Lumbar Segments Of Spinal Cord Research Articles

Exercise role on inflammatory and MCH-1 plasticity markers in severely contused spinal cord injured rodents

Approximately 17,500 new cases of Spinal cord Injury (SCI) occur annually in the United States. Growing evidence suggests that mobilizing the body following SCI through physical therapy regimens can help patients regain locomotor function. The immune response and the role of inflammation following SCI represents an important factor contributing to plasticity and recovery of function post-injury. The immune response uses both pro and anti-inflammatory factors to facilitate healing at the primary and secondary injury. In the severe case of SCI, inflammation can lead to maladaptive plasticity and that can hinder locomotor recovery. Pro-inflammatory cytokines such as Tumor necrosis factor alpha (TNF-a) can enhance the C-fiber activation in the dorsal horn resulting in sensitization and maladaptive plasticity. Exercise can also be pro-inflammatory. Exercise has been shown to decrease TNF-a levels and increases Interleukin-10 (IL-10) in human skeletal muscle. Bodyweight treadmill training in SCI animal has shown improvements in locomotor function. Interleukin-10 (IL-10), demonstrates potent anti-inflammatory effects in which it down-regulates TNF-alpha and its secondary effects. In addition, major histocompatibility complex class I proteins (MHC1), play a vital role in maintaining the structural integrity of neurons which is critical for fostering an environment for spinal plasticity to occur. In this study, we focus on the role of training paradigms to be used to facilitate locomotor recovery in spinally contused rats. The Body-Weight Supported Treadmill Training (BWSTT) and circular Bodyweight-Supported Ambulatory Rodent Trainer (cBART) both represent locomotor training for SCI animals. Mid-thoracic spinal cord contusion was performed, and the animals were allowed to recover for two weeks. The contused BWSTT alone, or cBART alone or combined BWSTT and cBART training groups were initiated at beginning of the third week for a total of 8-weeks of training. Controls include contused rats that received no training, and intact group (SHAM). At 10 weeks, the spinal cord was dissected for qPCR study. We measure the levels of TNF-alpha, MHC1, and IL-10 in the thoracic segment above the injury site, the injury site, Lumbar 1-3 and Lumbar 3-5 segment. Results show that in the cBART trained group, the MHC-1 expression in the L3-L5 segment was significantly greater when compared to the contused injury with no training intervention. No training effect was realized in MHC-1 in the L1-L3 trained groups. The training paradigms did not modulate TNF-a levels in the lumbar spinal cord segments. Lastly, IL-10 expression was undetectable in all the spinal cord segments. In this severe spinal cord contused model, we were only able to show modulation in the MHC-1 expression in the trained lumbar segment as function of exercise. Additional work will be required to consider the intensity of locomotor training and the severity of spinal cord contusion impact on these plasticity markers. Future understanding of these mechanisms may be useful in developing better locomotor training methods and drug targets for facilitation of recovery following SCI. CSULA RSCA 2020 MINI Grant- Dr. Joseph CSULA MORE Programs Offce, MBRS-URISE (1734GM145503) Bridges to Doctorate 5T23GM146700 NIH R25HD097701-04 - Dr. Ray De Leon. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peripheral Branch Injury Induces Oxytocin Receptor Expression at the Central Axon Terminals of Primary Sensory Neurons.

Considerable evidence suggests that oxytocin, as a regulatory nonapeptide, participates in modulatory mechanisms of nociception. Nonetheless, the role of this hypothalamic hormone and its receptor in the sensory pathway has yet to be fully explored. The present study performed immunohistochemistry, enzyme-linked immunosorbent assay, and RT-qPCR analysis to assess changes in the expression of the neuronal oxytocin receptor in female rats following tight ligation of the sciatic nerve after 1, 3, and 7 days of survival. Oxytocin receptor immunoreactivity was present in both dorsal root ganglia and lumbar spinal cord segments, but not accumulated at the site of the ligation of the peripheral nerve branch. We found a time-dependent change in the expression of oxytocin receptor mRNA in L5 dorsal root ganglion neurons, as well as an increase in the level of the receptor protein in the lumbar segment of the spinal cord. A peak in the expression was observed on day 3, which downturned slightly by day 7 after the nerve ligation. These results show that OTR expression is up-regulated in response to peripheral nerve lesions. We assume that the importance of OTR is to modify spinal presynaptic inputs of the sensory neurons upon injury-induced activation, thus to be targets of the descending oxytocinergic neurons from supraspinal levels. The findings of this study support the concept that oxytocin plays a role in somatosensory transmission.

Neurophysiological approach to the assessment of spinal cord function in kyphotic spinal deformity

The results of a comprehensive (clinical-radiological and neurophysiological) study performed in two clinical groups of patients (n = 64) with kyphotic spinal deformity of II–IV degree of severity of the thoracic and lumbar spine of various etiologies were analysed. With the progression of kyphotic spine deformity, the spinal cord (SC) located inside the spinal canal becomes involved in the pathological process. This period of dysfunction is preceded by a state of latent SM changes that are not detected clinically (prodromal period). The presence of neurological disorders is considered as a factor of increased risk of intra- and postoperative complications in the surgical treatment of kyphosis. Based on the registration and analysis of the parameters of somatosensory evoked potentials and motor responses during transcranial magnetic stimulation, electrophysiological signs of sensorimotor insufficiency of the SС nerve tracts were revealed, which correlate with the kyphotic arch value. The established electrophysiological criteria allow to verify the level of conductive lesion at the level of thoracic and lumbar SC segments, to determine its severity, to monitor recovery processes and to predict the functional outcome.

DISTRIBUTION OF PARVALBUMIN-EXPRESSING NEURONAL POPULATIONS IN THE CAT CERVICAL AND LUMBAR SPINAL CORD GRAY MATTER

Parvalbumin is a classical marker of interneuronal populations in the central nervous system. Analyzing the cervical and lumbar spinal cord segments of cats (Felis catus), both individual cells and entire populations of neurons expressing parvalbumin were identified in most of the gray matter laminae. These populations have strict laminar and nuclear localization. Numerous neuronal clusters are located in the medial part of lamina V–VI and in laminae VII of cervical and lumbar enlargements. We believe that the first one located in segments C4–C8 and L4–L7 may participate in the modulatory mechanisms of locomotor activity via the convergence of cutaneous and proprioceptive afferentation from the limbs. Neuronal populations in lamina VII consist of Ia interneurons and Renshaw interneurons that participate in the motoneuron inhibition. Less numerous populations of parvalbumin-immunopositive cells found in laminae III possibly participated in the regulation of cutaneous sensitivity. Another population located in lamina VIII possibly forms commissural and propriospinal connections and participates in modulating the activity of motoneurons. Immunopositive interneurons also revealed in the precerebellar nuclei: central cervical nucleus and Clarke’s nucleus; unlike the general population of these nuclei, neurons revealed are interneurons. Scarce immunopositive cells are found in lamina I of L6–L7 segments, as well as in laminae II, IV, and X of all segments investigated.

A candidate protective factor in amyotrophic lateral sclerosis: heterogenous nuclear ribonucleoprotein G.

Heterogenous nuclear ribonucleoprotein G is down-regulated in the spinal cord of the Tg(SOD1*G93A)1Gur (TG) amyotrophic lateral sclerosis mouse model. However, most studies have only examined heterogenous nuclear ribonucleoprotein G expression in the amyotrophic lateral sclerosis model and heterogenous nuclear ribonucleoprotein G effects in amyotrophic lateral sclerosis pathogenesis such as in apoptosis are unknown. In this study, we studied the potential mechanism of heterogenous nuclear ribonucleoprotein G in neuronal death in the spinal cord of TG and wild-type mice and examined the mechanism by which heterogenous nuclear ribonucleoprotein G induces apoptosis. Heterogenous nuclear ribonucleoprotein G in spinal cord was analyzed using immunohistochemistry and western blotting, and cell proliferation and proteins (TAR DNA binding protein 43, superoxide dismutase 1, and Bax) were detected by the Cell Counting Kit-8 and western blot analysis in heterogenous nuclear ribonucleoprotein G siRNA-transfected PC12 cells. We analyzed heterogenous nuclear ribonucleoprotein G distribution in spinal cord in the amyotrophic lateral sclerosis model at various time points and the expressions of apoptosis and proliferation-related proteins. Heterogenous nuclear ribonucleoprotein G was mainly localized in neurons. Amyotrophic lateral sclerosis mice were examined at three stages: preonset (60-70 days), onset (90-100 days) and progression (120-130 days). The number of heterogenous nuclear ribonucleoprotein G-positive cells was significantly higher in the anterior horn of the lumbar spinal cord segment of TG mice at the preonset stage than that of control group but lower than that of the control group at the onset stage. The number of heterogenous nuclear ribonucleoprotein G-positive cells in both central canal and surrounding gray matter of the whole spinal cord of TG mice at the onset stage was significantly lower than that in the control group, whereas that of the lumbar spinal cord segment of TG mice was significantly higher than that in the control group at preonset stage and significantly lower than that in the control group at the progression stage. The numbers of heterogenous nuclear ribonucleoprotein G-positive cells in the posterior horn of cervical and thoracic segments of TG mice at preonset and progression stages were significantly lower than those in the control group. The expression of heterogenous nuclear ribonucleoprotein G in the cervical spinal cord segment of TG mice was significantly higher than that in the control group at the preonset stage but significantly lower at the progression stage. The expression of heterogenous nuclear ribonucleoprotein G in the thoracic spinal cord segment of TG mice was significantly increased at the preonset stage, significantly decreased at the onset stage, and significantly increased at the progression stage compared with the control group. heterogenous nuclear ribonucleoprotein G expression in the lumbar spinal cord segment of TG mice was significantly lower than that of the control group at the progression stage. After heterogenous nuclear ribonucleoprotein G gene silencing, PC12 cell survival was lower than that of control cells. Both TAR DNA binding protein 43 and Bax expressions were significantly increased in heterogenous nuclear ribonucleoprotein G-silenced cells compared with control cells. Our study suggests that abnormal distribution and expression of heterogenous nuclear ribonucleoprotein G might play a protective effect in amyotrophic lateral sclerosis development via preventing neuronal death by reducing abnormal TAR DNA binding protein 43 generation in the spinal cord.

Integrative transcriptomic analysis of the amyotrophic lateral sclerosis spinal cord implicates glial activation and suggests new risk genes.

Amyotrophic lateral sclerosis (ALS) is a progressively fatal neurodegenerative disease affecting motor neurons in the brain and spinal cord. In this study, we investigated gene expression changes in ALS via RNA sequencing in 380 postmortem samples from cervical, thoracic and lumbar spinal cord segments from 154 individuals with ALS and 49 control individuals. We observed an increase in microglia and astrocyte gene expression, accompanied by a decrease in oligodendrocyte gene expression. By creating a gene co-expression network in the ALS samples, we identified several activated microglia modules that negatively correlate with retrospective disease duration. We mapped molecular quantitative trait loci and found several potential ALS risk loci that may act through gene expression or splicing in the spinal cord and assign putative cell types for FNBP1, ACSL5, SH3RF1 and NFASC. Finally, we outline how common genetic variants associated with splicing of C9orf72 act as proxies for the well-known repeat expansion, and we use the same mechanism to suggest ATXN3 as a putative risk gene.

Astrocyte Reactivity in Female Rats with Persistent Low Back Pain Following Spinal Mobilization

Low back pain (LBP) is a physiologically complex and highly disabling condition with poorly understood pathophysiology. Glial reactivity has been reported in patients with chronic LBP and astrocyte reactivity has been suggested as a potential contributor to LBP chronicity.1 Spinal mobilization (SM) is a non‐pharmacological approach with mild to moderate efficacy in treating LBP but physiological mechanisms responsible are unknown.PurposeTo investigate changes in astrocyte reactivity in the upper lumbar spinal cord following SM treatment in a Nerve‐Growth Factor (NGF)‐induced LBP pre‐clinical model. We hypothesize that the administration of trunk NGF will increase astrocyte reactivity, while SM treatment will decrease this reactivity in the upper lumbar spinal cord of female rats.MethodsFemale Sprague Dawley rats (212‐252g) were unilaterally injected with either NGF (50μl;2.0μM) or phosphate buffer saline ‐ PBS (50μl; control) on Days 0 and 5 in the left multifidus muscle under isoflurane anesthesia (1‐2%/2L O2). Ten days lapsed allowing for muscular LBP to develop in NGF animals. Treatment groups (NGF + SM [N=5]; PBS + SM [N=4]) received daily passive SM (10 min) via a feedback motor (1.2Hz, 0.9N) from Days 10 to 16, while the NGF only (N=5) group received no SM treatment. On Day 17, lumbar spinal cord (L1–L3) segments were harvested, fixated, and sectioned at 14μm. Sections were blocked and permeabilized (0.1% Triton X‐100 in 5% donkey serum) for 20 min at room temperature (RT), rinsed in PBS (6 x 5 min), and incubated with primary (Polyclonal Rabbit Anti‐Glial Fibrillary Acidic Protein ‐ 3h at RT) and secondary antibodies (IgG Highly Cross‐Adsorbed Donkey anti‐Rabbit, Alexa Fluor® 488 ‐ 1h at RT). Sections were then mounted on Fluorogel II containing 4′,6‐diamidino‐2‐phenylindole. Images of the superficial dorsal‐horn (laminas I‐III) and deep dorsal‐horn (lamina V) were captured at 20x magnification (Nikon Eclipse). Astrocyte mean fluorescent intensity (MFI) was quantified using FIJI/ImageJ® by blinded lab personnel. Analysis of variance was used to assess the differences in MFI between groups on the left and right sides of the spinal cord dorsal‐horn.ResultsNo significant differences between groups at the superficial horn level was found (p>0.05). Astrocyte MFI was statistically significantly higher in the NGF‐only group when compared to the PBS +SM group at the L3 spinal cord deep‐horn level (p=0.03). SM did not appear to decrease astrocyte reactivity at superficial or deep‐horn spinal cord levels.ConclusionOverall, these preliminary findings suggest that superficial‐horn astrocyte reactivity does not play a large mechanistic role in this female NGF‐induced LBP model despite astrocytic hyperreactivity being demonstrated among male rats with NGF‐induced LBP. Biological sex differences in glial signaling have been reported in other pre‐clinical pain models which may provide an explanation for the non‐significant NGF findings among females. Whether SM acts to decrease astrocyte reactivity in male NGF‐induced LBP rats remains to be investigated.

Spared Nerve Injury Causes Sexually Dimorphic Mechanical Allodynia and Differential Gene Expression in Spinal Cords and Dorsal Root Ganglia in Rats

Neuropathic pain is more prevalent in women. However, females are under-represented in animal experiments, and the mechanisms of sex differences remain inadequately understood. We used the spared nerve injury (SNI) model in rats to characterize sex differences in pain behaviour, unbiased RNA-Seq and proteomics to study the mechanisms. Male and female rats were subjected to SNI- and sham-surgery. Mechanical and cold allodynia were assessed. Ipsilateral lumbar dorsal root ganglia (DRG) and spinal cord (SC) segments were collected for RNA-seq analysis with DESeq2 on Day 7. Cerebrospinal fluid (CSF) samples for proteomic analysis and DRGs and SCs for analysis of IB-4 and CGRP, and IBA1 and GFAP, respectively, were collected on Day 21. Females developed stronger mechanical allodynia. There were no differences between the sexes in CGRP and IB-4 in the DRG or glial cell markers in the SC. No CSF protein showed change following SNI. DRG and SC showed abundant changes in gene expression. Sexually dimorphic responses were found in genes related to T-cells (cd28, ctla4, cd274, cd4, prf1), other immunological responses (dpp4, c5a, cxcr2 and il1b), neuronal transmission (hrh3, thbs4, chrna4 and pdyn), plasticity (atf3, c1qc and reg3b), and others (bhlhe22, mcpt1l, trpv6). We observed significantly stronger mechanical allodynia in females and numerous sexually dimorphic changes in gene expression following SNI in rats. Several genes have previously been linked to NP, while some are novel. Our results suggest gene targets for further studies in the development of new, possibly sex-specific, therapies for NP.

Effect of L-carnitine and atorvastatin on a rat model of ischemia-reperfusion injury of spinal cord

ABSTRACT Pro-inflammatory cytokines and reactive oxygen species (ROS) are produced in acute spinal cord injury, leading to myelin breakdown, inflammation, mitochondrial dysfunction, and apoptosis of neurons and glial cells. The aim of the present study was to investigate possible protective effects of L-carnitine (carn) or atorvastatin (ator) on spinal cord ischemia-reperfusion injury (IRI). Rats were randomized into nine equal groups (n = 8): control and control taking carn (100 mg/kg BW), ator (2.5 mg/kg BW) or both, as well as sham-operation, IRI and IRI taking same doses of carn, ator or both. Neurological assessments were done 48 hours after IRI, and serum nitrite/nitrate was measured. Finally, lumbar segments of spinal cord were excised, and part was homogenized and prepared for measuring tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), malondialdehyde (MDA), advanced oxidation protein products (AOPP), reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase. The other part was sectioned for evaluation of histopathological changes and for immunostaining by glial fibrillary acidic protein (GFAP), Bax and Bcl-2. The IRI increased ROS (nitrite/nitrate, MDA, AOPP) and pro-inflammatory cytokines (TNF-α, IL-1β), and decreased antioxidants (GSH, GPx, SOD, catalase) with impaired sensory and motor functions. Astrogliosis was detected by GFAP, and increased apoptosis was demonstrated by increasing Bax and decreasing Bcl-2. Treatment with carn or ator alone decreased TNF-α, IL-1β, nitrite/nitrate, MDA and AOPP, and increased GSH, GPx, SOD, and catalase with improvement of neurological functions and histological studies. Combination of carn and ator improved most of measured IRI-affected parameters better than isolated carn or ator administration.

Spinal motor mapping by epidural stimulation of lumbosacral posterior roots in humans.

SummaryEpidural electrical stimulation of the spinal cord is an emergent strategy for the neurological recovery of lower-extremity motor function. Motoneuron pools are thought to be recruited by stimulation of posterior roots. Here, we linked electromyographic data of epidurally evoked lower-extremity responses of 34 individuals with upper motoneuron disorders to a population model of the spinal cord constructed using anatomical parameters of thousands of individuals. We identified a relationship between segmental stimulation sites and activated spinal cord segments, which made spinal motor mapping from epidural space possible despite the complex anatomical interface imposed by the posterior roots. Our statistical approach provided evidence for low-threshold sites of posterior roots and effects of monopolar and bipolar stimulation previously predicted by computer modeling and allowed us to test the impact of different upper motoneuron disorders on the evoked responses. Finally, we revealed a statistical association between intraoperative and postoperative mapping of the spinal cord.

Possible Mechanisms of Axonal Transport Disturbances in Mouse Spinal Motoneurons Induced by Hypogravity.

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.

Nonopioid GTS-21 Mitigates Burn Injury Pain in Rats by Decreasing Spinal Cord Inflammatory Responses.

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. 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). 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. 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.

Differences in distribution of anterior segmental medullary arteries in the cervical and thoracolumbar spinal cord: the \u201cinseln\u201d were characteristics in the cervical spinal cord

Anterior segmental medullary arteries cause spinal cord infarction due to circulatory disturbance, but are difficult to identify in diagnostic images. This study investigated the arterial distribution from the cervical to lumbar segments of the dissecting spinal cord in 100 cadavers. The 488 arteries were distributed from C2 to L2, of which 252 arteries from C2 to C8 were slightly dominant on the right side, but 236 arteries from Th1 to L2 were obviously dominant on the left side. This change occurred at the C8 and Th1 segments. Of the 252 arteries, 37.30% (94 arteries) were divided into ascending and descending branches. Both branches formed loops by anastomosis with the anterior spinal arteries. The loops, called “insel”, have an unclear distribution and form. We focused on the features of inseln and found 63 of them in the cervical spinal cord of 45 cadavers. Their numbers and forms differed depending on whether the 94 arteries were bilateral (type A) or unilateral (type B), but the long axis of the insel was limited to one cervical segment. 90.63% of type A were bilateral at the same level, and 70% of type B were on the right side. The former always formed the insel. Further, 94 arteries were distributed from C2 to C7, 82.98% of which were concentrated at C3–C5. Therefore, the arterial blood supply of the spinal cord may differ between the cervical spinal cord and the thoracolumbar cord.

Microglial-induced apoptosis is potentially responsible for hyperalgesia variations during CFA-induced inflammation.

Activated microglia is known as a main mediator of inflammatory pain, but the possible mechanisms of its operation are poorly understood. Microglial cells have considered as one of the main sources of pro-inflammatory cytokines in the CNS. PTEN is one of the important targets of pro-inflammatory cytokines and the main mediator of apoptotic cell death. In this study, we investigated the possible effect of microglial activation on PTEN/PI3K/Akt signaling pathway and apoptosis in an inflammatoryrat modelofComplete Freund's adjuvant(CFA). Persistent peripheral inflammation was induced by a subcutaneous injection of CFA into the rats' right hind paw on day 0. Minocycline (a potent selective inhibitor of microglial) was administered intraperitoneally during days1-21 after CFA injection. Hyperalgesia was assessed on days 0, 7, and 21 using plantar test, then lumbar spinal cord segments were isolated, and the amount of spinal Iba1 (microglial marker), PTEN, P.Akt, and cleaved caspase-3 (a marker of apoptosis activation) were analyzed using Western blot. The spinal TNF-α levels were assayed by ELISA and the microglia numbers were determined using immunohistochemical technique. Results revealed that increased hyperalgesia was concurrent with an increment of Iba1 (P < 0.001), TNF-α (P < 0.001), PTEN (P < 0.01), cleaved caspase-3 (P < 0.001), and a decrement of P.Akt (P < 0.01) during the acute phase of CFA-induced inflammation, while, at the same time as decreasing hyperalgesia during the chronic phase of study, Iba1 and TNF-α expression significantly decreased and PTEN, cleaved caspase-3, and P.Akt restored to baseline on day0. Minocycline administration reduced the elevation of spinal Iba1 (P < 0.001), TNF-α (0.001), PTEN (P < 0.01), and cleaved caspase-3 (P < 0.001) expression induced by CFA injection, and also restored Akt activity to the baseline on day 0 (P < 0.001). These results suggest that microglial-mediated pain following CFA injection might be related in part to increased spinal cell apoptosis which probably is mediated by PTEN/PI3K/Akt deregulation.

Rostral lumbar segments are the key controllers of hindlimb locomotor rhythmicity in the adult spinal rat.

The precise location and functional organization of the spinal neuronal locomotor-related networks in adult mammals remain unclear. Our recent neurophysiological findings provided empirical evidence that the rostral lumbar spinal cord segments play a critical role in the initiation and generation of the rhythmic activation patterns necessary for hindlimb locomotion in adult spinal rats. Since added epidural stimulation at the S1 segments significantly enhanced the motor output generated by L2 stimulation, these data also suggested that the sacral spinal cord provides a strong facilitory influence in rhythm initiation and generation. However, whether L2 will initiate hindlimb locomotion in the absence of S1 segments, and whether S1 segments can facilitate locomotion in the absence of L2 segments remain unknown. Herein, adult rats received complete spinal cord transections at T8 and then at either L2 or S1. Rats with spinal cord transections at T8 and S1 remained capable of generating coordinated hindlimb locomotion when receiving epidural stimulation at L2 and when ensembles of locomotor related loadbearing input were present. In contrast, minimal locomotion was observed when S1 stimulation was delivered after spinal cord transections at T8 and L2. Results were similar when the nonspecific serotonergic agonists were administered. These results demonstrate in adult rats that rostral lumbar segments are essential for the regulation of hindlimb locomotor rhythmicity. In addition, the more caudal spinal networks alone cannot control locomotion in the absence of the rostral segments around L2 even when loadbearing rhythmic proprioceptive afferent input is imposed.NEW & NOTEWORTHY The exact location of the spinal neuronal locomotor-related networks in adult mammals remains unknown. The present data demonstrate that when the rostral lumbar spinal segments (~L2) are completely eliminated in thoracic spinal adult rats, hindlimb stepping is not possible with neurochemical modulation of the lumbosacral cord. In contrast, eliminating the sacral cord retains stepping ability. These observations highlight the importance of rostral lumbar segments in generating effective mammalian locomotion.

A Conditioning Sciatic Nerve Lesion Triggers a Pro-regenerative State in Primary Sensory Neurons Also of Dorsal Root Ganglia Non-associated With the Damaged Nerve.

The primary sensory neurons of dorsal root ganglia (DRG) are a very useful model to study the neuronal regenerative program that is a prerequisite for successful axon regeneration after peripheral nerve injury. Seven days after a unilateral sciatic nerve injury by compression or transection, we detected a bilateral increase in growth-associated protein-43 (GAP-43) and superior cervical ganglion-10 (SCG-10) mRNA and protein levels not only in DRG neurons of lumbar spinal cord segments (L4-L5) associated with injured nerve, but also in remote cervical segments (C6-C8). The increase in regeneration-associated proteins in the cervical DRG neurons was associated with the greater length of regenerated axons 1 day after ulnar nerve crush following prior sciatic nerve injury as compared to controls with only ulnar nerve crush. The increased axonal regeneration capacity of cervical DRG neurons after a prior conditioning sciatic nerve lesion was confirmed by neurite outgrowth assay of in vitro cultivated DRG neurons. Intrathecal injection of IL-6 or a JAK2 inhibitor (AG490) revealed a role for the IL-6 signaling pathway in activating the pro-regenerative state in remote DRG neurons. Our results suggest that the pro-regenerative state induced in the DRG neurons non-associated with the injured nerve reflects a systemic reaction of these neurons to unilateral sciatic nerve injury.

Sirtuin 1 (SIRT1) and activated caspase 3 expression on rat spinal cord in acute phase after Sciatic nerve injury

Background: Increase of SIRT1 expression inhibit the increase of activated Caspase 3 expression in acute phase of traumatic brain injury (TBI). However, the SIRT1 expression on rat spinal cord in acute phase after peripheral nerve injury was unknown.&#x0D; Objective: To reveal SIRT1and activated Caspase 3 expression on rat spinal cord in acute phase after sciatic nerve injury and to reveal the correlation between SIRT1 and activated Caspase 3 expression after sciatic nerve injury.&#x0D; Method: Thirty male Wistar rats aged 3 months were divided into sham operated and crush injury group. Termination was performing at days 3, 7 and 14. Lumbar spinal cord segments 4-5 (VTh12-VL1 high) embedded in paraffin blocks. Imunohistochemistry staining was performed using antibody of SIRT1 nuclear marker and activated Caspase 3. Observations were performed by two double blinded observers in 400x magnification. The data were analyzed using unpaired t-test and Pearson correlation. All data were processed using statistical analysis with confidence interval (CI) 95% and limit of significance (p-values)&lt;0.05.&#x0D; Results: SIRT1 expression on the anterior horn was higher compared with control at days 7 and 14 (p&lt;0.05), but no differences were found on posterior horn at all termination days(p&gt;0.05). Activated Caspase 3 expression on the anterior horn was higher at all termination days(p&lt;0.05) and at day 7 on posterior horn (p&lt;0.05). Thereis a positive correlation between SIRT1 and activated Caspase 3 expression on anterior horn at day 7 (p&lt;0.05).&#x0D; Conclusion: SIRT1 expression on the anterior horn was higher compared with the control at days 7 and 14. Activated Caspase 3 expression on the anterior horn was higher on all termination days and at day 7 on posterior horn. There is a positive correlation between SIRT1 and activated Caspase 3 expression on anterior horn on day 7.&#x0D; Bangladesh Journal of Medical Science Vol.18(1) 2019 p.50-56

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis.

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.

Synaptophysin and caspase-3 expression on lumbar segments of spinal cord after sensorimotor restriction during early postnatal period and treadmill training.

The purpose of the current study was to investigate whether locomotor stimulation training could have beneficial effects on spinal cord plasticity consequent to sensorimotor restriction (SR). Male Wistar rats were exposed to SR from postnatal day 2 (P2) to P28. Control and experimental rats underwent locomotor stimulation training in a treadmill from P31 to P52. The intensity of the synaptophysin and caspase-3 immunoreaction was determined on ventral horn of spinal cord. The synaptophysin immunoreactivity was lower in the ventral horn of sensorimotor restricted rats compared to controls animals and was accompanied by an increased caspase-3 immunoreactivity. Those alterations were reversed at the end of the training period. Our results suggest that immobility affects the normal developmental process that spinal cord undergoes in early postnatal life influencing both pro-apoptotic and synapse markers. Also, we demonstrated that this phenomenon was reversed by 3 weeks of treadmill training.

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.