Biodistribution of Adeno-Associated Virus Serotype 5 Viral Vectors Following Intrathecal Injection.

The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief.

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

Semi-quantitative distribution of excitatory amino acid (glutamate) transporters 1-3 (EAAT1-3) and the cystine-glutamate exchanger (xCT) in the adult murine spinal cord.

Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system xc- with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1-4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1-3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1-3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters.

Dysregulation of Steroid Hormone Receptors in Motor Neurons and Glia Associates with Disease Progression in ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting motor neurons which shows sexual dimorphism in its incidence, age of onset, and progression rate. All steroid hormones, including androgens, estrogens, and progestogens, have been implicated in modulating ALS. Increasing evidence suggests that steroid hormones provide neuroprotective and neurotrophic support to motor neurons, either directly or via surrounding glial cell interactions, by activating their respective nuclear hormone receptors and initiating transcriptional regulatory responses. The SOD1G93A transgenic mouse also shows sex-specific differences in age of onset and progression, and remains the most widely used model in ALS research. To provide a more comprehensive understanding of the influences of steroid hormone signaling in ALS, we systemically characterized sex hormone receptor expression at transcript and protein levels, cellular localization, and the impact of disease course in lumbar spinal cords of male and female SOD1G93A mice. We found that spinal motor neurons highly express nuclear androgen receptor (AR), estrogen receptor (ER)α, ERß, and progesterone receptor with variations in glial cell expression. AR showed the most robust sex-specific difference in expression and was downregulated in male SOD1G93A mouse spinal cord, in association with depletion in 5α-reductase type 2 isoform, which primarily metabolizes testosterone to 5α-dihydrotestosterone. ERα was highly enriched in reactive astrocytes of SOD1G93A mice and ERß was strongly upregulated. The 5α-reductase type 1 isoform was upregulated with disease progression and may influence local spinal cord hormone levels. In conclusion, steroid hormone receptor expression is dynamic and cell-type specific in SOD1G93A mice which may provide targets to modulate progression in ALS.

SOD1 Suppression with Adeno-Associated Virus and MicroRNA in Familial ALS.

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.

Chronic morphine induces cyclic adenosine monophosphate formation and hyperpolarization-activated cyclic nucleotide-gated channel expression in the spinal cord of mice.

Chronic morphine exposure persistently activates Gαi/o protein-coupled receptors and enhances adenylyl cyclase (AC) activity, which can increase cyclic adenosine monophosphate (cAMP) production. Direct binding of cAMP to the cytoplasmic site on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels increases the probability of channel opening. HCN channels play a prominent role in chronic pain the disease that shares some common mechanisms with opioid tolerance. This compensatory AC activation may be responsible for the induction of morphine-induced analgesic tolerance. We investigated spinal cAMP formation and expression of HCN2 in the spinal cord, and observed the effect of AC inhibition on the induction of morphine analgesic tolerance. We found that chronic morphine-induced antinociceptive tolerance increased spinal cAMP formation and the expression of spinal HCN2. Inhibition of spinal AC partially blocked chronic morphine-induced cAMP formation and prevented the induction of morphine-induced analgesic tolerance. Inhibition of HCN2 also showed a partial preventive effect on morphine-induced tolerance, hypothermia tolerance and also the right-shift of the dose-response curve. We conclude that repeated morphine treatment increases AC activity and cAMP formation, and also spinal HCN2 expression, blockade of AC or HCN2 can prevent the development of morphine-induced analgesic tolerance.

Distribution and chemical coding of phoenixin-immunoreactive nerve structures in the spinal cord of the pig.

Phoenixin (PNX) is a newly described peptide found in both neural and non-neural tissues. Until now, no attempts have been made to investigate the expression of PNX in the nervous system of animals other than laboratory rodents, in which an enzyme immunoassay revealed the highest quantity of the substance in the spinal cord. Since the domestic pig, due to its anatomical and histological resemblance to humans, is often used as an animal model in biomedical investigations, the present study was designed to examine PNX-immunoreactivity in the spinal cords of female pigs (n=5). The spinal cords were dissected and divided into the cervical, thoracic, lumbar, sacral and coccygeal segments, which were sectioned transversally into 10-µm-thick serial sections. The sections from each spinal cord segment were processed for double-labelling immunohistochemistry using antibodies against PNX in a mixture with those against calcitonin gene-related peptide (CGRP), substance P (SP) or choline acetyltransferase (CHAT). The PNX-immunoreactivity had a similar distribution in the grey matter of all the spinal cord sections examined and was mainly observed in varicose nerve fibres (NF) that formed a dense plexus in laminae I and II of the dorsal horn. Nearly all of the PNX-immunoreactive NF stained also for CGRP or SP and, interestingly, many of them were CHAT-positive. The present study has provided for the first time the detailed information on the arrangement and chemical features of nerve structures expressing PNX-immunoreactivity in the spinal cord of a large mammal. The exact function of PNX in the spinal cord is not known yet. However, the distribution pattern and immunohistochemical characteristics of PNX-IR NF clearly suggest that this peptite most likely plays a role in spinal noxious signalling.

Comprehensive analysis of the differential expression profile of microRNAs in rats with spinal cord injury treated by electroacupuncture.

Abnormal microRNA (miRNA) expression has been implicated in spinal cord injury (SCI), but the underlying mechanisms are poorly understood. To observe the effect of electroacupuncture (EA) on miRNA expression profiles in SCI rats and investigate the potential mechanisms involved in this process, Sprague­Dawley rats were divided into sham, SCI and SCI+EA groups (n=6 each). Basso, Beattie and Bresnahan (BBB) scoring and hematoxylin­eosin staining of cortical tissues were used to evaluate spinal cord recovery with EA treatment 21 days post­surgery across the three groups. To investigate miRNA expression profiles, 6 Sprague­Dawley rats were randomly divided into SCI and SCI+EA groups (n=3 in each group) and examined using next­generation sequencing. Integrated miRNA­mRNA­pathway network analysis was performed to elucidate the interaction network of the candidate miRNAs, their target genes and the involved pathways. Behavioral scores suggested that hindlimb motor functions improved with EA treatments. Apoptotic indices were lower in the SCI+EA group compared with the SCI group. It was also observed that 168 miRNAs were differentially expressed between the SCI and SCI+EA groups, with 29 upregulated and 139 downregulated miRNAs in the SCI+EA group. Changes in miRNA expression are involved in SCI physiopathology, including inflammation and apoptosis. Reverse transcription­quantitative PCR measurement of the five candidate miRNAs, namely rno­miR­219a­5p, rno­miR­486, rno­miR­136­5p, rno­miR­128­3p, and rno­miR­7b, was consistent with RNA sequencing data. Integrated miRNA­mRNA­pathway analysis suggested that the MAPK, Wnt and NF­κB signaling pathways were involved in EA­mediated recovery from SCI. The present study evaluated the miRNA expression profiles involved in EA­treated SCI rats and demonstrated the potential mechanism and functional role of miRNAs in SCI in rats.

Activity of Antioxidant Defense Enzymes in Rats with Experimental Allergic Encephalomyelitis.

We studied activities of antioxidant system enzymes in tissues of rats with experimental allergic encephalomyelitis. It was shown that the development of pathology is accompanied by deformation of the neurons and axonal degeneration, intensification of free radical oxidation, exhaustion of the reduced glutathione pool, and multidirectional changes in activities of antioxidant enzymes in rat tissues. The observed imbalance in the antioxidant defense system can be associated with excessive glutathione utilization in the glutathione transferase reaction and different severity of the pathological process in the brain and spinal cord. The received data necessitate the search for compounds that can prevent inhibition of antioxidant system components in order to analyze the possibility of their use in the treatment of multiple sclerosis.

Prevention of clinical and histological signs of MOG-induced experimental allergic encephalomyelitis by prolonged treatment with recombinant human EGF.

Epidermal growth factor (EGF) represents the prototype of the group I EGF family. The pleiotropic effects of the EGF have attracted attention to the possibility that it could be implicated in autoimmune diseases, such as Multiple Sclerosis (MS). We show here that treatment with EGF, as a late prophylactic regime, improved the clinical and histological features of EAE, a preclinical model of MS. In silico analysis further corroborated these findings by demonstrating that EGF receptors are less expressed in CNS from patients with MS as compared to controls. Taken together these data provide clear-cut in vivo proof of concept for a beneficial role of exogenously administered EGF in MS, that may, therefore, represent a novel therapeutic approach.

Neuropathic Pain Up-Regulates Hypothalamo-Neurohypophysial and Hypothalamo-Spinal Oxytocinergic Pathways in Oxytocin-Monomeric Red Fluorescent Protein 1 Transgenic Rat.

Despite the high incidence of neuropathic pain, its mechanism remains unclear. Oxytocin (OXT) is an established endogenous polypeptide produced in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. OXT, which is synthesized by OXT neurons in the SON and the magnocellular part of the PVN (mPVN), is delivered into the posterior pituitary (PP), then released into the systemic blood circulation. Meanwhile, OXT-containing neurosecretory cells in the parvocellular part of the PVN (pPVN) are directly projected to the spinal cord and are associated with sensory modulation. In this study, the OXT system in the hypothalamo-neurohypophysial and hypothalamo-spinal pathway was surveyed using a rat neuropathic pain model induced by partial sciatic nerve ligation (PSL). In the present study, we used transgenic rats expressing an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene. In a neuropathic pain model, mechanical allodynia was observed, and glial cell activation was also confirmed via immunohistochemistry. In this neuropathic pain model, a significant increase in the OXT-mRFP1 expression was observed in the PP, the SON, mPVN, and pPVN. Furthermore, OXT-mRFP1 granules with positive fluorescent reaction were remarkably increased in laminae I and II of the ipsilateral dorsal horn. Although the plasma concentrations of OXT did not significantly change, a significant increase of the mRNA levels of OXT and mRFP1 in the SON, mPVN, and pPVN were observed. These results suggest that neuropathic pain induced by PSL upregulates hypothalamic OXT synthesis and transportation to the OXTergic axon terminals in the PP and spinal cord.

S1PR2 deficiency enhances neuropathic pain induced by partial sciatic nerve ligation

Background/aim: Sphingosine 1-phosphate receptor 2 (S1PR2), a member of the seven-transmembrane receptor family, can be activated by its natural ligand sphingosine 1-phosphate (S1P) to initiate signal transduction and is involved in a wide range of biological effects such as immune cell migration and vascular permeability. Its relationship with neuropathic pain (NP) has not been reported. In this study, the effects of S1PR2 on the development of NP were studied. Materials and methods: We generated a model of NP by partial sciatic nerve ligation (pSNL). The 50% paw withdrawal threshold of the wild-type (WT) group and the S1PR2 deficiency group were measured at several time points after surgery. The inflammatory factor levels of the two groups were measured by real-time quantitative polymerase chain reaction (RT-PCR). Neutrophil infiltration and glial cell activation were detected by immunofluorescence. Matrix metalloproteinase 9 (MMP9) and its substrate myelin basic protein (MBP) were measured by RT-PCR, western blotting, and immunofluorescence. Result: The S1PR2 deficiency group showed a reduction in 50% paw withdrawal threshold compared with WT mice (P < 0.05) at 3 days after the operation. In the ligated sciatic nerve of the S1PR2 deficiency group, the mRNA expression of IL-1ß was increased; the numbers of infiltrating neutrophils and activated astrocytes were also increased. The expression of MMP9 was elevated while MBP was decreased. Conclusion: S1PR2 deficiency could increase the pain sensitivity of a NP mouse model and promote the development of NP

Exosomes derived from bone marrow mesenchymal stem cells overexpressing microRNA-25 protect spinal cords against transient ischemia.

OBJECTIVE: We investigated the neuroprotection of exosomes derived from bone marrow mesenchymal stem cells overexpressing microRNA-25 on ischemic spinal cords. METHODS: Cultured mesenchymal stem cells were transfected with lentivirus vectors containing pre-microRNA-25 or control vectors. Exosomes were isolated and harvested by centrifugation. Spinal cord ischemia was induced in rats by crossclamping the descending aorta just distal to the left subclavian artery for 15 minutes. Exosomes from mesenchymal stem cells, mesenchymal stem cells transfected with control vector, or pre-microRNA-25 were administered by intrathecal injection before ischemia. Hind-limb motor function was assessed with the motor deficit index. Contents of interleukin-1ß, tumor necrosis factor-α, malondialdehyde, and superoxide dismutase activity were measured using commercial kits. Expressions of NADPH oxidase 2, NADPH oxidase 4, and microRNA-25 were detected by Western blot and quantitative reverse transcription polymerase chain reaction. Lumbar spinal cords were harvested for histologic examination. RESULTS: Transfection of pre-microRNA-25 significantly enhanced microRNA-25 levels in mesenchymal stem cells and their exosomes (P < .001). All exosome-pretreating groups exhibited lower levels of interleukin-1ß and tumor necrosis factor-α (P < .001), more intact motor neurons (P < .001), and lower motor deficit index scores (P < .005) than those of controls. Compared with exosomes, microRNA-25-enriched exosomes markedly enhanced microRNA-25 level (P < .001), inhibited NADPH oxidase 4 expression (P = .012), but not NADPH oxidase 2 expression, decreased malondialdehyde content (P = .022), increased superoxide dismutase activity (P < .001) in spinal cords, and had additional neuroprotective effects as evidenced by lower motor deficit index scores (P < .005) and more survival neurons (P = .002). CONCLUSIONS: The neuroprotection of exosomes from mesenchymal stem cells on ischemic spinal cords can be enhanced by genetic modification of the exosomes to contain elevated microRNA-25.

A versatile quantitative microdroplet elemental imaging method optimised for integration in biochemical workflows for low-volume samples.

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis of µ-droplets is becoming an attractive alternative for detecting and quantifying elements in biological samples. With minimal sample preparation required and detection limits comparable to solution nebulisation ICP-MS, µ-droplets have substantial advantages over traditional elemental detection, particularly for low volumes, such as aliquots taken from samples required for multiple independent biochemical assays, or fluids and tissues where elements of interest exist at native concentrations not suited to the necessary dilution steps required for solution nebulisation ICP-MS. However, the characteristics of µ-droplet residue deposition are heavily dependent on the matrix, and potential effects on signal suppression or enhancement have not been fully characterised. We present a validated and flexible high-throughput method for quantification of elements in µ-droplets using LA-ICP-MS imaging and matrix-matched external calibrants. Imaging the entire µ-droplet area removes analytical uncertainty arising from the often-heterogenous distribution when compared to radial or bisecting line scans that capture only a small portion of the droplet residue. We examined the effects of common matrices found in a standard biochemistry workflow, including native protein and salt contents, as well as reagents used in typical preparation steps for concurrent biochemical assays, such as total protein quantification and enzyme activity assays. We found that matrix composition results in systemic, concentration-dependent signal enhancement and suppression for carbon, whereas high sodium content has a specific space-charge-like suppression effect on high masses. We confirmed the accuracy of our method using both a certified serum standard (Seronorm™ L1) and independent measurements of analysed samples by solution nebulisation ICP-MS, then tested the specificity and reproducibility by examining spinal cord tissue homogenates from SOD1-G93A transgenic mice with a known molecular phenotype of increased copper- and zinc-binding superoxide dismutase-1 expression and altered copper-to-zinc stoichiometry. The method presented is rapid and transferable to multiple other biological matrices and allows high-throughput analysis of low-volume samples with sensitivity comparable to standard solution nebulisation ICP-MS protocols. Graphical Abstract ᅟ.

Docosahexaenoic acid induces changes in microglia/macrophage polarization after spinal cord injury in rats.

Docosahexaenoic acid (DHA, 22:6 (n-3)) leads to recovery of locomotor functions observed of spinal cord injury (SCI) in rats. In present study, we characterized the expression of iba-1, CD86, CD163 in microglia/macrophages, to assess activation state and M1 (pro-inflammatory)/M2 (anti-inflammatory) phenotypes respectively, in the rostral, central and caudal segment of the spinal cord on 7 and 35 days after SCI. We found that DHA treatment leads to: (1) an increased activation and proliferation of microglial cells; (2) an alteration in the dynamics between M1 and M2 microglia/macrophages phenotypes (3) and increased production of an antioxidant enzymes. Overall, our data demonstrates that DHA has a complex effect in post-traumatic process within the central nervous system, and supports the therapeutic potential of DHA-based drugs.

RNA and Protein Interactors with TDP-43 in Human Spinal-Cord Lysates in Amyotrophic Lateral Sclerosis.

The TAR DNA-binding protein of 43 kDa (TDP-43) is a dual function RNA- and DNA-binding protein with varied cellular functions. In degenerating motor neurons in amyotrophic lateral sclerosis (ALS), TDP-43 relocalizes from the nucleus to the cytosol, where it is sequestered into inclusions. It is likely that the pathogenic role of TDP-43 in ALS can involve either a gain or a loss of function, depending on the nature of its RNA or protein interactor. However, while TDP-43 binding partners have been identified in a range of model systems and from the human brain, interactors from human spinal-cord tissue have not. In this study, we have characterized both protein and RNA TDP-43 interactors from neuropathologically normal (control) and ALS-affected ventral lumbar spinal cord, including sporadic ALS (sALS) and familial cases harboring either a A4T mutant SOD1 or a 3' UTR *c.41G>A mutant FUS/TLS or expressing pathological c9orf72 expanded repeats. RNA interactors with TDP-43 were similar between the control and ALS spinal cords examined regardless of genotype. In contrast, protein interactors with TDP-43 did demonstrate differences, with the sALS and mtSOD1 harboring cases examined differing from the protein interactors identified in the FUS 3' UTR mutation and c9orf72 repeat-positive cases.

Optimized induction of beta common receptor enhances the neuroprotective function of erythropoietin in spinal cord ischemic injury.

BACKGROUND: Paraplegia remains the most feared complication of complex thoracoabdominal aortic intervention. Although erythropoietin (EPO) has demonstrated neuroprotective effects in spinal cord ischemia, it does not work until expression of the beta common receptor subunit of the EPO receptor (ßcR) is induced by ischemia. We hypothesized that the ßcR can be induced by diazoxide (DZ), amplifying the neuroprotective effects of EPO in spinal cord ischemia-reperfusion injury. METHODS: For the DZ time trial, adult male C57/BL6 mice received DZ (20 mg/kg) by oral gavage. Spinal cords were harvested after 0, 12, 24, 36, and 48 hours of administration. To evaluate optimal dosing, DZ was administered at 0, 5, 10, 20, and 40 mg/kg. The expression of ßcR was assessed by Western blot analysis. Five groups were studied: PBS (pretreatment)+PBS (immediately before), PBS+EPO, DZ+PBS, DZ+EPO, and sham (without cross-clamping). Spinal cord ischemia was induced by 4 minutes of thoracic aortic cross-clamping. Functional scoring (Basso Mouse Score) was done at 12-hour intervals for 48 hours, and spinal cords were harvested for histological analysis. RESULTS: Western blot analysis demonstrated that optimal ßcR up-regulation occurred at 36 hours after DZ administration, and the optimal DZ dosage for ßcR induction was 20 mg/kg. Motor function at 48 hours after treatment was significantly better preserved in the DZ+EPO group compared with all other groups, and was significantly better preserved in the DZ only and EPO only groups compared with control (PBS+PBS). CONCLUSIONS: Pharmacologic up-regulation of ßcR with DZ can increase the efficacy of EPO in preventing spinal cord ischemia and reperfusion injury. Improved understanding of this synergetic mechanism may serve to further prevent ischemic complications for high-risk aortic intervention.

A cellular spinal cord scaffold seeded with rat adipose­derived stem cells facilitates functional recovery via enhancing axon regeneration in spinal cord injured rats.

Spinal cord injury (SCI), usually resulting in severe sensory and motor deficits, is a major public health concern. Adipose­derived stem cells (ADSCs), one type of adult stem cell, are free from ethical restriction, easily isolated and enriched. Therefore, ADSCs may provide a feasible cell source for cell­based therapies in treatment of SCI. The present study successfully isolated rat ADSCs (rADSCs) from Sprague­Dawley male rats and co­cultured them with acellular spinal cord scaffolds (ASCs). Then, a rat spinal cord hemisection model was built and rats were randomly divided into 3 groups: SCI only, ASC only, and ASC + ADSCs. Furthermore, behavioral tests were conducted to evaluate functional recovery. Hematoxylin & Eosin staining and immunofluorence were carried out to assess histopathological remodeling. In addition, biotinylated dextran amines anterograde tracing was employed to visualize axon regeneration. The data demonstrated that harvested cells, which were positive for cell surface antigen cluster of differentiation (CD) 29, CD44 and CD90 and negative for CD4, detected by flow cytometry analysis, held the potential to differentiate into osteocytes and adipocytes. Rats that received transplantation of ASCs seeded with rADSCs benefited greatly in functional recovery through facilitation of histopathological rehabilitation, axon regeneration and reduction of reactive gliosis. rADSCs co­cultured with ASCs may survive and integrate into the host spinal cord on day 14 post­SCI.

Expression pattern of high-affinity tyrosine kinase Aduring the development of human fetal spinal cord.

High-affinity tyrosine kinase A (TrkA) is responsible for the biological activities of nerve growth factor. Most studies of the molecular mechanisms of TrkA that underlie the development of the spinal cord have been conducted in animals and the expression pattern of TrkA during the development of the human fetal spinal cord is not well characterized. We investigated 45 3-28-week-old (G3W-G28W) human fetuses. We assessed the expression pattern of TrkA in the human fetal spinal cord using immunohistochemistry, western blot and reverse transcription polymerase chain reaction to clarify the spatiotemporal developmental changes and to determine the role TrkA plays in development. TrkA immunoreactive products were detected widely in the alar and basal plates, ependyma, glial cells, gray and white matter, internal limiting membrane, mantle layer, marginal layer, neuroepithelium and neurons during this period of development. Expression levels of TrkA mRNA and protein peaked at G12W and G16W, respectively. The strong expression of TrkA was closely related to the formation of the dorsal and ventral horns, and the differentiation of somatic motor neurons during late embryonic development. Our findings suggest that TrkA receptors play crucial roles during the development of human fetal spinal cord. The characteristic expression patterns may clarify the developmental characteristics of the human spinal cord.