Activation of Three Major Signaling Pathways After Endurance Training and Spinal Cord Injury.

We aimed to investigate the effects of endurance training on expression of growth factors (GFs) and stimulation of neurotrophin-dependent signaling pathways (PI3k/Akt, PLCγ/PKC, PLCγ/CAMKII, Ras-Erk1/2 and Rac1-Cdc42) responsible for neuroplasticity, neuroregeneration, survival and growth after spinal cord injury (SCI). Wistar rats were divided into four groups: (i) intact controls; (ii) 6 weeks of endurance training; (iii) SCI; (iv) pre-training + SCI. The animals survived for 6 weeks after SCI. Firstly, endurance training markedly upregulated mRNA expression and protein levels (up to four times) of growth factors (BDNF, GDNF) and their receptors (TrkB, Gfrα) in low thoracic segments (Th8-Th10) compared to levels in untrained animals. Secondly, we found that spontaneous neuroplasticity seen in the SCI alone group was GF-specific and was activated through both PLCγ-PKC and PLC-CAMKII signaling pathways. In addition, training prior to SCI markedly increased the activity of PLCγ-PKC signaling at both transcript and protein levels at and around the lesion site. Similar effects were seen in expression of PI3k/Akt and Ras/Erk1/2 signaling responsible for cell survival and regeneration. Thirdly, rats which underwent physical activity prior to SCI were more active and had significantly better neurological scores at the 14th and 42nd days of survival. These results suggest that regular physical activity could play an important role after SCI, as it maintains increased expression of GFs in spinal cord tissue 6 weeks post-SCI. The BDNF- and/or BDNF + GDNF-dependent signaling pathways were significantly affected in pre-trained SCI animals. In contrast, GDNF-dependent Rac1-Cdc42 signaling was not involved in training-affected SCI response.

Activation of Neuroprotective Microglia and Astrocytes at the Lesion Site and in the Adjacent Segments Is Crucial for Spontaneous Locomotor Recovery after Spinal Cord Injury.

Microglia and astrocytes play an important role in the regulation of immune responses under various pathological conditions. To detect environmental cues associated with the transformation of reactive microglia (M1) and astrocytes (A1) into their polarization states (anti-inflammatory M2 and A2 phenotypes), we studied time-dependent gene expression in naive and injured spinal cord. The relationship between astrocytes and microglia and their polarization states were studied in a rat model after Th9 compression (40 g/15 min) in acute and subacute stages at the lesion site, and both cranially and caudally. The gene expression of microglia/macrophages and M1 microglia was strongly up-regulated at the lesion site and caudally one week after SCI, and attenuated after two weeks post-SCI. GFAP and S100B, and A1 astrocytes were profoundly expressed predominantly two weeks post-SCI at lesion site and cranially. Gene expression of anti-inflammatory M2a microglia (CD206, CHICHI, IL1rn, Arg-1), M2c microglia (TGF-ß, SOCS3, IL4R α) and A2 astrocytes (Tgm1, Ptx3, CD109) was greatly activated at the lesion site one week post-SCI. In addition, we observed positive correlation between neurological outcome and expression of M2a, M2c, and A2 markers. Our findings indicate that the first week post-injury is critical for modulation of reactive microglia/astrocytes into their neuroprotective phenotypes.

A Single Dose of Atorvastatin Applied Acutely after Spinal Cord Injury Suppresses Inflammation, Apoptosis, and Promotes Axon Outgrowth, Which Might Be Essential for Favorable Functional Outcome.

The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) using Atorvastatin (ATR), a potent inhibitor of cholesterol biosynthesis. Adult Wistar rats were divided into five experimental groups: one control group, two Th9 compression (40 g/15 min) groups, and two Th9 compression + ATR (5 mg/kg, i.p.) groups. The animals survived one day and six weeks. ATR applied in a single dose immediately post-SCI strongly reduced IL-1ß release at 4 and 24 h and considerably reduced the activation of resident cells at one day post-injury. Acute ATR treatment effectively prevented the excessive infiltration of destructive M1 macrophages cranially, at the lesion site, and caudally (by 66%, 62%, and 52%, respectively) one day post-injury, whereas the infiltration of beneficial M2 macrophages was less affected (by 27%, 41%, and 16%). In addition, at the same time point, ATR visibly decreased caspase-3 cleavage in neurons, astrocytes, and oligodendrocytes. Six weeks post-SCI, ATR increased the expression of neurofilaments in the dorsolateral columns and Gap43-positive fibers in the lateral columns around the epicenter, and from day 30 to 42, significantly improved the motor activity of the hindlimbs. We suggest that early modulation of the inflammatory response via effects on the M1/M2 macrophages and the inhibition of caspase-3 expression could be crucial for the functional outcome.

Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation.

INTRODUCTION: Intraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury. METHODS: Three-month-old female Sprague-Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies. RESULTS: Intraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons. CONCLUSIONS: Peri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation.

BACKGROUND: Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1(G93A) rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1(G93A) rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1(G93A) animals. METHODS/PRINCIPAL FINDINGS: Presymptomatic SOD1(G93A) rats (60-65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1(G93A) rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1(G93A) rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1(G93A) rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50-65%) loss of large caliber descending motor axons. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.

Spinal cord transection modifies neuronal nitric oxide synthase expression in medullar reticular nuclei and in the spinal cord and increases parvalbumin immunopositivity in motoneurons below the site of injury in experimental rabbits.

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III-IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.

Effects of short-duration electromagnetic radiation on early postnatal neurogenesis in rats: Fos and NADPH-d histochemical studies.

The immediate effects of whole body electromagnetic radiation (EMR) were used to study postnatal neurogenesis in the subventricular zone (SVZ) and rostral migratory stream (RMS) of Wistar rats of both sexes. Newborn postnatal day 7 (P7) and young adult rats (P28) were exposed to pulsed electromagnetic fields (EMF) at a frequency of 2.45 GHz and mean power density of 2.8 mW/cm(2) for 2 h. Post-irradiation changes were studied using immunohistochemical localization of Fos and NADPH-d. We found that short-duration exposure induces increased Fos immunoreactivity selectively in cells of the SVZ of P7 and P28 rats. There were no Fos positive cells visible within the RMS of irradiated rats. These findings indicate that some differences exist in prerequisites of proliferating cells between the SVZ and RMS regardless of the age of the rats. Short-duration exposure also caused praecox maturation of NADPH-d positive cells within the RMS of P7 rats. The NADPH-d positive cells appeared several days earlier than in age-matched controls, and their number and morphology showed characteristics of adult rats. On the other hand, in the young adult P28 rats, EMR induced morphological signs typical of early postnatal age. These findings indicate that EMR causes age-related changes in the production of nitric oxide (NO), which may lead to different courses of the proliferation cascade in newborn and young adult neurogenesis.

Immunohistochemical study of postnatal neurogenesis after whole-body exposure to electromagnetic fields: evaluation of age- and dose-related changes in rats.

It is well established that strong electromagnetic fields (EMFs) can give rise to acute health effects, such as burns, which can be effectively prevented by respecting exposure guidelines and regulations. Current concerns are instead directed toward the possibility that long-term exposure to weak EMF might have detrimental health effects due to some biological mechanism, to date unknown. (1) The possible risk due to pulsed EMF at frequency 2.45 GHz and mean power density 2.8 mW/cm(2) on rat postnatal neurogenesis was studied in relation to the animal's age, duration of the exposure dose, and post-irradiation survival. (2) Proliferating cells marker, BrdU, was used to map age- and dose-related immunohistochemical changes within the rostral migratory stream (RMS) after whole-body exposure of newborn (P7) and senescent (24 months) rats. (3) Two dose-related exposure patterns were performed to clarify the cumulative effect of EMF: short-term exposure dose, 2 days irradiation (4 h/day), versus long-term exposure dose, 3 days irradiation (8 h/day), both followed by acute (24 h) and chronic (1-4 weeks) post-irradiation survival. (4) We found that the EMF induces significant age- and dose-dependent changes in proliferating cell numbers within the RMS. Our results indicate that the concerns about the possible risk of EMF generated in connection with production, transmission, distribution, and the use of electrical equipment and communication sets are justified at least with regard to early postnatal neurogenesis.

Induction of mesenchymal stem cells leads to HSP72 synthesis and higher resistance to oxidative stress.

The phenomenon of neuronal transdifferentiation performed on bone marrow mesenchymal stem cells (MSCs) has been criticized by recent studies indicating that acquired neuron-like morphology of induced MSCs is caused by cellular stress. Therefore, to test this hypothesis we have investigated whether exposure of rat MSCs (rMSCs) to chemical inducer 2 mM beta-mercaptoethanol (BME) for 1-3 h followed by 24 h incubation leads to HSP72 synthesis, thus suggesting higher resistance of rMSCs to oxidative damage. Present data from immunohistochemistry clearly indicate development of time-dependent sub-cellular HSP72 distribution, initially seen in nuclei at 1 h followed by its translocation to surrounding central cytoplasm and processes at 2-3 h after BME stimulation. Western blot (WB) analysis confirmed the expression of HSP72 protein in induced rMSCs at both stimulation periods. Furthermore, preconditioned rMSCs with BME for 1 h expressing HSP72 positivity at 24 h showed higher resistance (78 +/- 10% of survival cells) to oxidative stress caused by 1 mM H(2)O(2) when compared to those preconditioned for 3 h (59 +/- 8% of survival cells) or control-unconditioned rMSCs exposed to the same stressor conditions (56 +/- 6% of survival cells). Thus, the cellular protection was lost if the duration of BME preconditioning was increased as far as possible (3 h) (while still remaining sub-lethal). This suggests that exposure of rMSCs to the optimal concentration of BME (2 mM) during optimal induction period (1 h) mediate their protection and increases resistance to oxidative injury, while over crossing these limits is in-effective. In addition, our findings confirm that cultured rMSCs remain competent to be preconditioned by BME, through a pathway that may increase the antioxidant balance or involve activation of HSP72 protein induced tolerance.