Impact of Endurance Training on Regeneration of Axons, Glial Cells, and Inhibitory Neurons after Spinal Cord Injury: A Link between Functional Outcome and Regeneration Potential within the Lesion Site and in Adjacent Spinal Cord Tissue.

Endurance training prior to spinal cord injury (SCI) has a beneficial effect on the activation of signaling pathways responsible for survival, neuroplasticity, and neuroregeneration. It is, however, unclear which training-induced cell populations are essential for the functional outcome after SCI. Adult Wistar rats were divided into four groups: control, six weeks of endurance training, Th9 compression (40 g/15 min), and pretraining + Th9 compression. The animals survived six weeks. Training alone increased the gene expression and protein level of immature CNP-ase oligodendrocytes (~16%) at Th10, and caused rearrangements in neurotrophic regulation of inhibitory GABA/glycinergic neurons at the Th10 and L2 levels, known to contain the interneurons with rhythmogenic potential. Training + SCI upregulated markers for immature and mature (CNP-ase, PLP1) oligodendrocytes by ~13% at the lesion site and caudally, and increased the number of GABA/glycinergic neurons in specific spinal cord regions. In the pretrained SCI group, the functional outcome of hindlimbs positively correlated with the protein levels of CNP-ase, PLP1, and neurofilaments (NF-l), but not with the outgrowing axons (Gap-43) at the lesion site and caudally. These results indicate that endurance training applied before SCI potentiates the repair in damaged spinal cord, and creates a suitable environment for neurological outcome.

Epidural oscillating field stimulation increases axonal regenerative capacity and myelination after spinal cord trauma.

Oscillating field stimulation (OFS) with regular alterations in the polarity of electric current is a unique, experimental approach to stimulate, support, and potentially guide the outgrowth of both sensory and motor nerve fibers after spinal cord injury (SCI). In previous experiments, we demonstrated the beneficial effects of OFS in a 4-week survival period after SCI. In this study, we observed the major behavioral, morphological, and protein changes in rats after 15 minutes of T9 spinal compression with a 40 g force, followed by long-lasting OFS (50 µA), over a 8-week survival period. Three groups of rats were analyzed: rats after T9 spinal compression (SCI group); SCI rats subjected to implantation of active oscillating field stimulator (OFS + SCI group); and SCI rats subjected to nonfunctional OFS (nOFS + SCI group). Histopathological analysis of spinal tissue indicated a strong impact of epidural OFS on the reduction of tissue and myelin loss after SCI in the segments adjacent to the lesion site. Quantitative fluorescent analysis of the most affected areas of spinal cord tissue revealed a higher number of spared axons and oligodendrocytes of rats in the OFS + SCI group, compared with rats in the SCI and nOFS + SCI groups. The protein levels of neurofilaments (NF-l), growth-associated protein-43 (marker for newly sprouted axons), and myelin basic protein in rats were signifiantly increased in the OFS + SCI group than in the nOFS + SCI and SCI groups. This suggests a supporting role of the OFS in axonal and myelin regeneration after SCI. Moreover, rats in the OFS + SCI group showed great improvements in sensory and motor functions than did rats in the nOFS + SCI and SCI groups. All these findings suggest that long-lasting OFS applied immediately after SCI can provide a good microenviroment for recovery of damaged spinal tissue by triggering regenreative processes in the acute phase of injury.

The Histopathology of Severe Graded Compression in Lower Thoracic Spinal Cord Segment of Rat, Evaluated at Late Post-injury Phase.

Spontaneous recovery of lost motor functions is relative fast in rodent models after inducing a very mild/moderate spinal cord injury (SCI), and this may complicate a reliable evaluation of the effectiveness of potential therapy. Therefore, a severe graded (30 g, 40 g and 50 g) weight-compression SCI at the Th9 spinal segment, involving an acute mechanical impact followed by 15 min of persistent compression, was studied in adult female Wistar rats. Functional parameters, such as spontaneous recovery of motor hind limb and bladder emptying function, and the presence of hematuria were evaluated within 28 days of the post-traumatic period. The disruption of the blood-spinal cord barrier, measured by extravasated Evans Blue dye, was examined 24 h after the SCI, when maximum permeability occurs. At the end of the survival period, the degradation of gray and white matter associated with the formation of cystic cavities, and quantitative changes of glial structural proteins, such as GFAP, and integral components of axonal architecture, such as neurofilaments and myelin basic protein, were evaluated in the lesioned area of the spinal cord. Based on these functional and histological parameters, and taking the animal's welfare into account, the 40 g weight can be considered as an upper limit for severe traumatic injury in this compression model.

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.

Glial-Neuronal Interactions in Pathogenesis and Treatment of Spinal Cord Injury.

Traumatic spinal cord injury (SCI) elicits an acute inflammatory response which comprises numerous cell populations. It is driven by the immediate response of macrophages and microglia, which triggers activation of genes responsible for the dysregulated microenvironment within the lesion site and in the spinal cord parenchyma immediately adjacent to the lesion. Recently published data indicate that microglia induces astrocyte activation and determines the fate of astrocytes. Conversely, astrocytes have the potency to trigger microglial activation and control their cellular functions. Here we review current information about the release of diverse signaling molecules (pro-inflammatory vs. anti-inflammatory) in individual cell phenotypes (microglia, astrocytes, blood inflammatory cells) in acute and subacute SCI stages, and how they contribute to delayed neuronal death in the surrounding spinal cord tissue which is spared and functional but reactive. In addition, temporal correlation in progressive degeneration of neurons and astrocytes and their functional interactions after SCI are discussed. Finally, the review highlights the time-dependent transformation of reactive microglia and astrocytes into their neuroprotective phenotypes (M2a, M2c and A2) which are crucial for spontaneous post-SCI locomotor recovery. We also provide suggestions on how to modulate the inflammation and discuss key therapeutic approaches leading to better functional outcome after SCI.

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.

Is Innervation of the Neuromuscular Junction at the Diaphragm Modulated by sGC/cGMP Signaling?

We previously reported NO/sGC signaling in the upper respiratory pathway, receiving input from the respiratory neurons of the brainstem to phrenic motoneurons in the C3-C6 spinal cord. In order to assess whether innervation of the neuromuscular junction (NMJ) at the diaphragm is modulated by sGC/cGMP signaling, we performed unilateral 8-day continuous ligation of the phrenic nerve in rats. We examined sGCß1 within the lower bulbospinal pathway (phrenic motoneurons, phrenic nerves and NMJs at the diaphragm) and the cGMP level in the contra- and ipsilateral hemidiaphragm. Additionally, we characterized the extent of phrenic nerve axonal degeneration and denervation at diaphragm NMJs. The results of our study show that continuous 8-day phrenic nerve ligation caused a marked increase in sGCß1 (immunoreactivity and the protein level) in the ipsilateral phrenic motor pool. However, the protein sGCß1 level in the phrenic nerve below its ligation and the cGMP level in the ipsilateral hemidiaphragm were evidently decreased. Using confocal analysis we discovered a reduction in sGCß1-IR boutons/synaptic vesicles at the ipsilateral MNJs. These findings are consistent with the marked axonal loss (∼47%) and significant NMJs degeneration in the ipsilateral diaphragm muscle. The remarkable unilateral decrease in cGMP level in the diaphragm and the failure of EMG recordings in the ipsilateral hemidiaphragm muscle can be attributed to the fact that sGC is involved in transmitter release at the diaphragm NMJs via the sGC-cGMP pathway.

Epidural oscillating field stimulation as an effective therapeutic approach in combination therapy for spinal cord injury.

BACKGROUND: Traumatic spinal cord injury (SCI) causes partial or total loss of sensory and motor functions. Despite enormous efforts, there is still no effective treatment which might improve patients' neurological status.The application of electric current to the injured spinal cord is known to promote healing and tissue regeneration. The use of this modality in treating the injured spinal cord to improve neurological recovery has been introduced as a potential treatment. NEW METHOD: Here we describe the method of epidural implantation of a miniature oscillating field (OF) stimulator designed in our laboratory immediately after Th9 spinal compression in Wistar rats. Three groups of animals were analyzed (intact; SCI only; OFS + SCI; n = 8 each). Histological, immunohistological and behavioral analysis were used to show the favorable effect of epidural OF stimulation on axonal regeneration and modulation of astrogliosis. RESULTS: Our study revealed considerable differences in white matter integrity in animals with an implanted OF stimulator. Moreover, we detected significantly increased numbers of neurofilaments and massive reduction in activated forms of astrocytes in the group of stimulated animals compared to the animals without stimulation. COMPARISON WITH EXISTING METHOD(S): Compared with previous research, our study revealed that epidural implantation of an OF stimulator immediately after spinal compression effectively reduced the expression of inflammatory response and suppressed activated astrocyte formation. CONCLUSIONS: Our finding confirms that implanting an OF stimulator is safe, stable and suitable for future combined therapy which could effectively promote and accelerate regeneration and functional restoration after spinal trauma.

Hypothermic treatment after computer-controlled compression in minipig: A preliminary report on the effect of epidural vs. direct spinal cord cooling.

The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (beginning 30 min post-injury persisting for 5 h) on tissue preservation along the rostro-caudal axis of the spinal cord (3 cm cranially and caudally from the lesion site), and the prevention of injury-induced functional loss in a newly developed computer-controlled compression model in minipig (force of impact 18N at L3 level), which mimics severe spinal cord injury (SCI). Minipigs underwent SCI with two post-injury modifications (durotomy vs. intact dura mater) followed by hypothermia through a perfusion chamber with cold (epidural t≈15°C) saline, DMEM/F12 or enriched DMEM/F12 (SCI/durotomy group) and with room temperature (t≈24°C) saline (SCI-only group). Minipigs treated with post-SCI durotomy demonstrated slower development of spontaneous neurological improvement at the early postinjury time points, although the outcome at 9 weeks of survival did not differ significantly between the two SCI groups. Hypothermia with saline (t≈15°C) applied after SCI-durotomy improved white matter integrity in the dorsal and lateral columns in almost all rostro-caudal segments, whereas treatment with medium/enriched medium affected white matter integrity only in the rostral segments. Furthermore, regeneration of neurofilaments in the spinal cord after SCI-durotomy and hypothermic treatments indicated an important role of local saline hypothermia in the functional outcome. Although saline hypothermia (24°C) in the SCI-only group exhibited a profound histological outcome (regarding the gray and white matter integrity and the number of motoneurons) and neurofilament protection in general, none of the tested treatments resulted in significant improvement of neurological status. The findings suggest that clinically-proven medical treatments for SCI combined with early 5 h-long saline hypothermia treatment without opening the dural sac could be more beneficial for tissue preservation and neurological outcome compared with hypothermia applied after durotomy.

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.

Neuroprotective effect of local hypothermia in a computer-controlled compression model in minipig: Correlation of tissue sparing along the rostro-caudal axis with neurological outcome.

This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of spinal cord injury (SCI) induced by a computer-controlled impactor device. The tissue integrity observed at the injury epicenter, and up to 3 cm cranially and caudally from the lesion site correlated with motor function. A computer-controlled device produced contusion lesions at L3 level with two different degrees of tissue sparing, depending upon pre-set impact parameters (8N- and 15N-force impact). Hypothermia with cold (4°C) saline or Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium was applied 30 min after SCI (for 5 h) via a perfusion chamber (flow 2 ml/min). After saline hypothermia, the 8N-SCI group achieved faster recovery of hind limb function and the ability to walk from one to three steps at nine weeks in comparison with non-treated animals. Such improvements were not observed in saline-treated animals subjected to more severe 15N-SCI or in the group treated with DMEM/F12 medium. It was demonstrated that the tissue preservation in the cranial and caudal segments immediately adjacent to the lesion, and neurofilament protection in the lateral columns may be essential for modulation of the key spinal microcircuits leading to a functional outcome. Tissue sparing observed only in the caudal sections, even though significant, was not sufficient for functional improvement in the 15N-SCI model.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.