Isolated spinal cord contusion in rats induces chronic brain neuroinflammation,neurodegeneration, and cognitive impairment

Cognitive dysfunction has been reported in patients with spinal cord injury (SCI), but it has been questioned whether such changes may reflect concurrent head injury, and the issue has not been addressed mechanistically or in a well-controlled experimental model. Our recent rodent studies examining SCI-induced hyperesthesia revealed neuroinflammatory changes not only in supratentorial pain-regulatory sites, but also in other brain regions, suggesting that additional brain functions may be impacted following SCI. Here we examined effects of isolated thoracic SCI in rats on cognition, brain inflammation, and neurodegeneration. We show for the first time that SCI causes widespread microglial activation in the brain, with increased expression of markers for activated microglia/macrophages, including translocator protein and chemokine ligand 21 (C–C motif). Stereological analysis demonstrated significant neuronal loss in the cortex, thalamus, and hippocampus. SCI caused chronic impairment in spatial, retention, contextual, and fear-related emotional memory—evidenced by poor performance in the Morris water maze, novel objective recognition, and passive avoidance tests. Based on our prior work implicating cell cycle activation (CCA) in chronic neuroinflammation after SCI or traumatic brain injury, we evaluated whether CCA contributed to the observed changes. Increased expression of cell cycle-related genes and proteins was found in hippocampus and cortex after SCI. Posttraumatic brain inflammation, neuronal loss, and cognitive changes were attenuated by systemic post-injury administration of a selective cyclin-dependent kinase inhibitor. These studies demonstrate that chronic brain neurodegeneration occurs after isolated SCI, likely related to sustained microglial activation mediated by cell cycle activation.     

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.     

EBF proteins participate in transcriptional regulation of Xenopus muscle development

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells.     

Differential expression of metabolic genes essential for glucose and lipid metabolism in skeletal muscle from spinal cord injured subjects

Skeletal muscle plays an important role in the regulation of energy homeostasis; therefore, the ability of skeletal muscle to adapt and alter metabolic gene expression in response to changes in physiological demands is critical for energy balance. Individuals with cervical spinal cord lesions are characterized by tetraplegia, impaired thermoregulation, and altered skeletal muscle morphology. We characterized skeletal muscle metabolic gene expression patterns, as well as protein content, in these individuals to assess the impact of spinal cord injury on critical determinants of skeletal muscle metabolism. Our results demonstrate that mRNA levels and protein expression of skeletal muscle genes essential for glucose storage are reduced, whereas expression of glycolytic genes is reciprocally increased in individuals with spinal cord injury. Furthermore, expression of genes essential for lipid oxidation is coordinately reduced in spinal cord injured subjects, consistent with a marked reduction of mitochondrial proteins. Thus spinal cord injury resulted in a profound and tightly coordinated change in skeletal muscle metabolic gene expression program that is associated with the aberrant metabolic features of the tissue.     

Differential sensitivity to apoptosis between the human small and large intestinal mucosae: linkage with segment-specific regulation of BCL-2 homologs and involvement of signaling pathways.

The small and large intestines differ in their expression profiles of Bcl-2 homologs. Intestinal segment-specific Bcl-2 homolog expression profiles are acquired as early as by mid-gestation (18-20 weeks) in man. In the present study, we examined the question whether such distinctions underlie segment-specific control mechanisms of intestinal cell survival. Using mid-gestation human jejunum and colon organotypic cultures, we analyzed the impact of growth factors (namely insulin; 10 microg/ml) and pharmacological compounds that inhibit signal transduction molecules/pathways (namely tyrosine kinases, Fak, P13-K/Akt, and MEK/Erk) on cell survival and Bcl-2 homolog expression (anti-apoptotic: Bcl-2, Bcl-X(L), Mcl-1; pro-apoptotic: Bax, Bak, Bad). The relative activation levels of p125Fak, p42Erk-2, and p57Akt were analyzed as well. Herein, we report that (1) the inhibition of signal transduction molecules/pathways revealed striking differences in their impact on cell survival in the jejunum and colon (e.g., the inhibition of p125Fak induced apoptosis with a significantly greater extent in the jejunum [approximately 43%] than in the colon [approximately 24%]); (2) sharp distinctions between the two segments were noted in the modulatory effects of the various treatments on Bcl-2 homolog steady-state levels (e.g., inhibition of tyrosine kinase activities in the jejunum down-regulated all anti-apoptotics analyzed while increasing Bax, whereas the same treatment in the colon down-regulated Bcl-X(L) only and increased all pro-apoptotics); and (3) in addition to their differential impact on cell survival and Bcl-2 homolog expression, the MEK/Erk and P13-K/Akt pathways were found to be distinctively regulated in the jejunum and colon mucosae (e.g., insulin in the jejunum increased p42Erk-2 activation without affecting that of p57Akt, whereas the same treatment in the colon decreased p42Erk-2 activation while increasing that of p57Akt). Altogether, these data show that intestinal cell survival is characterized by segment-specific susceptibilities to apoptosis, which are in turn linked with segmental distinctions in the involvement of signaling pathways and the regulation of Bcl-2 homolog steady-state levels. Therefore, these indicate that cell survival is subject to segment-specific control mechanisms along the proximal-distal axis of the intestine.     

Identification of key pathways and RNAs associated with skeletal muscle atrophy after spinal cord injury

Objective:This study was performed to investigate the potential key molecules involved in the progression of skeletal muscle atrophy after SCI.Methods:Based on {"type":"entrez-geo","attrs":{"text":"GSE21497","term_id":"21497"}}GSE21497 dataset, the DEmRNAs and DElncRNAs were screened after differentially expressed analysis. Then the enrichment analyses were performed on DEmRNAs. Then the PPI network and ceRNA network were constructed. Finally, the DGIdb was utilized to predict drug-gene interactions.Results:A total of 412 DEmRNAs and 21 DElncRNAs were obtained. The DEmRNAs were significantly enriched in MAPK signaling pathway and FoxO signaling pathway. In addition, UBE2D1, JUN, and FBXO32 had higher node degrees in PPI network, and the top 20 genes with high degree were significantly enriched in FoxO signaling pathway and Endometrial cancer. Moreover, FOXO3 was regulated by hsa-miR-1207-5p and hsa-miR-1207-5p was regulated by lncRNA RP11-253E3.3 in ceRNA network. Finally, 37 drug-gene interactions were obtained based on the 26 genes in ceRNA network.Conclusion:UBE2D1, JUN, and FBXO32 are likely to be related to the progression of skeletal muscle atrophy after SCI, and activating of MAPK signaling pathway, Endometrial cancer and FoxO signaling pathway may induce skeletal muscle inflammation, apoptosis, autophagy and atrophy after SCI. Moreover, RP11-253E3.3-hsa-miR-1207-5p-FOXO3 axis may be a promising therapeutic target for skeletal muscle atrophy after SCI.     

Human spinal GABA neurons alleviate spasticity and improve locomotion in rats with spinal cord injury

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Delayed dedifferentiation and retention of properties in dissociated adult skeletal muscle fibers in vitro

Adult skeletal muscle fibers can be isolated and cultured but tend to dedifferentiate and sprout with time in culture. We examined isolated adult mouse flexor digitorum brevis muscle fibers under various culture conditions by monitoring maintenance of the same fibers at 2-d intervals using survival analysis. Fibers plated on laminin and cultured in serum-free media did not show sprouting and exhibited significantly (P < 0.0001) longer survival (median survival time, T(50) = 10.2 d) than fibers in serum-containing media (T(50) = 3.3 d). Cell proliferation was markedly suppressed in serum-free cultures. Multiple or delayed Ca(2+) transients in response to brief field stimulation were often observed in dedifferentiated fibers after several d in serum-containing media but were not observed in fibers in serum-free media. The addition of cytosine arabinoside to serum-containing cultures did not prolong fiber survival (P = 0.39) and did not eliminate sprouting but did greatly suppress proliferation of nonmuscle cells. Fibers cultured in agarose gel with serum exhibited small, bud-like extensions but no sprouts and did not survive as long (T(50) = 6.2 d) as fibers plated on laminin and cultured in serum-free media (T(50) = 10.2 d) did. These results demonstrate that both morphological and physiological properties of fibers become modified in serum-containing media but can be retained by culturing without serum.     

Differential activation of stress-responsive signalling proteins associated with altered loading in a rat skeletal muscle

Skeletal muscle undergoes a significant reduction in tension upon unloading. To explore intracellular signalling mechanisms underlying this phenomenon, we investigated twitch tension, the ratio of actin/myosin filaments, and activities of key signalling molecules in rat soleus muscle during a 3-week hindlimb suspension and 2-week reloading. Twitch tension and myofilament ratio (actin/myosin) gradually decreased during unloading but progressively recovered to initial levels during reloading. To study the involvement of stress-responsive signalling proteins during these changes, the activities of protein kinase C alpha (PKCalpha) and three mitogen-activated protein kinases (MAPKs)--c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 MAPK--were examined using immunoblotting and immune complex kinase assays. PKCalpha phosphorylation correlated positively with the tension (Pearson's r = 0.97, P < 0.001) and the myofilament ratio (r = 0.83, P < 0.01) over the entire unloading and reloading period. Treatment of the soleus muscle with a PKC activator resulted in a similar paralleled increment in both PKCalpha phosphorylation and the alpha-sarcomeric actin expression. The three MAPKs differed in the pattern of activation in that JNK activity peaked only for the first hours of reloading, whereas ERK and p38 MAPK activities remained elevated during reloading. These results suggest that PKCalpha may play a pivotal role in converting loading stress to intracellular changes in contractile proteins that determine muscle tension. Differential activation of MAPKs may also help alleviate muscle damage, modulate energy transport and/or regulate the expression of contractile proteins upon altered loading.     

Hepatoma-derived growth factor, a new trophic factor for motor neurons, is up-regulated in the spinal cord of PQBP-1 transgenic mice before onset of degeneration

Hepatoma-derived growth factor (HDGF) is a nuclear protein homologous to the high-mobility group B1 family of proteins. It is known to be released from cells and to act as a trophic factor for dividing cells. In this study HDGF was increased in spinal motor neurons of a mouse model of motor neuron degeneration, polyglutamine-tract-binding protein-1 (PQBP-1) transgenic mice, before onset of degeneration. HDGF promoted neurite extension and survival of spinal motor neurons in primary culture. HDGF repressed cell death of motor neurons after facial nerve section in newborn rats in vivo. We also found a significant increase in p53 in spinal motor neurons of the transgenic mice. p53 bound to a sequence in the upstream of the HDGF gene in a gel mobility shift assay, and promoted gene expression through the cis-element in chloramphenicol acetyl transfer (CAT) assay. Finally, we found that HDGF was increased in CSF of PQBP-1 transgenic mice. Collectively, our results show that HDGF is a novel trophic factor for motor neurons and suggest that it might play a protective role against motor neuron degeneration in PQBP-1 transgenic mice.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

Trophic effects of skeletal muscle extracts on ventral spinal cord neurons in vitro: separation of a protein with morphologic activity from proteins with cholinergic activity

Protein factors derived from skeletal muscle separately promote neurite elongation and acetylcholine synthesis in cultured rat ventral spinal neurons. Morphologic factor activity (neurite-inducing activity) is specifically found in rat skeletal muscle and cord neuron extracts, decreases with the postnatal age of the rats from which muscle extract is prepared, and increases in rat hindlimb muscle after 5 d of denervation. Cholinergic factor activity (acetylcholine synthesis-stimulating activity) is found in extracts of rat cerebral cortex and cardiac muscle in addition to spinal cord and skeletal muscle, increases with animal age, and decreases following 5 d of denervation. Biochemically, the factors responsible for these activities differ in their lability to denaturing conditions, apparent molecular weights, isoelectric points, and lectin-binding specificities. Under reducing conditions, morphologic activity is isolated in a single acidic glycoprotein with an Mr of 35,000, while acetylcholine synthesis-stimulating activity is found in multiple species of different molecular weights. Thus, acetylcholine synthesis-promoting activities and neurite growth-promoting activity appear to reside in different molecules. Significant purification of several of these factors has been achieved.     

Elongation of mouse spinal cord axons in isogenic grafts of the sciatic nerve, skeletal muscle and submaxillary gland

Isografts of sciatic nerve, skeletal muscle, submaxillary gland and, as control experiments, of optice nerve, were transplanted into the non transected spinal cord of young albino mice, through a punctiform pial aperture. Under these conditions, local cellular reactions were reduced and the sensori motor behavior of the operated animals remained apparently undisturbed throughout the experimental period. Within a few days, axonal sprouts issuing mainly from the terminal clubs of intraspinal nerve fibres severed by the grafting procedure were seen elongating and growing into--and presumably throughout--the nervous as well as the muscular and glandular transplants. The Schwann cells of these grafts, either sedentary or migrating towards the cord and intermingling with host reactive glial cells, appeared to guide the growth of the axonal sprouts they ensheathed (from day 3 to day 10) and generally myelinated (as early as day 6). Optic nerve transplants, lacking Schwann cells, were never reinnervated. Furthermore, in control microinjuries without grafting, limited growth of axonal sprouts was observed only when a few host Schwann cells were present. Mouse spinal neurons, therefore, demonstrate a marked capacity for regrowth when minimal damage to the spinal cord is associated with an adequate supply of Schwann cells. In contrast, host as well as transplanted glial cells, were unable, at least when they were not associated with Schwannian elements, to promote regenerative expression of these central neurons.     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Effects of cisplatin on mitochondrial function and autophagy-related proteins in skeletal muscle of rats

Cisplatin is widely known as an anti-cancer drug. However, the effects of cisplatin on mitochondrial function and autophagy-related proteins levels in the skeletal muscle are unclear. The purpose of this study was to investigate the effect of different doses of cisplatin on mitochondrial function and autophagy-re-lated protein levels in the skeletal muscle of rats. Eight-week-old male Wistar rats (n = 24) were assigned to one of three groups; the first group was administered a saline placebo (CON, n = 10), and the second and third groups were given 0.1 mg/kg body weight (BW) (n = 6), and 0.5 mg/kg BW (n = 8) of cisplatin, respectively. The group that had been administered 0.5 mg cisplatin exhibited a reduced BW, skeletal muscle tissue weight, and mitochondrial function and upregulated levels of autophagy-related proteins, including LC3II, Beclin 1, and BNIP3. Moreover, this group had a high LC3 II/I ratio in the skeletal muscle; i.e., the administration of a high dose of cisplatin decreased the muscle mass and mitochondrial function and increased the levels of autophagy-related proteins. These results, thus, suggest that reducing mitochondrial dysfunction and autophagy pathways may be important for preventing skeletal muscle atrophy following cisplatin administration.     

Differential expression of skeletal muscle proteins in high‐fat diet‐fed rats in response to capsaicin feeding

In this study, the effects of capsaicin on expression of skeletal muscle proteins in Sprague–Dawley rats fed with a high‐fat diet (HFD) were investigated. Rats were fed a HFD with or without capsaicin treatment for 8 wk. After HFD feeding, capsaicin‐treated rats weighed an average of 8% less than those of the HFD control group. Gastrocnemius muscle tissue from lean and obese rats with or without capsaicin treatment was arrayed using 2‐DE for detection of HFD‐associated markers. Proteomic analysis using 2‐DE demonstrated that 36 spots from a total of approximately 600 matched spots showed significantly different expression; 27 spots were identified as gastrocnemius muscle proteins that had been altered in response to capsaicin feeding, and 6 spots could not be identified by mass fingerprinting. Expression of various muscle proteins was determined by immunoblot analysis for the determination of molecular mechanisms, whereby capsaicin caused inhibition of adipogenesis. Immunoblot analysis revealed increased uncoupling protein 3 (UCP3) protein expression in HFD‐fed rats, whereas contents were reduced with capsaicin treatment. Compared with the HFD control group, capsaicin treatment increased phosphorylation of AMP‐activated protein kinase (AMPIC) CP3 and acetyl‐CoA carboxylase (ACC). To support this result, we also analyzed in vitro differential protein expression in L6 skeletal muscle cells. These data suggest that the AMPK‐ACC‐malonyl‐CoA metabolic signaling pathway is one of the targets of capsaicin action. To the best of our knowledge, this is the first proteomic study to report on analysis of diet‐induced alterations of protein expression that are essential for energy expenditure in rat muscle.