal cord, with the highest expression in the dorsal horn area. EA application (2 Hz) to LI4 and LI11 attenuated GNTI-induced scratching behavior and repressed GNTI-induced Iba1 expression and Iba1 (+) microglia in the dorsal horn. In contrast, EA at the ST36 acupoint had no such effects. Confocal image analysis revealed co-expression of phosphorylated p38 and Iba1 in microglia with EA at the ST36 acupoint, but not at the LI4 or LI11 acupoints. In Western blot analysis, s.c. injection of GNTI to the back of the neck increased Iba1 and phospho-p38 expression in the spinal cord as compared with injection of saline, while EA at LI4 and LI11 reduced GNTI-induced expression of Iba1 and phospho-p38. These findings indicate that EA at LI4 and LI11, but not at ST36, reduces GNTI-induced microglial activation in the mouse spinal cord. [ABSTRACT FROM AUTHOR]

Pregabalin is thought to exert its therapeutic effect in neuropathic pain via binding to α2δ−1 subunits of voltage-gated calcium (Ca 2+ ) channels. However, the exact analgesic mechanism after its binding to α2δ−1 subunits remains largely unknown. Whether a clinical concentration of pregabalin (≈10 μM) can cause acute inhibition of dorsal horn neurons in the spinal cord is controversial. To address this issue, we undertook intracellular Ca 2+ -imaging studies using spinal cord slices with an intact attached L5 dorsal root, and examined if pregabalin acutely inhibits the primary afferent stimulation-evoked excitation of dorsal horn neurons in normal rats and in rats with streptozotocin-induced painful diabetic neuropathy. Under normal conditions, stimulation of a dorsal root evoked Ca 2+ signals predominantly in the superficial dorsal horn. Clinically relevant (10 μM) and a very high concentration of pregabalin (100 μM) did not affect the intensity or spread of dorsal root stimulation-evoked Ca 2+ signals, whereas an extremely high dose of pregabalin (300 μM) slightly but significantly attenuated Ca 2+ signals in normal rats and in diabetic neuropathic (DN) rats. There was no difference between normal rats and DN rats with regard to the extent of signal attenuation at all concentrations tested. These results suggest that the activity of dorsal horn neurons in the spinal cord is not inhibited acutely by clinical doses of pregabalin under normal or DN conditions. It is very unlikely that an acute inhibitory action in the dorsal horn is the main analgesic mechanism of pregabalin in neuropathic pain states. [ABSTRACT FROM AUTHOR]

The neural pathways underlying the respiratory variation dependent on vigilance states remain unsettled. In the present study, we examined the orexinergic innervation of Kölliker-Fuse nucleus (KFN) neurons sending their axons to the rostral ventral respiratory group (rVRG) and phrenic nucleus (PhN) as well as to the hypoglossal nucleus (HGN) by using a combined retrograde tracing and immunohistochemistry. After injection of cholera toxin B subunit (CTb) into the KFN, CTb-labeled neurons that are also immunoreactive for orexin (ORX) were found prominently in the perifornical and medial regions and additionally in the lateral region of the hypothalamic ORX field. After injection of fluorogold (FG) into the rVRG, PhN or HGN, we found an overlapping distribution of ORX-immunoreactive axon terminals and FG-labeled neurons in the KFN. Within the neuropil of the KFN, asymmetrical synaptic contacts were made between these terminals and neurons. We further demonstrated that many neurons labeled with FG injected into the rVRG, PhN, or HGN are immunoreactive for ORX receptor 2. Present data suggest that rVRG-, PhN- and HGN-projecting KFN neurons may be under the excitatory influence of the ORXergic neurons for the state-dependent regulation of respiration. [ABSTRACT FROM AUTHOR]

Lamina X of the spinal cord is a functionally diverse area with roles in locomotion, autonomic control and processing of mechano and nociceptive information. It is also a neurochemically diverse region. However, the different populations of cells in lamina X remain to be fully characterised. To determine the co-localisation of the enzymes responsible for the production of GABA and acetylcholine (which play major roles in the spinal cord) in lamina X of the adult and juvenile mouse, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurons, combined with choline acetyltransferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurons were observed in lamina X of both adult and juvenile mice and in both age groups a population of cells containing both ChAT-IR and GAD67-GFP were observed in lumbar, thoracic and cervical spinal cord. Such dual labelled cells were predominantly located ventral to the central canal. Immunohistochemistry for vesicular acetylcholine transporter (VAChT) and GAD67 revealed a small number of double labelled terminals located lateral, dorsolateral and ventrolateral to the central canal. This study therefore describes in detail a population of ChAT-IR/GAD67-GFP neurons predominantly ventral to the central canal of the cervical, thoracic and lumbar spinal cord of adult and juvenile mice. These cells potentially correspond to a sub-population of the cholinergic central canal cluster cells which may play a unique role in controlling spinal cord circuitry. [ABSTRACT FROM AUTHOR]

The role of P2X 2/3 , P2X 3 , P2X 4 or P2X 7 and P2Y 2 , P2Y 6 , and P2Y 12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y 1 receptors is scarcely studied. In this study we assessed the role of P2Y 1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y 1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y 1 receptor antagonist on the expression of P2Y 1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y 1 receptors in DRG but not in the dorsal part of the spinal cord at 1–3 days after injury. Intrathecal injection of the selective P2Y 1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1–3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y 1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y 1 receptors up-regulation. Our results suggest that P2Y 1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the effects of hindlimb unloading (HU) on the expression of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), together with the expression of their high-affinity receptors tropomyosin receptor kinase C (TrkC) and tropomyosin receptor kinase B (TrkB), in lumbar (L4–6) segment of the spinal cord and in the soleus muscle. The mRNA and protein levels of the genes of interest were compared using quantitative PCR and western blot assays. Immunohistochemistry for NT-3 and BDNF was used to detect the levels of protein in the motoneurons in the lateral motor column. In this study, NT-3 and BDNF mRNA and protein expression were significantly increased in the spinal cord and soleus muscle after HU. NT-3 immunoreactivity, but not BDNF immunoreactivity, was significantly increased in the large motoneurons located in lateral motor column after 14 days of HU. The level of TrkC protein in the spinal cord and soleus muscle were significantly elevated after both 7 days and 14 days of HU. However, TrkC mRNA, TrkB mRNA and TrkB protein levels did not change significantly. Elevated BDNF, NT-3 and TrkC levels in the neuromuscular system indicate that neurotrophins are involved in HU-induced neuromuscular plasticity. NT-3 is a candidate to mediate the synaptic efficacy between alpha motoneurons and group Ia afferents. [ABSTRACT FROM AUTHOR]

[Display omitted] • Incision enhanced Hevin level and neurexin1β/neuroligin1 interaction in dorsal horn. • Intrathecal Hevin siRNA inhibited postoperative pain hypersensitivity. • Intrathecal Hevin siRNA reduced incision-induced neurexin1β/neuroligin1 interaction. • Intrathecal Hevin siRNA reduced synaptic target of AMPA receptor GluA1 subunit. The astrocytes-secreted active molecule, Hevin considerably contributes in the transsynaptic bridge of neurexin1β/neuligin1 in excitatory synapse. Previous studies have demonstrated that activity-dependent synaptic recruitment of spinal neuroligin1 and GluA1-containing AMPA receptors (AMPARs) is involved in incisional, inflammatory and neuropathic pain. Here, we hypothesized that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1β/neuroligin1-mediated synaptic targeting of GluA1-containing AMPARs in spinal dorsal horns (DH). Our results showed that plantar incision induced significant postoperative pain behavior, which was described by the cumulative pain scores. At 1 d and 3 d post-incision, Hevin expression was considerably elevated in ipsilateral DHs, although it recovered to baseline value at 5 d following the incision. At 1 d post plantar incision, the neurexin1β/neuroligin1 interactions significantly increased in ipsilateral DHs in rats subjected to incision when compared with those in control rats. Intrathecal pretreatments of small interference RNA targeting Hevin substantially suppressed postoperative pain hypersensitivity and reduced the neurexin1β/neurolgin1 interaction as well as the synaptic targeting of GluA1 in ipsilateral spinal DHs. These data suggest that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1β/neuroligin1 interaction and subsequent synaptic targeting of GluA1-containing AMPARs in ipsilateral spinal DHs. It provides new insights into the role of Hevin-mediated trans -synaptic regulation in postoperative pain hypersensitivity, which would help develop a novel therapeutic strategy. [ABSTRACT FROM AUTHOR]

Serotonin (5-HT) participates in pain modulation by interacting with different 5-HT receptors. The role of 5-HT 5A receptor in neuropathic pain has not previously studied. The purpose of this study was to investigate: A) the role of 5-HT 5A receptors in rats subjected to spinal nerve injury; B) the expression of 5-HT 5A receptors in dorsal spinal cord and dorsal root ganglia (DRG). Neuropathic pain was induced by L5/L6 spinal nerve ligation. Tactile allodynia in neuropathic rats was assessed with von Frey filaments. Western blot methodology was used to determine 5-HT 5A receptor protein expression. Intrathecal administration (on day 14th) of 5-HT (10–100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03–0.3 nmol) reversed nerve injury-induced tactile allodynia. Intrathecal non-selective (methiothepin, 0.1–0.8 nmol) and selective (SB-699551, 1–10 nmol) 5-HT 5A receptor antagonists reduced, by ~60% and ~25%, respectively, the antiallodynic effect of 5-HT (100 nmol) or 5-CT (0.3 nmol). Moreover, both selective 5-HT 1A and 5-HT 1B/1D receptor antagonists, WAY-100635 (0.3–1 nmol) and GR-127935 (0.3–1 nmol), respectively, partially diminished the antiallodynic effect of 5-HT or 5-CT by about 30%. Injection of antagonists, by themselves, did not affect allodynia. 5-HT 5A receptors were expressed in the ipsilateral dorsal lumbar spinal cord and DRG and L5/L6 spinal nerve ligation did not modify 5-HT 5A receptor protein expression in those sites. Results suggest that 5-HT 5A receptors reduce pain processing in the spinal cord and that 5-HT and 5-CT reduce neuropathic pain through activation of 5-HT 5A and 5-HT 1A/1B/1D receptors. These receptors could be an important part of the descending pain inhibitory system. [ABSTRACT FROM AUTHOR]

Paraplegia following complex aortic intervention relies on crude evaluation of lower extremity strength such as whether the patient can lift their legs or flex the ankle. Little attention has been given to the possible long-term neurologic sequelae following these procedures in patients appearing functionally normal. We hypothesize that mice subjected to minimal ischemic time will have functional and histological changes despite the gross appearance of normal function. Male mice underwent 3 min of aortic occlusion ( n =14) or sham surgery ( n =4) via a median sternotomy. Neurologic function was graded by Basso Motor Score (BMS) preoperatively and at 24 h intervals after reperfusion. Mice appearing functionally normal and sham mice were placed on a walking beam and recorded on high-definition, for single-frame motion analysis. After 96hrs, spinal cords were removed for histological analysis. Following 3 min of ischemia, functional outcomes were split evenly with either mice displaying almost normal function n =7 or near complete paraplegia n =7. Additionally, single-frame motion analysis revealed significant changes in gait. Histologically, there was a significant stepwise reduction of neuronal viability, with even the normal function ischemic group demonstrating significant loss of neurons. Despite the appearance of normal function, temporary ischemia induced marked cyto-architectural changes and neuronal degeneration. Furthermore high-definition gait analysis revealed significant changes in gait and activity following thoracic aortic occlusion. These data suggest that all patients undergoing procedures, even with short ischemic times, may have spinal cord injury that is not evident clinically. [ABSTRACT FROM AUTHOR]

The present study was designed to further compare the stepping-like movements generated via epidural (ES) and/or intraspinal (IS) stimulation. We examined the ability to generate stepping-like movements in response to ES and/or IS of spinal lumbar segments L1–L7 in decerebrate cats. ES (5–10 Hz) of the dorsal surface of the spinal cord at L3–L7 induced hindlimb stepping-like movements on a moving treadmill belt, but with no rhythmic activity in the forelimbs. IS (60 Hz) of the dorsolateral funiculus at L1–L3 (depth of 0.5–1.0 mm from the dorsal surface of the spinal cord) induced quadrupedal stepping-like movements. Forelimb movements appeared first, followed by stepping-like movements in the hindlimbs. ES and IS simultaneously enhanced the rhythmic performance of the hindlimbs more robustly than ES or IS alone. The differences in the stimulation parameters, site of stimulation, and motor outputs observed during ES vs. IS suggest that different neural mechanisms were activated to induce stepping-like movements. The effects of ES may be mediated more via dorsal structures in the lumbosacral region of the spinal cord, whereas the effects of IS may be mediated via more ventral propriospinal networks and/or brainstem locomotor areas. Furthermore, the more effective facilitation of the motor output during simultaneous ES and IS may reflect some convergence of pathways on the same interneuronal populations involved in the regulation of locomotion. [ABSTRACT FROM AUTHOR]

We assessed the effects of simulated weightlessness, hindlimb unloading (HU) by 7 days of tail suspension, on noxious mechanically and heat evoked spinal withdrawal reflexes and spinal Fos expression during muscle nociception elicited by intramuscular (i.m.) injection of hypertonic (HT; 5.8%) saline into gastrocnemius muscle in rats. In HU rats, i.m. HT saline-induced secondary mechanical hyperalgesia was enhanced, and secondary heat hypoalgesia was significantly delayed. After 7 days of HU, basal Fos expression in spinal L4-6 segments was bilaterally enhanced only in superficial (I–II) but not middle and deep laminae (III–VI) of the spinal dorsal horn, which finding was not influenced by tail denervation. Unilateral i.m. HT saline injection increased spinal Fos expression bilaterally in both the control rats and 7 days of HU rats. The HT saline-induced bilateral increase of spinal Fos occurred within 0.5 h and reached its peak within 1 h, after which it gradually returned to the control levels within 8 h. Spatial patterns of spinal Fos expression differed between the control group and 7 days of HU group. In superficial laminae, the HT saline-induced increases in Fos expression were higher and in the middle and deep laminae V–VI lower in the 7 days of HU than control rats. It is suggested that supraspinal mechanisms presumably underlie the effects of HU on spinally-organized nociception. Simulated weightlessness may enhance descending facilitation and weaken descending inhibition of nociception. [ABSTRACT FROM AUTHOR]