Continuous intrathecal orexin delivery inhibits cataplexy in a murine model of narcolepsy.

Narcolepsy-cataplexy is a chronic neurological disorder caused by loss of orexin (hypocretin)-producing neurons, associated with excessive daytime sleepiness, sleep attacks, cataplexy, sleep paralysis, hypnagogic hallucinations, and fragmentation of nighttime sleep. Currently, human narcolepsy is treated by providing symptomatic therapies, which can be associated with an array of side effects. Although peripherally administered orexin does not efficiently penetrate the blood-brain barrier, centrally delivered orexin can effectively alleviate narcoleptic symptoms in animal models. Chronic intrathecal drug infusion through an implantable pump is a clinically available strategy to treat a number of neurological diseases. Here we demonstrate that the narcoleptic symptoms of orexin knockout mice can be reversed by lumbar-level intrathecal orexin delivery. Orexin was delivered via a chronically implanted intrathecal catheter at the upper lumbar level. The computed tomographic scan confirmed that intrathecally administered contrast agent rapidly moved from the spinal cord to the brain. Intrathecally delivered orexin was detected in the brain by radioimmunoassay at levels comparable to endogenous orexin levels. Cataplexy and sleep-onset REM sleep were significantly decreased in orexin knockout mice during and long after slow infusion of orexin (1 nmol/1 µL/h). Sleep/wake states remained unchanged both quantitatively as well as qualitatively. Intrathecal orexin failed to induce any changes in double orexin receptor-1 and -2 knockout mice. This study supports the concept of intrathecal orexin delivery as a potential therapy for narcolepsy-cataplexy to improve the well-being of patients.

Unusual Quadrupedal Locomotion in Rat during Recovery from Lumbar Spinal Blockade of 5-HT7 Receptors.

Coordination of four-limb movements during quadrupedal locomotion is controlled by supraspinal monoaminergic descending pathways, among which serotoninergic ones play a crucial role. Here we investigated the locomotor pattern during recovery from blockade of 5-HT7 or 5-HT2A receptors after intrathecal application of SB269970 or cyproheptadine in adult rats with chronic intrathecal cannula implanted in the lumbar spinal cord. The interlimb coordination was investigated based on electromyographic activity recorded from selected fore- and hindlimb muscles during rat locomotion on a treadmill. In the time of recovery after hindlimb transient paralysis, we noticed a presence of an unusual pattern of quadrupedal locomotion characterized by a doubling of forelimb stepping in relation to unaffected hindlimb stepping (2FL-1HL) after blockade of 5-HT7 receptors but not after blockade of 5-HT2A receptors. The 2FL-1HL pattern, although transient, was observed as a stable form of fore-hindlimb coupling during quadrupedal locomotion. We suggest that modulation of the 5-HT7 receptors on interneurons located in lamina VII with ascending projections to the forelimb spinal network can be responsible for the 2FL-1HL locomotor pattern. In support, our immunohistochemical analysis of the lumbar spinal cord demonstrated the presence of the 5-HT7 immunoreactive cells in the lamina VII, which were rarely 5-HT2A immunoreactive.

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.

Repeated electroacupuncture treatment attenuated hyperalgesia through suppression of spinal glial activation in chronic neuropathic pain rats.

BACKGROUND: Cumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown. METHODS: A total of 120 male Wistar rats were used in the present study. The neuropathic pain model was established by chronic constrictive injury (CCI) of the sciatic nerve. The rats were randomly divided into sham group, CCI group, and sham CCI + EA group, and CCI + EA group. EA was applied to bilateral Zusanli (ST36)-Yanlingquan (GB34). The mechanical (both time and force responses) and thermal pain thresholds (PTs) of the bilateral hind-paws were measured. The number of microgliacytes and activity of astrocytes in the dorsal horns (DHs) of lumbar spinal cord (L4-5) were examined by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) protein was detected by western blot. RESULTS: Following CCI, both mechanical and thermal PTs of the ipsilateral hind-paw were significantly decreased beginning from the 3rd day after surgery (P < 0.05), and the mechanical PT of the contralateral hind-paw was considerably decreased from the 6th day on after surgery (P < 0.05). CCI also significantly upregulated the number of Iba-1 labeled microgliacytes and the fluorescence intensity of glial fibrillary acidic protein (GFAP) -labeled astrocyte in the superficial laminae of DHs on bilateral sides (P < 0.05). After repeated EA, the mechanical and thermal PTs at bilateral hind-paws were significantly relieved (P < 0.05). The increased of number of microgliacytes was markedly suppressed by 2 days' EA intervention, and the average fluorescence intensity was suppressed by 2 weeks' EA. The expression of GFAP protein were down-regulated by 1 and 2 weeks' EA treatment, respectively (P < 0.05). CONCLUSIONS: Repeated EA can relieve neuropathic pain and mirror-image pain in chronic neuropathic pain rats, which is probably associated with its effect in downregulating glial cell activation of the lumbar spinal cord, the microgliacyte first and astrocyte later.

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.

The cumulative analgesic effect of repeated electroacupuncture is modulated by Adora3 in the SCDH of mice with neuropathic pain.

BACKGROUND: Existing remedial approaches for relieving neuropathic pain (NPP) are challenging and open the way for alternative therapeutic measures such as electroacupuncture (EA). The mechanism underlying the antinociceptive effects of repeated EA sessions, particularly concerning the regulation of the Adora3 receptor and its associated enzymes, has remained elusive. METHODS: This study used a mouse model of spared nerve injury (SNI) to explore the cumulative analgesic effects of repeated EA at ST36 (Zusanli) and its impact on Adora3 regulation in the spinal cord dorsal horn (SCDH). Forty-eight male mice underwent SNI surgery for induction of neuropathic pain and were randomly assigned to the SNI, SNI + 2EA, SNI + 4EA, and SNI + 7EA groups. Spinal cord (L4-L6) was sampled for immunofluorescence, adenosine (ADO) detection and for molecular investigations following repeated EA treatment. RESULTS: Following spared nerve injury (SNI), there was a significant decrease in mechanical withdrawal thresholds (PWTs) and thermal nociceptive withdrawal latency (TWL) in the ipsilateral hind paw on the third day post-surgery, while the contralateral hind paw PWTs showed no significant changes. On subsequent EA treatments, the SNI + EA groups led to a significant increase in pain thresholds (p < 0.05). Repeated EA sessions in SNI mice upregulated Adenosine A3 (Adora3) and cluster of differentiation-73 (CD73) expression while downregulating adenosine deaminase (ADA) and enhancing neuronal instigation in the SCDH. Colocalization analysis of Neun-treated cells revealed increased Adora3 expression, particularly in the SNI + 7EA group. CONCLUSIONS: In conclusion, cumulative electroacupuncture treatment reduced neuropathic pain by regulating Adora3 and CD73 expression, inhibiting ADA and most likely increasing neuronal activation in the SCDH. This study offers a promising therapeutic option for managing neuropathic pain, paving the way for further research.

Morphometric Evaluation of the Thoracic and Lumbar Vertebral Canal and Spinal Cord Using Computed Tomography in Healthy Korean Shorthair Cats.

This study aimed to evaluate the spinal morphometry of the thoracic and lumbar regions in normal Korean Shorthair cats using computed tomography (CT) and to investigate the relationship with variables such as sex, age and body weight. Fifteen clinically healthy Korean Shorthair cats (eight males, seven females) from Seoul National University Veterinary Medical Teaching Hospital were included in this retrospective study. Measurements of the height, width and area of the vertebral canal and spinal cord on CT images were taken at the cranial, middle and caudal points of the thoracic and lumbar vertebrae by three observers, and the ratios of the spinal cord area to the vertebral canal area were calculated. The significance of the differences in measurements between sexes and correlations with age and body weight were analysed. The mean age of the cats was 7 years (range: 2-12 years), with a mean weight of 5.27 kg (range: 2.6-8.3 kg). The height, width and area of the vertebral canal and spinal cord were significantly greater in males than in females (p < 0.05). The ratios of the spinal cord area to the vertebral canal area showed no significant difference between sexes (p > 0.05), and no significant correlations were found between the ratios of the spinal cord area to the vertebral canal area and age or body weight. This study provides useful reference intervals for spinal morphometry in the thoracic and lumbar regions of healthy Korean Shorthair cats and investigate the relationship with variables such as sex, age and body weight. This anatomical information may assist in the diagnosis and prognosis of thoracic, lumbar vertebral and spinal cord diseases using CT.

Involvement of cannabinoid receptors in depression of the putative nociceptive response in spinal cord preparations isolated from neonatal rats.

A metabolite of acetaminophen, AM404, which is an anandamide transporter inhibitor, induces analgesia mainly via activation of transient receptor potential channel 1 in the spinal cord, although the role of cannabinoid receptors remains to be studied. The ventral root reflex response induced by stimulation of the dorsal root in in vitro preparations of rat spinal cord is useful to assess the effect of analgesics. We analyzed the effects of AM404 and cannabinoid receptor antagonist AM251 on reflex responses in lumbar spinal cord preparations from newborn rats and found that the amplitude of the slow ventral root potential after administration of 10 µM AM404 was not significantly changed, whereas 10 µM AM251 significantly increased the amplitude. Administration of the cannabinoid receptor 1 agonist WIN55,212-2 (10 µM) did not significantly affect the reflex response. We suggest that endogenous cannabinoids in the spinal cord are involved in the antinociceptive mechanism through suppressive effects.

Intraperitoneal 5-Azacytidine Alleviates Nerve Injury-Induced Pain in Rats by Modulating DNA Methylation.

To investigate the role of DNA methylation in modulating chronic neuropathic pain (NPP), identify possible target genes of DNA methylation involved in this process, and preliminarily confirm the medicinal value of the DNA methyltransferases (DNMTs) inhibitor 5-azacytidine (5-AZA) in NPP by targeting gene methylation. Two rat NPP models, chronic constriction injury (CCI) and spinal nerve ligation (SNL), were used. The DNA methylation profiles in the lumbar spinal cord were assayed using an Arraystar Rat RefSeq Promoter Array. The underlying genes with differential methylation were then identified and submitted to Gene Ontology and pathway analysis. Methyl-DNA immunoprecipitation quantitative PCR (MeDIP-qPCR) and quantitative reverse transcription-PCR (RT-qPCR) were used to confirm gene methylation and expression. The protective function of 5-AZA in NPP and gene expression were evaluated via behavioral assays and RT-qPCR, respectively. Analysis of the DNA methylation patterns in the lumbar spinal cord indicated that 1205 differentially methylated fragments in CCI rats were located within DNA promoter regions, including 638 hypermethylated fragments and 567 hypomethylated fragments. The methylation levels of Grm4, Htr4, Adrb2, Kcnf1, Gad2, and Pparg, which are associated with long-term potentiation (LTP) and glutamatergic synapse pathways, were increased with a corresponding decrease in their mRNA expression, in the spinal cords of CCI rats. Moreover, we found that the intraperitoneal injection of 5-AZA (4 mg/kg) attenuated CCI- or SNL-induced mechanical allodynia and thermal hyperalgesia. Finally, the mRNA expression of hypermethylated genes such as Grm4, Htr4, Adrb2, Kcnf1, and Gad2 was reversed after 5-AZA treatment. CCI induced widespread methylation changes in the DNA promoter regions in the lumbar spinal cord. Intraperitoneal 5-AZA alleviated hyperalgesia in CCI and SNL rats, an effect accompanied by the reversed expression of hypermethylated genes. Thus, DNA methylation inhibition represents a promising epigenetic strategy for protection against chronic NPP following nerve injury. Our study lays a theoretical foundation for 5-AZA to become a clinical targeted drug.

Proteomics analysis indicates the involvement of immunity and inflammation in the onset stage of SOD1-G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease, and the pathogenic mechanism that underlies ALS is still unclear. We analyzed the differentially expressed proteins (DEPs) in the spinal cord between SOD1-G93A transgenic mice at the onset stage and non-transgenic (NTG) littermates based on 4D label-free quantitative proteomics (4D-LFQ) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In our study, 189 DEPs were screened, of which 166 were up-regulated and 23 down-regulated. Clusters of Orthologous Groups (COG)/ EuKaryotic Orthologous Groups (KOG) classification, subcellular localization annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, clustering analysis and protein-protein interaction (PPI) network analyses were performed. Parallel reaction monitoring (PRM) analysis validated 48 proteins from immunity and inflammation-related pathways of KEGG. We described the function and distribution of DEPs, most of which were involved in the following pathways: complement and coagulation cascades, antigen processing and presentation, NF-kappa B signaling pathway, Retinoic acid-inducible gene I (RIG) -I-like receptor signaling pathway, the extracellular matrix-receptor (ECM-receptor) interaction, focal adhesion, phagosome and lysosome. PPI network analysis identified Fn1, Fga, Serpina1e and Serpina3n as potential biomarkers. Our discoveries broaden the view and expand our understanding of immunity and inflammation in ALS. SIGNIFICANCE: This study gives a comprehensive description of DEPs in the spinal cord proteomics of SOD1-G93A mice at the onset period. Compared with a previous study focusing on progressive stage, we showed that immunity and inflammation play an important role at the onset stage of ALS. Several pathways validated by PRM bring new insight to the pathological mechanisms of ALS. The participation of RIG-I-like signaling pathway in ALS and potential biomarkers Fga, Fn1, Serpina1e and Serpina3n are supplements to existing knowledge.

Expression and Kinetics of Endogenous Cannabinoids in the Brain and Spinal Cord of a Spare Nerve Injury (SNI) Model of Neuropathic Pain.

The role of endogenous cannabinoids in neuropathic pain has been actively studied, among which 2-arachidonoyl glycerol (2-AG) has received the most attention. However, owing to its chemical properties, direct detection of 2-AG distribution in tissues is difficult. Moreover, although desorption electrospray ionization mass spectrometry imaging (DESI-MSI) has enabled the detection of 2-AG, its distribution in the brain and spinal cord of neuropathic pain models has not been reported. In this study, the expression and distribution of 2-AG in the brain and spinal cord of a spare nerve injury (SNI) mice model of neuropathic pain was examined using DESI-MSI. The brain and lumbar spinal cord were collected and analyzed on days 3, 7, and 21 after treatment. On days 3 and 7 after treatment, 2-AG expression in the SNI model was decreased in the hypothalamus, midbrain, and especially in the periaqueductal gray (PAG) region but increased in the lumbar spinal cord. On day 21, the SNI model showed decreased 2-AG expression in the hypothalamus, but the difference from the control was not significant. Furthermore, there were no differences in 2-AG expression between the lumbar spinal cord, midbrain, or PAG. These data suggest that 2-AG might be involved in pain control.

Adapting Human-Based Transcutaneous Spinal Cord Stimulation to Develop a Clinically Relevant Animal Model.

Transcutaneous spinal cord stimulation (tSCS) as a neuromodulatory strategy has received great attention as a method to promote functional recovery after spinal cord injury (SCI). However, due to the noninvasive nature of tSCS, investigations have primarily focused on human applications. This leaves a critical need for the development of a suitable animal model to further our understanding of this therapeutic intervention in terms of functional and neuroanatomical plasticity and to optimize stimulation protocols. The objective of this study is to establish a new animal model of thoracolumbar tSCS that (1) can accurately recapitulate studies in healthy humans and (2) can receive a repeated and stable tSCS treatment after SCI with minimal restraint, while the electrode remains consistently positioned. We show that our model displays bilateral evoked potentials in multisegmental leg muscles characteristically comparable to humans. Our data also suggest that tSCS mainly activates dorsal root structures like in humans, thereby accounting for the different electrode-to-body-size ratio between the two species. Finally, a repeated tSCS treatment protocol in the awake rat after a complete spinal cord transection is feasible, tolerable, and safe, even with minimal body restraint. Additionally, repeated tSCS was capable of modulating motor output after SCI, providing an avenue to further investigate stimulation-based neuroplasticity and optimize treatment.

Coherence analysis of the calcium activity of putative astrocytic and neuronal cells on the L5 ventral horn and neural output in activated lumbar CPG networks.

Astrocytes are thought to play a crucial role in providing structure to the spinal cord and maintaining efficient synaptic function and metabolism because their fine processes envelop the synapses of neurons and form many neuronal networks within the central nervous system (CNS). To investigate whether putative astrocytes and putative neurons distributed on the ventral horn play a role in the modulation of lumbar locomotor central pattern generator (CPG) networks, we used extracellular recording and optical imaging techniques and recorded the neural output from the left L5 ventral root and the calcium activity of putative astrocytes and neurons in the L5 ventral horn at the same time when activating an isolated L1-L5 spinal cord preparation from rats aged 0-2 days. Optical measurements detected cells that showed a fluorescence intensity change under all experimental conditions, namely, (1) 5-HT + NMDA, (2) TTX, and (3) TTX + Low K+. These cells were semiautomatically identified using an in-house MATLAB-based program, as putative astrocytes and neurons according to the cell classification, i.e., increased or decreased fluorescence intensity change (ΔF/F0), and subjective judgment based on their soma size. Coherence and its phase were calculated according to the calcium activity of the putative astrocytes and putative neurons, and neural output was calculated during fictive locomotion with in-house MATLAB-based programs. We found that the number of putative astrocytes activated by applying low K+ tends not to differ from that activated by applying the protease-activated receptor 1 (PAR1) selective agonist TFLLR-NH2 (TFLLR). Moreover, the calcium activity of several putative astrocytes and neurons synchronized with locomotor-like activity at a frequency range below 0.5 Hz and the time lag between peaks of cellular calcium activity and locomotor-like activity ranged from -1000 to + 1000 ms. These findings presumably indicates that these putative astrocytes and neurons in the left L5 ventral horn require -1000 to + 1000 ms to communicate with lumbar CPG networks and maintain efficient synaptic function and metabolism in activated lumbar CPG networks. This finding suggests the possibility that putative astrocytic and neuronal cells in the L5 ventral horn contribute to generating the rhythms and patterns of locomotor-like activity by activated CPG networks in the first to fifth lumbar spinal cord.

Hypothalamic A11 Nuclei Regulate the Circadian Rhythm of Spinal Mechanonociception through Dopamine Receptors and Clock Gene Expression.

Several types of sensory perception have circadian rhythms. The spinal cord can be considered a center for controlling circadian rhythms by changing clock gene expression. However, to date, it is not known if mechanonociception itself has a circadian rhythm. The hypothalamic A11 area represents the primary source of dopamine (DA) in the spinal cord and has been found to be involved in clock gene expression and circadian rhythmicity. Here, we investigate if the paw withdrawal threshold (PWT) has a circadian rhythm, as well as the role of the dopaminergic A11 nucleus, DA, and DA receptors (DR) in the PWT circadian rhythm and if they modify clock gene expression in the lumbar spinal cord. Naïve rats showed a circadian rhythm of the PWT of almost 24 h, beginning during the night-day interphase and peaking at 14.63 h. Similarly, DA and DOPAC's spinal contents increased at dusk and reached their maximum contents at noon. The injection of 6-hydroxydopamine (6-OHDA) into the A11 nucleus completely abolished the circadian rhythm of the PWT, reduced DA tissue content in the lumbar spinal cord, and induced tactile allodynia. Likewise, the repeated intrathecal administration of D1-like and D2-like DA receptor antagonists blunted the circadian rhythm of PWT. 6-OHDA reduced the expression of Clock and Per1 and increased Per2 gene expression during the day. In contrast, 6-OHDA diminished Clock, Bmal, Per1, Per2, Per3, Cry1, and Cry2 at night. The repeated intrathecal administration of the D1-like antagonist (SCH-23390) reduced clock genes throughout the day (Clock and Per2) and throughout the night (Clock, Per2 and Cry1), whereas it increased Bmal and Per1 throughout the day. In contrast, the intrathecal injection of the D2 receptor antagonists (L-741,626) increased the clock genes Bmal, Per2, and Per3 and decreased Per1 throughout the day. This study provides evidence that the circadian rhythm of the PWT results from the descending dopaminergic modulation of spinal clock genes induced by the differential activation of spinal DR.

Comparison of effect of crush or transection peripheral nerve lesion on lumbar spinal cord synaptic plasticity and microglial dynamics.

In an injury to the peripheral nervous system, the spinal cord and brain structure reorganize connections to optimize the function of the remaining parts. Many cell events are triggered in the spinal cord to support changes in the synaptic connections around motoneurons, where old connections are removed, and new ones created. Microglial cells are primitive macrophages that invade the central nervous system in early stages of neurodevelopment and have several functions, such as eliminating synapses. We investigated the synaptic plasticity after different types of peripheral (sciatic) nerve injury (crush or total transection), as well as the behavior of microglial cells for 2 weeks after a peripheral lesion. As expected, sciatic-nerve injury reduced motor performance in mice, but crushed animals regained partial motor control. Because of sciatic-nerve injury, pre-synaptic inputs decreased around the motoneurons in the ventro-lateral horn, while microglial cells increased around these cells. Microglial cells also exhibited altered morphology in both types of peripheral lesion, indicating a similar underlying mechanism of plasticity. To investigate the involvement of microglia in this scenario, microglial activation was modulated by daily administration of minocycline. The minocycline treatment directly affected the microglial response and impacted the synapse rearrangement in the spinal cord. Together, these results demonstrate that microglia cells are involved in synaptic plasticity in the lumbar spinal cord in both nerve-injury scenarios. SUMMARY OF STATEMENT: Here, we demonstrated that acute plasticity in the lumbar spinal cord (LSC) did not differ between crush and transection of peripheral nerve, and that microglial reactivity in the LSC was important after both injury types.

Spinal Cord Injury Causes Reduction of Galanin and Gastrin Releasing Peptide mRNA Expression in the Spinal Ejaculation Generator of Male Rats.

Spinal cord injury (SCI) in men is commonly associated with sexual dysfunction, including anejaculation, and chronic mid-thoracic contusion injury in male rats also impairs ejaculatory reflexes. Ejaculation is controlled by a spinal ejaculation generator consisting of a population of lumbar spinothalamic (LSt) neurons that control ejaculation through release of four neuropeptides including galanin and gastrin releasing peptide (GRP) onto lumbar and sacral autonomic and motor nuclei. It was recently demonstrated that spinal contusion injury in male rats caused reduction of GRP-immunoreactivity, but not galanin-immunoreactivity in LSt cells, indicative of reduced GRP peptide levels, but inconclusive results for galanin. The current study further tests the hypothesis that contusion injury causes a disruption of GRP and galanin mRNA in LSt cells. Male rats received mid-thoracic contusion injury and galanin and GRP mRNA were visualized 8 weeks later in the lumbar spinal cord using fluorescent in situ hybridization. Spinal cord injury significantly reduced GRP and galanin mRNA in LSt cells. Galanin expression was higher in LSt cells compared to GRP. However, expression of the two transcripts were positively correlated in LSt cells in both sham and SCI animals, suggesting that expression for the two neuropeptides may be co-regulated. Immunofluorescent visualization of galanin and GRP peptides demonstrated a significant reduction in GRP-immunoreactivity, but not galanin in LSt cells, confirming the previous observations. In conclusion, SCI reduced GRP and galanin expression in LSt cells with an apparent greater impact on GRP peptide levels. GRP and galanin are both essential for triggering ejaculation and thus such reduction may contribute to ejaculatory dysfunction following SCI in rats.

Observations on the recovering lumbar spinal cord of lizards show multiple origins of the cells forming the bridge region including immune cells.

After transection the lumbar spinal cord of lizards forms a bridge of connective and nervous tissues between the severed proximal and distal ends of the cord. The types of proliferating cells activated in the injured spinal cord have been analyzed using light and ultrastructural immunolabeling for 5BrdU and nestin from 11 to 34 days after injury, when recovery of some hindlimb movements has occurred. At 11-22 days post-transection an intense proliferation of glial, immune and meningeal cells takes place. Nestin is almost absent in the normal spinal cord but becomes detectable at 11-34 days postinjury in ependymal and sparse glial cells located in the bridge region. At 11-22 days postinjury also numerous macrophages, lymphocytes, and some plasma cells appear proliferating during the intense inflammatory and antimicrobial phase. Phagocytosis within the injured spinal cord probably decreases inflammation and may indirectly promote axonal regeneration. Proliferating cells likely derive from precursor or stem elements of the reactive ependymal epithelium, but also from glial cells and meningeal fibroblasts. This is indicated by the presence of 5BrdU-long retaining labeling cells of glial and fibroblast types located in the stumps of the spinal cord and in the bridge. The present observations suggest that meningeal, ependymal, and numerous glial cells are the precursors of those forming the bridge region. Among glial cells, sparse oligodendrocytes myelinating the few axons present at 34 day after the injury also appear capable to proliferate. The myelinated axons are probably involved in the limited but important functional recovery of limb movements observed after 30-90 days postinjury.

Therapeutic Hypothermia Improves Hind Limb Motor Outcome and Attenuates Oxidative Stress and Neuronal Damage in the Lumbar Spinal Cord Following Cardiac Arrest.

: Hypothermia enhances outcomes of patients after resuscitation after cardiac arrest (CA). However, the underlying mechanism is not fully understood. In this study, we investigated effects of hypothermic therapy on neuronal damage/death, microglial activation, and changes of endogenous antioxidants in the anterior horn in the lumbar spinal cord in a rat model of asphyxial CA (ACA). A total of 77 adult male Sprague-Dawley rats were randomized into five groups: normal, sham ACA plus (+) normothermia, ACA + normothermia, sham ACA + hypothermia, and ACA + hypothermia. ACA was induced for 5 min by injecting vecuronium bromide. Therapeutic hypothermia was applied after return of spontaneous circulation (ROSC) via rapid cooling with isopropyl alcohol wipes, which was maintained at 33 ± 0.5 °C for 4 h. Normothermia groups were maintained at 37 ± 0.2 °C for 4 h. Neuronal protection, microgliosis, oxidative stress, and changes of endogenous antioxidants were evaluated at 12 h, 1 day, and 2 days after ROSC following ACA. ACA resulted in neuronal damage from 12 h after ROSC and evoked obvious degeneration/loss of spinal neurons in the ventral horn at 1 day after ACA, showing motor deficit of the hind limb. In addition, ACA resulted in a gradual increase in microgliosis with time after ACA. Therapeutic hypothermia significantly reduced neuronal loss and attenuated hind limb dysfunction, showing that hypothermia significantly attenuated microgliosis. Furthermore, hypothermia significantly suppressed ACA-induced increases of superoxide anion production and 8-hydroxyguanine expression, and significantly increased superoxide dismutase 1 (SOD1), SOD2, catalase, and glutathione peroxidase. Taken together, hypothermic therapy was found to have a substantial impact on changes in ACA-induced microglia activation, oxidative stress factors, and antioxidant enzymes in the ventral horn of the lumbar spinal cord, which closely correlate with neuronal protection and neurological performance after ACA.

Chronic Spinal Cord Injury Reduces Gastrin-Releasing Peptide in the Spinal Ejaculation Generator in Male Rats.

Spinal cord injury (SCI) causes sexual dysfunction, including anejaculation in men. Likewise, chronic mid-thoracic contusion injury impairs ejaculatory reflexes in male rats. Ejaculation is controlled by a spinal ejaculation generator (SEG) comprised of a population of lumbar spinothalamic (LSt) neurons. LSt neurons co-express four neuropeptides, including gastrin-releasing peptide (GRP) and galanin and control ejaculation via release of these peptides in lumbar and sacral autonomic and motor nuclei. Here, we tested the hypothesis that contusion injury causes a disruption of the neuropeptides that are expressed in LSt cell bodies and axon terminals, thereby causing ejaculatory dysfunction. Male Sprague Dawley rats received contusion or sham surgery at spinal levels T6-7. Five to six weeks later, animals were perfused and spinal cords were immunoprocessed for galanin and GRP. Results showed that numbers of cells immunoreactive for galanin were not altered by SCI, suggesting that LSt cells are not ablated by SCI. In contrast, GRP immunoreactivity was decreased in LSt cells following SCI, evidenced by fewer GRP and galanin/GRP dual labeled cells. However, SCI did not affect efferent connections of LSt, cells as axon terminals containing galanin or GRP in contact with autonomic cells were not reduced following SCI. Finally, no changes in testosterone plasma levels or androgen receptor expression were noted after SCI. In conclusion, chronic contusion injury decreased immunoreactivity for GRP in LSt cell soma, but did not affect LSt neurons per se or LSt connections within the SEG. Since GRP is essential for triggering ejaculation, such loss may contribute to ejaculatory dysfunction following SCI.

Bioinformatic Study of Transcriptome Changes in the Mice Lumbar Spinal Cord After the 30-Day Spaceflight and Subsequent 7-Day Readaptation on Earth: New Insights Into Molecular Mechanisms of the Hypogravity Motor Syndrome.

The hypogravity motor syndrome (HMS) is one of the deleterious impacts of weightlessness on the human body in orbital space missions. There is a hypothesis that disorders of musculoskeletal system as part of HMS arise in consequence of changes in spinal motor neurons. The study was aimed at bioinformatic analysis of transcriptome changes in lumbar spinal cords of mice after a 30-day spaceflight aboard biosatellite Bion-M1 (space group, S) and subsequent 7-day readaptation to the Earth's gravity (recovery group, R) when compared with control mice (C group) housed in simulated biosatellite conditions on the Earth. Gene ontology and human phenotype ontology databases were used to detect biological processes, molecular functions, cellular components, and human phenotypes associated with HMS. Our results suggest resemblance of molecular changes developing in space orbit and during the postflight recovery to terrestrial neuromuscular disorders. Remarkably, more prominent transcriptome changes were revealed in R vs. S and R vs. C comparisons that are possibly related to the 7-day recovery period in the Earth's gravity condition. These data may assist with establishment of HMS pathogenesis and proposing effective preventive and therapeutic options.