Search

Training of impaired motor functions forms activity-dependent plasticity aimed to compensate the functional deficiency. In individuals with chronic motor-complete spinal cord injury (SCI), the existence of potential for neuroplasticity and the possibility to mobilize it are debatable. The study aimed at assessing the existence, frequency, and magnitude of positive changes in locomotor ability and sensory and motor characteristics of patients with motor-complete SCI during repeated courses of complex neurorehabilitation. For analysis we selected 37 patients (16 men, 19 women, aged 18–55) with thoracic or lumbar cord injuries with a post-injury period of more than 1 year (1–32 years) and the severity of AIS A (20) and B (15) according to the American Spinal Injury Association (ASIA) impairment scale. All participants completed 3 inpatient courses of complex rehabilitation with exoskeleton-induced walk training (two 22-day courses and one 14‑day course, with one-month intervals). Each course included exoskeleton-induced walk training (40 min/day), training of vertical posture with electrical stimulation of muscles, pneumatic stimulation of the supporting areas of the sole, classical manual and lymphatic drainage massage, and exercise therapy (3–4 h a day in total). The third course additionally included transcutaneous spinal cord electrical stimulation (SCES) performed in static mode and in phasic mode during exoskeleton-induced walking. The dynamics of tactile (AISLT) and pain (AISPP) sensitivity, as well as leg muscle strength (AISMOTOR) were evaluated before and after each course. Locomotor capabilities of participants were tested by forward and backward tetrapedal tests with control of the time spent and need for external assistance. The excitability of motor neurons of the lumbar enlargement was also evaluated. Over the observation period, an increase of AISLT was detected in 28 patients (76%) on average for 9.4 AIS points and of AISPP in 22 patients (60%) for 8.0 AIS points, and of AISMOTOR – in 15 patients (40.5%) for 3.9 AIS points. Neurological improvements (AIS) could begin during the first, second, or third courses, positive changes occurred during the course and between courses with comparable frequency and were cumulative. In some cases, a post-interval return to the initial level was observed but no cases of significant deterioration. Changes in sensitivity prevailed during the first and second courses, motor progress was observed in the third course that included SCES. Progress in locomotor capabilities, sensitivity, and muscle control, as well as changes in the excitability of lumbar enlargement motor neurons in patients with motor-complete SCI during intensive exoskeleton-induced walk training are estimated as a result of sensorimotor adjustments revealing of activity-dependent plasticity. The obtained data substantiate the possibility to mobilize the potential for neuroplasticity in adults with a chronic motor-complete SCI: powered exoskeleton-induced walk training with intense afferent stimulation shows high impact on locomotor and sensory functions, whereas the SCES improves motor ability.

BACKGROUND: It has been reported that neuropathic pain can be overcome by targeting the NR2B subunit of N-methyl-D-aspartate receptors (NR2B). This study aimed to investigate the effects of minocycline on phosphorylated and total expression of NR2B in the spinal cord of rats with diabetic neuropathic pain. METHODS: A total of 32 Sprague–Dawley male rats were randomly assigned into four groups (n = 8); control healthy, control diabetic (PDN), and PDN rats that received 80 µg or 160 µg intrathecal minocycline respectively. The rats were induced to develop diabetes and allowed to develop into the early phase of PDN for two weeks. Hot-plate and formalin tests were conducted. Intrathecal treatment of minocycline or normal saline was conducted for 7 days. The rats were sacrificed to obtain the lumbar enlargement region of the spinal cord (L4-L5) for immunohistochemistry and western blot analyses to determine the expression of phosphorylated (pNR2B) and total NR2B (NR2B). RESULTS: PDN rats showed enhanced flinching (phase 1: p

The overall goal of this work was to create a high-resolution MRI atlas of the lumbosacral enlargement of the spinal cord of the rat (Sprague–Dawley), cat, domestic pig, rhesus monkey, and human. These species were chosen because they are commonly used in basic and translational research in spinal cord injuries and diseases. Six spinal cord specimens from each of the studied species (total of 30 specimens) were fixed, extracted, and imaged. Sizes of the spinal cord segments, cross-sectional dimensions, and locations of the spinal cord gray and white matter were quantified and compared across species. The lumbar enlargement spans spinal cord levels L3-S1 in rats, L4-S1 in cats, L3-S1 in pigs, L2/L3-L7/S1 in monkeys, and T12/L1-S1/S2 in humans. The enlargements in pigs and humans are largest and most similar in size (length and cross-sectional area); followed by monkeys and cats; and followed by rats. The obtained atlas establishes a neuroanatomical reference for the intact lumbosacral spinal cord in these species. It can also be used to guide the planning of surgical procedures of the spinal cord and technology design and development of spinal cord neuroprostheses, as well as precise delivery of cells/drugs into target regions within the spinal cord parenchyma.

The spinal ganglia, which perform the function of the first link on the afferent impulses’ way from the receptors to the central nervous system, recognize internal and external irritations, and are the first to transform them into a nervous impulse. As the representatives of the peripheral nervous system, they are some of the main objects of the studies in contemporary neuromorphology. Based on the results of anatomic, neurohistological, histochemical, morphometric and statistical methods of the studies, we conducted a complex survey, revealing the morphology of spinal ganglia of different segmental levels in the domestic dog. In particular, we determined the differences in the microscopic structure and morphometric parameters of cervical, thoracic, lumbar and sacral spinal ganglia and the ganglia of the cervical and lumbar enlargements in mature domestic dogs. The study showed that the spinal ganglia of domestic dogs can have different skeletotopy, different shape and sizes due to their species peculiarity. Also, the surveyed animals, according to the results of our studies, had the cervical and thoracic spinal ganglia of oval, while the lumbar and sacral – spindle-like shapes. According to the results of morphometry, the area of the spinal ganglia in lengthwise section differed: the smallest area belonged to the thoracic, the largest to the sacral spinal ganglia. The density of neuronal arrangement per 0.1 mm2 of the area of the spinal ganglia correlated with their sizes: the highest parameter was identified for the thoracic spinal ganglia, the lowest – for the sacral. The conducted studies revealed that histo- and cyto-structure of the spinal ganglia is characteristic of notable differentiation of the nervous cells of small sizes. Therefore, we differentiated neurons of the spinal ganglia into large, medium and small. The highest quantity of large neurons was found in the sacral ganglia, and largest amount of medium-sized neurons – in the ganglia of the lumbar enlargement. In other ganglia, small neurons dominated. Correspondingly, different nuclear-cytoplasmic ratio in these neurons was determined, indicating different extent of morphofunctional condition of nervous cells. We determined content of localization and separation of nucleic acids in histostructure of the spinal cord at the tissue and cellular levels.

There are no published normal values for spinal cord and canal diameters in newborns. Spinal cord and spinal canal diameters are assessed subjectively by radiologists without any objective values for the upper limit of normal. To determine normal values for anteroposterior (AP) diameters of the spinal cord and spinal canal on sonography in healthy term newborns. We performed ultrasound of the entire spine on 37 healthy newborns (23 male, 14 female). The AP diameters of the spinal canal and spinal cord were measured at representative levels of the cervical (C4, C5, C6), thoracic (T5, T6, T7, T8) and lumbar spine (lumbar enlargement and above and below the lumbar enlargement level). Statistical analysis was performed to determine the mean and standard deviation of the spinal canal and spinal cord AP diameter at each aforementioned vertebral level, and their correlations with birth weight, length and head circumference. The mean AP spinal cord diameter was 4.1±0.5 mm at the cervical level, 3.3±0.3 mm at the thoracic level and 4.4±0.6 mm at the lumbar level. The mean AP spinal canal diameter was 7.7±0.7 mm at the cervical level, 6.2±0.8 mm at the thoracic level, and 8.4±0.7 mm at the lumbar level. In this prospective study, we have determined normal values for AP diameters of the spinal cord and spinal canal on sonography in healthy newborns at representative cervical, thoracic and lumbar levels. This data may assist in evaluating the neonatal spine in clinical situations such as suspected spinal cord injury.

Purpose: To study the analgesic effect of flurbiprofen ester in rats with incision pain, and its effect on serum inflammatory factors and β-endorphin expression. Methods: Seventy-five (75) healthy rats with foot contraction threshold induced by basic mechanical stimulation were randomly assigned to control, model control and treatment groups. Flurbiprofen was administered in 3 doses: 5, 10 and 15 mg/kg. Then, 3 mL of ventricular blood was taken from anesthetized rats and the serum levels of tumor necrosis factor-α (TNF- α), interleukin-1 β, interleukin-6 and β-endorphin were measured. The expression of β-endorphin in the spinal cord of rats with lumbar enlargement and ARC was determined. Results: The TNF- α, interleukin-1 β and interleukin-6 concentrations were significantly lower in treatment group than in model rats, and decreased with time and dose (p < 0.05). In the treatment group, the level of serum β-endorphin decreased with increase in dose at 1 h, but increased with increase in dose at 5 h and 10 h (p < 0.05). The levels of β-endorphin in the spinal cord, was significantly lower in model rats than in control rats (p < 0.05). Conclusion: Pre-administration of flurbiprofen ester reduces serum inflammatory factors and upregulates β-endorphin expression in rats with incision pain. Thus, it flurbiprofen exerts analgesic effect. Keywords: Flurbiprofen ester, Incision pain, Rat, Analgesia, Inflammatory factor, β-endorphin

We explored the relationship between population interneuronal network activation and motor output in the adult, in vivo, air-stepping, spinal cat. By simultaneously measuring the activity of large numbers of spinal interneurons, we explored ensembles of coherently firing interneurons and their relation to motor output. In addition, the networks were analyzed in relation to their spatial distribution along the lumbar enlargement for evidence of localized groups driving particular phases of the locomotor step cycle. We simultaneously recorded hindlimb EMG activity during stepping and extracellular signals from 128 channels across two polytrodes inserted within lamina V–VII of two separate lumbar segments. Results indicated that spinal interneurons participate in one of two ensembles that are highly correlated with the flexor or the extensor muscle bursts during stepping. Interestingly, less than half of the isolated single units were significantly unimodally tuned during the step cycle whereas >97% of the single units of the ensembles were significantly correlated with muscle activity. These results show the importance of population scale analysis in neural studies of behavior as there is a much greater correlation between muscle activity and ensemble firing than between muscle activity and individual neurons. Finally, we show that there is no correlation between interneurons’ rostrocaudal locations within the lumbar enlargement and their preferred phase of firing or ensemble participation. These findings indicate that spinal interneurons of lamina V–VII encoding for different phases of the locomotor cycle are spread throughout the lumbar enlargement in the adult spinal cord. NEW & NOTEWORTHY We report on the ensemble organization of interneuronal activity in the spinal cord during locomotor movements and show that lumbar intermediate zone interneurons organize in two groups related to the two major phases of walking: stance and swing. Ensemble organization is also shown to better correlate with muscular output than single-cell activity, although ensemble membership does not appear to be somatotopically organized within the spinal cord.

ObjectivesTraumatic and non-traumatic spinal cord injury produce neurodegeneration across the entire neuraxis. However, the spatiotemporal dynamics of spinal cord grey and white matter neurodegeneration above and below the injury is understudied.MethodsWe acquired longitudinal data from 13 traumatic and 3 non-traumatic spinal cord injury patients (8–8 cervical and thoracic cord injuries) within 1.5 years after injury and 10 healthy controls over the same period. The protocol encompassed structural and diffusion-weighted MRI rostral (C2/C3) and caudal (lumbar enlargement) to the injury level to track tissue-specific neurodegeneration. Regression models assessed group differences in the temporal evolution of tissue-specific changes and associations with clinical outcomes.ResultsAt 2 months post-injury, white matter area was decreased by 8.5% and grey matter by 15.9% in the lumbar enlargement, while at C2/C3 only white matter was decreased (−9.7%). Patients had decreased cervical fractional anisotropy (FA: −11.3%) and increased radial diffusivity (+20.5%) in the dorsal column, while FA was lower in the lateral (−10.3%) and ventral columns (−9.7%) of the lumbar enlargement. White matter decreased by 0.34% and 0.35% per month at C2/C3 and lumbar enlargement, respectively, and grey matter decreased at C2/C3 by 0.70% per month.ConclusionsThis study describes the spatiotemporal dynamics of tissue-specific spinal cord neurodegeneration above and below a spinal cord injury. While above the injury, grey matter atrophy lagged initially behind white matter neurodegeneration, in the lumbar enlargement these processes progressed in parallel. Tracking trajectories of tissue-specific neurodegeneration provides valuable assessment tools for monitoring recovery and treatment effects.

Implantable spinal-cord-neuroprostheses aiming to restore standing and walking after paralysis have been extensively studied in animal models (mainly cats) and have shown promising outcomes. This study aimed to take a critical step along the clinical translation path of these neuroprostheses, and investigated the organization of the neural networks targeted by these implants in a non-human primate. This was accomplished by advancing a microelectrode into various locations of the lumbar enlargement of the spinal cord, targeting the ventral horn of the gray matter. Microstimulation in these locations produced a variety of functional movements in the hindlimb. The resulting functional map of the spinal cord in monkeys was found to have a similar overall organization along the length of the spinal cord to that in cats. This suggests that the human spinal cord may also be organized similarly. The obtained spinal cord maps in monkeys provide important knowledge that will guide the very first testing of these implants in humans.

In spinal cord injury (SCI), timely therapeutic intervention is critical to inhibit the post-injury rapidly progressing degeneration of spinal cord. Towards that objective, we determined the accessibility of intravenously administered biodegradable nanoparticles (NPs) as a drug delivery system to the lesion site in rat and pig contusion models of SCI. Poly ( d , l -lactide co-glycolide, PLGA)-based NPs loaded with a near-infrared dye as a marker for NPs were used. To analyze and quantify localization of NPs to the lesion site, we mapped the entire spinal cord, segment-by-segment, for the signal count. Our objectives were to determine the NP dose effect and duration of retention of NPs at the lesion site, and the time window post-SCI within which NPs localize at the lesion site. We hypothesized that breakdown of the blood-spinal cord barrier following contusion injury could lead to more specific localization of NPs at the lesion site. The mapping data showed a dose-dependent increase and significantly greater localization of NPs at the lesion site than in the remaining uninjured segment of the spinal cord. Further, NPs were seen to be retained at the lesion site for more than a week. With delayed post-SCI administration, localization of NPs at the lesion site was reduced but still localize even at four weeks post-injury administration. Interestingly, in uninjured animals (sham control), greater accumulation of NPs was seen in the thoracic and lumbar enlargement regions of the spinal cord, which in animals with SCI changed to the lesion site, indicating drastic post-injury hemodynamic changes in the spinal cord. Similar to the rat results, pig contusion model of SCI showed greater NP localization at the lesion site. In conclusion, NPs could potentially be explored as a carrier for delivery of therapeutics to the lesion site to minimize the impact of post-SCI response.

ObjectiveTo determine tissue-specific neurodegeneration across the spinal cord in patients with mild-moderate degenerative cervical myelopathy (DCM).MethodsTwenty-four mild-moderate DCM and 24 healthy subjects were recruited. In patients, a T2-weighted scan was acquired at the compression site, while in all participants a T2*-weighted and diffusion-weighted scan was acquired at the cervical level (C2-C3) and in the lumbar enlargement (i.e. rostral and caudal to the site of compression). We quantified intramedullary signal changes, maximal canal and cord compression, white (WM) and grey matter (GM) atrophy, and microstructural indices from diffusion-weighted scans. All patients underwent clinical (modified Japanese Orthopaedic Association (mJOA)) and electrophysiological assessments. Regression analysis assessed associations between MRI readouts and electrophysiological and clinical outcomes.ResultsTwenty patients were classified with mild and four with moderate DCM using the mJOA scale. The most frequent site of compression was at C5-C6 level with maximum cord compression of 4.68±0.83 mm. Ten patients showed imaging evidence of cervical myelopathy. In the cervical cord, WM and GM atrophy and WM microstructural changes were evident, while in the lumbar cord only WM showed atrophy and microstructural changes. Remote cervical cord WM microstructural changes were pronounced in patients with radiological myelopathy and associated with impaired electrophysiology. Lumbar cord WM atrophy was associated with lower limb sensory impairments.ConclusionTissue-specific neurodegeneration revealed by quantitative MRI, already apparent across the spinal cord in mild-moderate DCM prior to the onset of severe clinical impairments. WM microstructural changes are particularly sensitive to remote pathologically and clinically eloquent changes in DCM.

Coupling between cervical and lumbar spinal networks (cervico-lumbar coupling) is vital during human locomotion. Impaired cervico-lumbar coupling after neural injuries or diseases can be reengaged via simultaneous arm and leg cycling training. Sensorimotor circuitry including cervico-lumbar coupling may further be enhanced by non-invasive modulation of spinal circuity using transcutaneous spinal cord stimulation (tSCS). This project aimed to determine the effect of cervical, lumbar, or combined tSCS on spinal reflex (Hoffmann [H-]) and corticospinal (motor evoked potential [MEP]) excitability during a static or cycling cervico-lumbar coupling task. Fourteen neurologically intact study participants were seated in a recumbent leg cycling system. H-reflex and MEP amplitudes were assessed in the left flexor carpi radialis (FCR) muscle during two tasks (Static and Cycling) and four conditions: (1) No tSCS, (2) tSCS applied to the cervical enlargement (Cervical); (3) tSCS applied to the lumbar enlargement (Lumbar); (4) simultaneous cervical and lumbar tSCS (Combined). While cervical tSCS did not alter FCR H-reflex amplitude relative to No tSCS, lumbar tSCS significantly facilitated H-reflex amplitude by 11.1%, and combined cervical and lumbar tSCS significantly enhanced the facilitation to 19.6%. Neither cervical nor lumbar tSCS altered MEP amplitude alone (+4.9 and 1.8% relative to legs static, No tSCS); however, combined tSCS significantly increased MEP amplitude by 19.7% compared to No tSCS. Leg cycling alone significantly suppressed the FCR H-reflex relative to static, No tSCS by 13.6%, while facilitating MEP amplitude by 18.6%. When combined with leg cycling, tSCS was unable to alter excitability for any condition. This indicates that in neurologically intact individuals where interlimb coordination and corticospinal tract are intact, the effect of leg cycling on cervico-lumbar coupling and corticospinal drive was not impacted significantly with the tSCS intensity used. This study demonstrates, for the first time, that tonic activation of spinal cord networks through multiple sites of tSCS provides a facilitation of both spinal reflex and corticospinal pathways. It remains vital to determine if combined tSCS can influence interlimb coupling after neural injury or disease when cervico-lumbar connectivity is impaired.

Cutamesine, a sigma-1 receptor agonist, functions in both neuroprotection and neurite outgrowth. We assessed the therapeutic effects of cutamesine in a rodent spinal cord injury (SCI) model to demonstrate pre-clinical proof-of-concept. First of all, in order to determine optimal cutamesine dose, cutamesine was administered to normal rats and BDNF protein levels in the lumbar spinal cord were assessed by Western blot. Next, for the SCI model, spinal cords of adult female Sprague-Dawley rats were contused using an Infinite Horizon Impactor. Two weeks post-injury, rats were randomly assigned to receive daily subcutaneous injections of either cutamesine (3.0 mg/kg/day) or saline (as a control) for another two weeks. Immunohistochemistry for BDNF and 5-HT was assessed at four and twelve weeks post-injury in the lumbar spinal cord. Locomotor function was assessed weekly using the BBB locomotor scale until twelve weeks after SCI and CatWalk XT 10.5 gait analysis was conducted at twelve weeks after SCI. In normal rats, cutamesine treatment (3.0 mg/kg/day) significantly up-regulated BDNF expression in the lumbar spinal cord. In SCI rats, cutamesine treatment (3.0 mg/kg/day) significantly increased the fluorescence intensity of neuronal BDNF and serotonin boutons in the injured spinal cord compared to saline. However, cutamesine treatment did not promote significant locomotor recovery. Recent work indicates that cutamesine treatment alone did not promote locomotor recovery in spite of immunohistological changes. Future work will explore the influence of combining cutamesine with other treatment promoting plasticity (e.g. rehabilitative training) in SCI rats.

After spinal cord injury (SCI), the majority of individuals develop spasticity, a debilitating condition involving involuntary movements, co-contraction of antagonistic muscles, and hyperreflexia. By acting on GABAergic and Ca2+-dependent signaling, current anti-spastic medications lead to serious side effects, including a drastic decrease in motoneuronal excitability which impairs motor function and rehabilitation efforts. Exercise, in contrast, decreases spastic symptoms without decreasing motoneuron excitability. These functional improvements coincide with an increase in expression of the chloride co-transporter KCC2 in lumbar motoneurons. Thus, we hypothesized that spastic symptoms can be alleviated directly through restoration of chloride homeostasis and endogenous inhibition by increasing KCC2 activity. Here, we used the recently developed KCC2 enhancer, CLP257, to evaluate the effects of acutely increasing KCC2 extrusion capability on spastic symptoms after chronic SCI. Sprague Dawley rats received a spinal cord transection at T12 and were either bike-trained or remained sedentary for 5 weeks. Increasing KCC2 activity in the lumbar enlargement improved the rate-dependent depression of the H-reflex and reduced both phasic and tonic EMG responses to muscle stretch in sedentary animals after chronic SCI. Furthermore, the improvements due to this pharmacological treatment mirror those of exercise. Together, our results suggest that pharmacologically increasing KCC2 activity is a promising approach to decrease spastic symptoms in individuals with SCI. By acting to directly to restore endogenous inhibition, this strategy has potential to avoid severe side effects and improve the quality of life of affected individuals.Significance StatementSpasticity is a condition that develops after spinal cord injury (SCI) and causes major complications for individuals. We have previously reported that exercise attenuates spastic symptoms after SCI through an increase in expression of the chloride co-transporter KCC2, suggesting that restoring chloride homeostasis contributes to alleviating spasticity. However, the early implementation of rehabilitation programs in the clinic is often problematic due to co-morbidities. Here, we demonstrate that pharmacologically enhancing KCC2 activity after chronic SCI reduces multiple signs of spasticity, without the need for rehabilitation.

Progress in regenerative medicine is realizing the possibility of neural regeneration and functional recovery in spinal cord injury (SCI). Recently, rehabilitation has attracted much attention with respect to the synergistic promotion of functional recovery in combination with neural stem/progenitor cell (NS/PC) transplantation, even in the chronic refractory phase of SCI. Nevertheless, sensory disturbance is one of the most prominent sequelae, even though the effects of combination or single therapies have been investigated mostly in the context of motor recovery. To determine how combination therapy with treadmill training (TMT) and NS/PC transplantation affects the manifestation of thermal allodynia and tactile hyperalgesia in chronic phase SCI, four groups of SCI mice were used to assess pain-related behavior and histological changes: combined transplantation and TMT therapy, transplantation only, TMT only, and control groups. Thermal allodynia and coarse touch-pressure hyperalgesia exhibited significant recovery in the combined therapy group in comparison with controls, whereas there were no significant differences with fine touch-pressure hyperalgesia and motor function. Further investigation revealed fewer fibers remaining in the posterior funiculus, which contained the tracts associated with the two modalities showing less recovery; that is, touch-pressure hyperalgesia and motor function. A significant correlation was only observed between these two modalities. Although no remarkable histological recovery was found within the lesion epicenter, changes indicating amelioration of pain were observed in the lumbar enlargement of the combination therapy group. Our results suggest that amelioration of thermal allodynia and tactile hyperalgesia can be brought about by the additive effect of NS/PC transplantation and TMT. The degree of recovery seems dependent on the distribution of damage.

Functional magnetic resonance imaging (fMRI) is based on neurovascular coupling, which allows inferring neuronal activity from hemodynamic changes. Spinal fMRI has been used to examine pain processes, although spinal neurovascular coupling has never been investigated. In addition, fluctuations in mean arterial pressure (MAP) occur during nociceptive stimulation and this may affect neurovascular coupling. The objective of this study was to examine neurovascular coupling in the rat spinal cord during nociceptive stimulation while MAP was manipulated by cervical spinal transection, which prevents nociception-related MAP increases. Six male Wistar rats were anesthetized with isoflurane (1.2-1.5%). Local field potentials (LFP) and spinal cord blood flow (SCBF) were recorded concurrently in the lumbar enlargement, where activity was evoked by electrical stimulation of the sciatic nerve. In intact conditions, stimulation of graded intensity produced proportional changes in SCBF and LFP that were paralleled by similar changes in MAP. However, spinal transection almost abolished MAP changes (p0.001), while SCBF and LFP responses were not significantly affected (p0.3) and remained similarly coupled before and after spinal transection. This indicates that spinal hemodynamic changes reflect neuronal activity even when large fluctuations in MAP occur. This contrasts with results from previous studies on cerebral neurovascular coupling and suggests that spinal autoregulation might allow better adaptation to sudden MAP changes than cerebral autoregulation. Although assessment of the coupling between spinal neuronal activity and BOLD signal remains to be investigated, this study supports the use of spinal fMRI, based on the tight coupling between SCBF and LFP.

Spinal cord injury (SCI) causes loss of normal sensation and often leads to debilitating neuropathic pain (NeP). Chronic NeP develops at or below the SCI lesion in as many as 80% of patients with SCI and may be induced by modulators of neuronal excitability released from activated microglia and macrophages. In the inflammatory response after SCI, different microglia/macrophage populations that are classically activated (M1 phenotype) or alternatively activated (M2 phenotype) have become of great interest. Chemokines have also recently attracted attention in neuron-microglia communication. CCL21 is a chemokine that activates microglia in the central nervous system (CNS) and is expressed only in neurons with an insult or mechanical injury. In this study using an SCI model in mutant (plt) mice with deficient CCL21 expression, we assessed post-SCI NeP and expression of microglia/macrophages and inflammatory cytokines at the injured site and lumbar enlargement. SCI-induced hypersensitivities to mechanical and thermal stimulation were relieved in plt mice compared with those in wild-type (C57BL/6) mice, although there was no difference in motor function. Immunohistochemistry and flow cytometry analysis showed that the phenotype of microglia/macrophages was M1 type-dominant in both types of mice at the lesion site and lumbar enlargement. A decrease of M1-type microglia/macrophages was seen in plt mice compared with wild-type, while the number of M2-type microglia/macrophages did not differ between these mice. In immunoblot analysis, expression of M1-induced cytokines [tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)] was decreased in plt mice, while that of M2-induced cytokines interleukin-4 (IL-4, IL-10) did not differ in the two types of mice. The results of this study indicate that suppression of expression of inflammatory cytokines by decreasing the number of M1-type microglia/macrophages at the injured site and lumbar enlargement is associated with provision of an environment for reduction of NeP. These findings may be useful for the design of new therapies to alleviate NeP after SCI.

Disturbance of glutamate homeostasis is a well-characterized mechanism of neuropathic pain. Vesicular glutamate transporters (VGLUTs) determine glutamate accumulation in synaptic vesicles and their roles in neuropathic pain have been suggested by gene-knockout studies. Here, we investigated the spatio-temporal changes in VGLUT expression during the development of neuropathic pain in wild-type rats. Spared nerve injury (SNI) induced mechanical allodynia from postoperative day 1 to at least day 14. Expression of VGLUT1 and VGLUT2 in dorsal root ganglia and spinal cord was examined by western blot analyses on different postoperative days. We observed that VGLUT2 were selectively upregulated in crude vesicle fractions from the ipsilateral lumbar enlargement on postoperative days 7 and 14, while VGLUT1 was transiently downregulated in ipsilateral DRG (day 4) and contralateral lumbar enlargement (day 1). Upregulation of VGLUT2 was not accompanied by alterations in vesicular expression of synaptotagmin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thus, VGLUTs expression, especially VGLUT2, is regulated following peripheral nerve injury. Temporal regulation of VGLUT2 expression in spinal cord may represent a novel presynaptic mechanism contributing to injury-induced glutamate imbalance and associated neuropathic pain.

Chronic intractable neuropathic pain after central or peripheral nervous system injury remains refractory to therapeutic intervention. Using microarray and RT-qPCR methods, we found that Noggin mRNA is downregulated in the lumbar enlargement 2 weeks after chronic constriction injury (CCI) in rats. Eight-week-old female Sprague Dawley rats were used for the CCI model. Two weeks after CCI, rats underwent a laminectomy at L5 under halothane anesthesia, and a silicone tube connected to an osmotic minipump was inserted intrathecally for 14 days. Rats were administered Noggin ranging from 10 ng/ml to 10 μg/ml. Phosphate buffered saline (PBS) was used as a control. The time course of mechanical allodynia was assessed for 5 weeks using von Frey filaments. An ANOVA showed that rats administered Noggin at 2 μg/ml had significantly less mechanical allodynia compared with controls. We next compared the effect of intrathecal administration (14 days) of Noggin (2 μg/ml), bone morphogenetic protein 4 (BMP4; 2 μg/ml), or BMP4 (μg/ml) + Noggin (μg/ml) with controls. Only Noggin administration significantly reduced mechanical allodynia in the CCI model. Fluorescence immunohistochemistry indicated that Noggin administration decreased astrocyte accumulation in the dorsal horn compared with PBS after administration for one week. BMP4-driven conversion of oligodendrocyte progenitor cells (OPCs) to type 2 astrocytes is inhibited by Noggin Hampton et al. (2007) . We speculated that Noggin administration inhibits the conversion of OPCs to astrocytes, and decreases glial fibrillar acidic protein expression. This histological condition could decrease neuropathic pain., Highlights • Noggin mRNA is significantly down-regulated two weeks after CCI in rats. • The mechanical allodynia was decreased in Noggin administration at seven days. • Noggin administration influenced GFAP expression and reduced mechanical allodynia.

Spinal cord injury (SCI) promotes inflammation along the neuroaxis that jeopardizes plasticity, intrinsic repair and recovery. While inflammation at the injury site is well-established, less is known within remote spinal networks. The presence of bone marrow-derived immune (myeloid) cells in these areas may further impede functional recovery. Previously, high levels of the gelatinase, matrix metalloproteinase-9 (MMP-9) occurred within the lumbar enlargement after thoracic SCI and impeded activity-dependent recovery. Since SCI-induced MMP-9 potentially increases vascular permeability, myeloid cell infiltration may drive inflammatory toxicity in locomotor networks. Therefore, we examined neurovascular reactivity and myeloid cell infiltration in the lumbar cord after thoracic SCI. We show evidence of region-specific recruitment of myeloid cells into the lumbar but not cervical region. Myeloid infiltration occurred with concomitant increases in chemoattractants (CCL2) and cell adhesion molecules (ICAM-1) around lumbar vasculature 24 h and 7 days post injury. Bone marrow GFP chimeric mice established robust infiltration of bone marrow-derived myeloid cells into the lumbar gray matter 24 h after SCI. This cell infiltration occurred when the blood-spinal cord barrier was intact, suggesting active recruitment across the endothelium. Myeloid cells persisted as ramified macrophages at 7 days post injury in parallel with increased inhibitory GAD67 labeling. Importantly, macrophage infiltration required MMP-9.

Multisegmental muscle responses (MMR) are reflexes in the leg muscles evoked by transcutaneous electrical spinal cord stimulation over the Th11–Th12 vertebrae. We have used MMR to evaluate the excitability of lumbosacral motor neurons in individuals having paraplegia of low limbs. Ten individuals were tested using H-reflexes and MMR bilaterally before (n 0 = 20) and during 4-weeks course of rehabilitation (n=76). The H-reflex and MMR of m. gastrocnemius lateralis were obtained in: 15 and 13 cases out of 20, respectively. Both reflexes were recorded in 11 and were absent in 3 cases, matched up to 70% of recordings. In dynamic, the both methods were 100% reproducible and the responses’ amplitude varied in similar directions in 67% of records. The data confirm the validity and reproducibility of the MMR for evaluation of the motor neurons excitability in lumbosacral cord. The H-reflex magnitude shows moderate correlation with MMR in m. gastrocnemius lateralis (r = 0.59, p < 0.001), and weak correlation with MMR in mm. rectus femoris, biceps femoris, and tibialis anterior (r < 0.40, p < 0.001). These findings do not allow extrapolate the results from the H-reflex measurement on the state of lumbosacral cord on the whole. At the same time, measurements of the MMR allow estimate simultaneously the excitability of motor pools innervating several muscle groups. This gives the possibility to assess the functional state of the motor neurons in the lumbosacral cord for clinical and experimental studies, including the spinal cord damage.

Spinal cord injury (SCI) causes loss of locomotor function and chronic neuropathic pain (NeP). Hematogenous macrophages and activated microglia are key monocytic lineage cell types in the response to SCI, and each has M1- and M2-phenotypes. To understand the roles of these cells in neuronal regeneration and chronic NeP after SCI, differences in distribution and phenotypes of activated microglia and infiltrated macrophages after SCI were examined at the injured site and the lumbar enlargement, as a remote region. Chimeric mice were used for differentiating activated microglia from hematogenous macrophages. The prevalences of activated microglia and infiltrating macrophages increased at day 14 after SCI, at the time of most severe pain hypersensitivity, with mainly M1-type hematogenous macrophages at the injured site and M2-type activated microglia at the lumbar enlargement. Peak expression of TNF-α, an M1-induced cytokine, occurred on day 4 post-SCI at the injured site, but not until day 14 at the lumbar enlargement. Expression of IL-4, a M2-induced cytokine, peaked at 4 days after SCI at both sites. These results suggest different roles of activated microglia and hematogenous macrophages, including both phenotypes of each cell, in neuronal regeneration and chronic NeP after SCI at the injured site and lumbar enlargement. The prevalence of the M1 over the M2 phenotype at the injured site until the subacute phase after SCI may be partially responsible for the lack of functional recovery and chronic NeP after SCI. Activation of M2-type microglia at the lumbar enlargement in response to inflammatory cytokines from the injured site might be important in chronic below-level pain. These findings are useful for establishment of a therapeutic target for prevention of motor deterioration and NeP in the time-dependent response to SCI.

The complications of diabetic polyneuropathy (DN) determines its level of severity. It may occur with distinctive clinical symptoms (painful DN) or appears undetected (painless DN). This study aimed to investigate microglia activation and signalling molecules brain-derived neurotrophic factor (BDNF) and downstream regulatory element antagonist modulator (DREAM) proteins in spinal cord of streptozotocin-induced diabetic neuropathy rats. Thirty male Sprague-Dawley rats (200-230 g) were randomly assigned into three groups: (1) control, (2) painful DN and (3) painless DN. The rats were induced with diabetes by single intraperitoneal injection of streptozotocin (60 mg/kg) whilst control rats received citrate buffer as a vehicle. Four weeks post-diabetic induction, the rats were induced with chronic inflammatory pain by intraplantar injection of 5% formalin and pain behaviour responses were recorded and assessed. Three days later, the rats were sacrificed and lumbar enlargement region of spinal cord was collected. The tissue was immunoreacted against OX-42 (microglia), BDNF and DREAM proteins, which was also quantified by western blotting. The results demonstrated that painful DN rats exhibited increased pain behaviour score peripherally and centrally with marked increase of spinal activated microglia, BDNF and DREAM proteins expressions compared to control group. In contrast, painless DN group demonstrated a significant reduction of pain behaviour score peripherally and centrally with significant reduction of spinal activated microglia, BDNF and DREAM proteins expressions. In conclusions, the spinal microglia activation, BDNF and DREAM proteins correlate with the pain behaviour responses between the variants of DN.

In the course of investigating how common clinical treatments and adaptive technologies affect recovery after spinal cord injury (SCI), we discovered that a clinically-modeled hindlimb stretching protocol dramatically, but transiently, reduces locomotor function. Nociceptive sensory input is capable of altering motor output at the spinal level, and nociceptive neurons are sensitized after SCI. Here we tested the hypotheses that stretch-induced locomotor deficits are dependent on nociceptive afferents by depleting TRPV1+ sensory afferents using capsaicin injections in neonatal rats. Following maturation, animals received 25g-cm contusive SCI at T10. After plateau of locomotor recovery at 6 weeks, daily stretching was performed for 3 weeks, followed by 2 weeks without stretch, and again for two additional weeks. Animals were sacrificed 2 h after the last stretching session for histological assessments. Consistent with previous findings, stretch-induced drops in locomotor function were observed in nociceptor-intact animals but were nearly absent in nociceptor-depleted animals. These functional changes were accompanied by corresponding increases in the number of c-Fos+ nuclei throughout the lumbar enlargement. As expected, nociceptor-depleted animals had very little CGRP+ axonal innervation of the dorsal horn. Nociceptor-intact stretched animals had significantly higher levels of CGRP+ as compared to non-stretched SCI rats, suggesting that stretching promoted intraspinal CGRP+ sprouting. These results indicate that stretch-induced locomotor dysfunction in animals with incomplete SCI involves C-fibers, adding a negative post-SCI role to their adaptive roles (e.g., bladder control), and suggesting that the clinical use of muscle stretching to combat contractures and spasticity may be unintentionally detrimental to locomotor function.

Nowadays there are various models of spinal cord injury (SCI) that recreate mechanisms of human SCI. The ex vivo modeling of injury is a robust approach, confronts with less experimental and ethical challenges. Currently almost all ex vivo models are obtained either from embryonic or postnatal animals, which can hardly mimic features of human SCI. This study was designed to develop SCI in slice culture of adult rats. Here, the lumbar enlargement of an adult rat was sliced and cultured. After seven days in vitro, a weight was dropped to simulate the injury. The result showed that although the rate of cell death in first days of in vitro was high, it reduced after 7 days and dropping a weight at the time caused significant rate of cell death in slices. It was shown that injury can disturb histological features and neuronal integrity in the slices. Treating the injured slices with valproic acid resulted in a significant decrease of TNF-α and increase of BDNF expression. Collected data revealed obtained slices from adult rat were able to adjust to the culture environment after 7 days and dropping a weight at the time point could simulate the injury. Besides mimicking the disturbing features of human SCI, this model can response to VPA pharmacological treatment.

Background: We compared functional, electrophysiological and morphological parameters after SCI in two groups of rats Sprague Dawley (SD) rats with normal vision and blind rats from a SD-substrain Royal College of Surgeons (SD/RCS) who lose their photoreceptor cells after birth due to a genetic defect in the retinal pigment epithelium. For these animals skin-, intramuscular-, and tendon receptors are major available means to resolve spatial information. Objective: The purpose of this study was to check whether increased sensitivity in SD/RCS rats would promote an improved recovery after SCI. Methods: All rats were subjected to severe compression of the spinal cord at vertebra Th8, spinal cord segment Th10. Recovery of locomotion was analyzed at 1, 3, 6, 9, and 12 weeks after SCI using video recordings of beam walking and inclined ladder climbing. Five functional parameters were studied: foot-stepping angle (FSA), rump-height index (RHI) estimating paw placement and body weight support, respectively, number of correct ladder steps (CLS) assessing skilled hindlimb movements, the BBB-locomotor score and an established urinary bladder score (BS). Sensitivity tests were followed by electrophysiological measurement of M- and H-wave amplitudes from contractions of the plantar musculature after stimulation of the tibial nerve. The closing morphological measurements included lesion volume and expression of astro- and microglia below the lesion. Results: Numerical assessments of BBB, FSA, BS, lesion volume and GFAP-expression revealed no significant differences between both strains. However, compared to SD-rats, the blind SD/RCS animals significantly improved RHI and CLS by 6 - 12 weeks after SCI. To our surprise the withdrawal latencies in the blind SD/RCS rats were longer and the amplitudes of M- and H-waves lower. The expression of IBA1-immunoreactivity in the lumbar enlargement was lower than in the SD-animals. Conclusion: The longer withdrawal latencies suggest a decreased sensitivity in the blind SD/RCS rats, which promotes better recovery after SCI. In this way our results provide indirect support to earlier work showing, that hypersensitivity and chronic pain after contusive SCI impair the recovery of locomotor function.

Spinal cord injury (SCI) can cause neuropathic pain (NeP), often reducing a patient's quality of life. We recently reported that granulocyte colony-stimulating factor (G-CSF) could attenuate NeP in several SCI patients. However, the mechanism of action underlying G-CSF-mediated attenuation of SCI-NeP remains to be elucidated. The purpose of the present study was to elucidate the therapeutic effect and mechanism of action of granulocyte colony-stimulating factor for SCI-induced NeP. T9 level contusive SCI was introduced to adult male Sprague Dawley rats. Three weeks after injury, rats received intraperitoneal recombinant human G-CSF (15.0 μg/kg) for 5 days. Mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry and western blot analysis were performed in spinal cord lumbar enlargement samples. Testing with von Frey filaments showed significant increase in the paw withdrawal threshold in the G-CSF group compared with the vehicle group 4 weeks, 5 weeks, 6 weeks and 7 weeks after injury. Immunohistochemistry for CD11b (clone OX-42) revealed that the number of OX-42-positive activated microglia was significantly smaller in the G-CSF group than that in the vehicle rats. Western blot analysis indicated that phosphorylated-p38 mitogen-activated protein kinase (p38MAPK) and interleukin-1β expression in spinal cord lumbar enlargement were attenuated in the G-CSF-treated rats compared with that in the vehicle-treated rats. The present results demonstrate a therapeutic effect of G-CSF treatment for SCI-induced NeP, possibly through the inhibition of microglial activation and the suppression of p38MAPK phosphorylation and the upregulation of interleukin-1β.

The superficial dorsal horn (laminae I and II) of the spinal cord contains numerous excitatory and inhibitory interneurons, and recent studies have shown that each of these groups can be divided into several neurochemically distinct populations. Although it has long been known that some neurons in this region have intersegmental (propriospinal) axonal projections, there have been conflicting reports concerning the number of propriospinal cells and the extent of their axons. In addition, little is known about the neurochemical phenotype of propriospinal neurons or about the termination pattern of their axons. In the present study we show, using retrograde tracing, that around a third of lamina I–II neurons in the lumbar enlargement project at least five segments cranially. Substance P-expressing excitatory neurons are over-represented among these cells, accounting for one-third of the propriospinal neurons. In contrast, inhibitory interneurons and excitatory PKCγ neurons are both under-represented among the retrogradely labelled cells. By combining viral vector-mediated Cre-dependent anterograde tracing with immunocytochemistry, we provide evidence that the lateral spinal nucleus (LSN), rather than the superficial dorsal horn, is the main target for axons belonging to propriospinal substance P-expressing neurons. These findings help to resolve the discrepancies between earlier studies and have implications for the role of the LSN in pain mechanisms.

BackgroundSpinal aneurysms are rare among spinal arteriovenous malformations (SAVMs). There are few reports of endovascular management of spinal aneurysms associated with SAVM.ObjectiveTo present endovascular management of aneurysms associated with SAVM.MethodsOf 91 patients with SAVMs,eight (9%) presented with aneurysms. Of these, three were male and five were female with a median age of 18 years (range 11–38). We evaluated the presenting pattern, lesion level, type of the target aneurysm related to the presenting pattern and AVM nidus, and the result obtained after embolization or open surgery. Clinical status was evaluated by Aminoff–Logue (ALS) gait and micturition scale scores.ResultsThe presenting patterns were subarachnoid hemorrhage (SAH, n=3) or mass effect caused by extrinsic (n=4) or intrinsic (n=1) cord compression. Aneurysms were located in four cervical, two thoracic, and two lumbar enlargement areas. There were two prenidal (arterial), three nidal, and three postnidal (venous) aneurysms. The mean diameter of the aneurysms was 9 mm (range 3–27). Glue embolization (n=6), open surgery (n=1), and combined surgery and embolization (n=1) was performed to obliterate the aneurysms. Obliteration of the target aneurysms resulted in improvement of symptoms and clinical stabilization of SAVMs in all patients during a mean of 55 months (range 7–228) of follow-up.ConclusionsIdentification of a symptomatic aneurysm should be associated with clinical presentation pattern. Targeted obliteration of the aneurysm by embolization and/or surgery resulted in improvement of symptoms and stabilization of SAVM.

Objective The overall goal of this study was to investigate the effects of various anesthetic protocols on the intraoperative responses to intraspinal microstimulation (ISMS). ISMS is a neuroprosthetic approach that targets the motor networks in the ventral horns of the spinal cord to restore function after spinal cord injury. In preclinical studies, ISMS in the lumbosacral enlargement produced standing and walking by activating networks controlling the hindlimb muscles. ISMS implants are placed surgically under anesthesia, and refinements in placement are made based on the evoked responses. Anesthesia can have a significant effect on the responses evoked by spinal neuroprostheses; therefore, in preparation for clinical testing of ISMS, we compared the evoked responses under a common clinical neurosurgical anesthetic protocol with those evoked under protocols commonly used in preclinical studies. Approach Experiments were conducted in seven pigs. An ISMS microelectrode array was implanted in the lumbar enlargement and responses to ISMS were measured under three anesthetic protocols: (1) isoflurane, an agent used pre-clinically and clinically, (2) total intravenous anesthesia (TIVA) with propofol as the main agent commonly used in clinical neurosurgical procedures, (3) TIVA with sodium pentobarbital, an anesthetic agent used mostly preclinically. Responses to ISMS were evaluated based on stimulation thresholds, movement kinematics, and joint torques. Motor evoked potentials (MEP) and plasma concentrations of propofol were also measured. Main results ISMS under propofol anesthesia produced large and functional responses that were not statistically different from those produced under pentobarbital anesthesia. Isoflurane, however, significantly suppressed the ISMS-evoked responses. Significance This study demonstrated that the choice of anesthesia is critical for intraoperative assessments of motor responses evoked by spinal neuroprostheses. Propofol and pentobarbital anesthesia did not overly suppress the effects of ISMS; therefore, propofol is expected to be a suitable anesthetic agent for clinical intraoperative testing of an intraspinal neuroprosthetic system.

(Sub)chronic local drug application is clearly superior to systemic administration, but may be associated with substantial obstacles, particularly regarding the applications to highly sensitive central nervous system (CNS) structures that are shielded from the outer environment by the blood-brain barrier. Violation of the integrity of the barrier and CNS tissues by a permanently implanted probe or cannula meant for prolonged administration of drugs into specific CNS structures can be a severe confounding factor because of the resulting inflammatory reactions. In this study, we tested the utility of a novel way for (sub)chronic local delivery of highly active (i.e., used in very low amounts) drugs to the rat spinal cord employing a non-woven nanofiber mat dressing. To this end, we compared the morphology and motoneuron (a + g) counts in spinal cord cervical and lumbar segments between rats with glutamate-loaded nanofiber mats applied to the lumbar enlargement and rats with analogical implants carrying no glutamate. Half of the rats with glutamate-loaded implants were given daily valproate treatment to test its potential for counteracting the detrimental effects of glutamate excess. The mats were prepared in-house by electrospinning of an emulsion made of a solution of the biocompatible and biodegradable poly(L-lactide-co-caprolactone) polymer in a mixture of organic solvents, an aqueous phase with or without monosodium glutamate, and sodium dodecyl sulfate as an emulsifier; the final glutamate content was 1.4 µg/mg of the mat. Three weeks after mat implantation there was no inflammation or considerable damage of the spinal cord motoneuron population in the rats with the subarachnoid dressing of a glutamate-free mat, whereas the spinal cords of the rats with glutamate-loaded nanofiber mats showed clear symptoms of excitotoxic damage and a substantial increase in dying/damaged motoneuron numbers in both segments studied. The rats given systemic valproate treatment showed significantly lower percentages of damaged/dying motoneurons in their lumbar enlargements. These results demonstrate the capacity of nanofiber mats for generation of neurotoxic glutamate in the rat CNS. However, the tested nanofiber mats need further improvements aimed at extending the period of effective drug release and rendering the release more steady.

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1–VGLUT3 transcripts in lumbar 4–5 (L4–5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4–5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III–IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III–IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury. Fil: Malet, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vieytes, C. A.. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina Fil: Lundgren, K. H.. University of Cincinnati; Estados Unidos Fil: Seal, R. P.. University of Pittsburgh; Estados Unidos Fil: Tomasella, María Eugenia. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Seroogy, K. B.. University of Cincinnati; Estados Unidos Fil: Hökfelt, T.. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia Fil: Gebhart, G. F.. University of Pittsburgh; Estados Unidos Fil: Brumovsky, Pablo Rodolfo. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados Unidos

TRESK gene recombinant adenovirus (10 IU/ml), which has been constructed successfully in our previous study, was implemented through an intrathecal injection. The fact that the method can effectively upregulate the expression of TRESK mRNA in the dorsal root ganglia of spared nerve injury in rats was verified. We also investigated the role of TRESK gene recombinant adenovirus in attenuating tactile allodynia and thermal hyperalgesia in spared nerve injury rats. Spared nerve injury to the sciatic nerve induced persistent tactile allodynia, but had no effect on thermal hyperalgesia. Intrathecal injection of TRESK gene recombinant adenovirus (25 µl) into the region of lumbar enlargement in advance reduced tactile allodynia. Moreover, intrathecal injection of TRESK gene recombinant adenovirus (25 µl) significantly alleviated the activation of astrocytes in spinal cord induced by spared nerve injury. The current study shows that an intrathecal injection of the TRESK gene recombinant adenovirus attenuated the activity of astrocytes in spinal cord, which contributed to relieving neuropathic pain in spared nerve injury rats. According to the result reported in our previous study, attenuating the expression of TRESK in dorsal root ganglia was involved in the development of neuropathic pain. On the basis of these results, we theorized that the therapeutic utility of upregulation of TRESK in dorsal root ganglia was effective in relieving neuropathic pain syndromes induced by peripheral nerve injury.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

OBJECTIVE Spinal cord injury causes a drastic loss of motor, sensory and autonomic function. The goal of this project was to investigate the use of intraspinal microstimulation (ISMS) for producing long distances of walking over ground. ISMS is an electrical stimulation method developed for restoring motor function by activating spinal networks below the level of an injury. It produces movements of the legs by stimulating the ventral horn of the lumbar enlargement using fine penetrating electrodes (≤50 μm diameter). APPROACH In each of five adult cats (4.2-5.5 kg), ISMS was applied through 16 electrodes implanted with tips targeting lamina IX in the ventral horn bilaterally. A desktop system implemented a physiologically-based control strategy that delivered different stimulation patterns through groups of electrodes to evoke walking movements with appropriate limb kinematics and forces corresponding to swing and stance. Each cat walked over an instrumented 2.9 m walkway and limb kinematics and forces were recorded. MAIN RESULTS Both propulsive and supportive forces were required for over-ground walking. Cumulative walking distances ranging from 609 to 835 m (longest tested) were achieved in three animals. In these three cats, the mean peak supportive force was 3.5 ± 0.6 N corresponding to full-weight-support of the hind legs, while the angular range of the hip, knee, and ankle joints were 23.1 ± 2.0°, 29.1 ± 0.2°, and 60.3 ± 5.2°, respectively. To further demonstrate the viability of ISMS for future clinical use, a prototype implantable module was successfully implemented in a subset of trials and produced comparable walking performance. SIGNIFICANCE By activating inherent locomotor networks within the lumbosacral spinal cord, ISMS was capable of producing bilaterally coordinated and functional over-ground walking with current amplitudes

Twenty-six cattle and 7 horses were diagnosed with rabies. Samples of brain and spinal cord were processed for hematoxylin and eosin staining and immunohistochemistry (IHC). In addition, refrigerated fragments of brain and spinal cord were tested by direct fluorescent antibody test and intracerebral inoculation in mice. Statistical analyses and Fisher exact test were performed by commercial software. Histologic lesions were observed in the spinal cord in all of the cattle and horses. Inflammatory lesions in horses were moderate at the thoracic, lumbar, and sacral levels, and marked at the lumbar enlargement level. Gitter cells were present in large numbers in the lumbar enlargement region. IHC staining intensity ranged from moderate to strong. Inflammatory lesions in cattle were moderate in all spinal cord sections, and gitter cells were present in small numbers. IHC staining intensity was strong in all spinal cord sections. Only 2 horses exhibited lesions in the brain, which were located mainly in the obex and cerebellum; different from that observed in cattle, which had lesions in 25 cases. Fisher exact test showed that the odds of detecting lesions caused by rabies in horses are 3.5 times higher when spinal cord sections are analyzed, as compared to analysis of brain samples alone.

Cerebral infarction can cause secondary damage to nonischemic brain regions. However, whether this phenomenon will appear in central nervous system regions outside the brain remains unclear. Here we investigated pathological changes in the spinal cord and ventral root after ischemic stroke. All rats exhibited apparent neurological deficits post-MCAO, which improved gradually but could still be detected 12-weeks. Neuronal filaments in the corticospinal tract (CST) and neurons in the ventral horn were significantly declined in the contralateral cervical and lumbar enlargement 1-week post-MCAO. These decreases remained stable until 12-weeks, accompanied by progressively increased glial activation in the ventral horn. Axonal degeneration and structural derangement were evident in the contralateral cervical and lumbar ventral root 1-week post-MCAO; these changes spontaneously attenuated over time, but abnormalities could still be observed 12-weeks. The number of neural fibers in the contralateral CST and neurons in the contralateral ventral horn were positively correlated with neurological scores 12-weeks post-MCAO. Additionally, GFAP+cell density in the contralateral CST and ventral horn was negatively correlated with neurological scores. Our results suggest that cerebral infarction can elicit secondary degeneration in the cervical and lumbar spinal cord, as well as the projecting ventral root, which may hamper functional recovery after stroke.

This study evaluated the role of spared axons on structural and behavioral neuroplasticity in the lumbar enlargement after a thoracic spinal cord injury (SCI). Previous work has demonstrated that recovery in the presence of spared axons after an incomplete lesion increases behavioral output after a subsequent complete spinal cord transection (TX). This suggests that spared axons direct adaptive changes in below-level neuronal networks of the lumbar cord. In response to spared fibers, we postulate that lumbar neuron networks support behavioral gains by preventing aberrant plasticity. As such, the present study measured histological and functional changes in the isolated lumbar cord after complete TX or incomplete contusion (SCI). To measure functional plasticity in the lumbar cord, we used an established instrumental learning paradigm. In this paradigm, neural circuits within isolated lumbar segments demonstrate learning by an increase in flexion duration that reduces exposure to a noxious leg shock. We employed this model using a proof-of-principle design to evaluate the role of sparing on lumbar learning and plasticity early (7 days) or late (42 days) after midthoracic SCI in a rodent model. Early after SCI or TX at 7d, spinal learning was unattainable regardless of whether the animal recovered with or without axonal substrate. Failed learning occurred alongside measures of cell soma atrophy and aberrant dendritic spine expression within interneuron populations responsible for sensorimotor integration and learning. Alternatively, exposure of the lumbar cord to a small amount of spared axons for 6 weeks produced near-normal learning late after SCI. This coincided with greater cell soma volume and fewer aberrant dendritic spines on interneurons. Thus, an opportunity to influence activity-based learning in locomotor networks depends on spared axons limiting maladaptive plasticity. Together, this work identifies a time dependent interaction between spared axonal systems and adaptive plasticity in locomotor networks and highlights a critical window for activity-based rehabilitation. □

Phosphate-based glass fibers (PGFs), due to characteristics such as biodegradability and directionality, could be effective as spatial cues for axonal outgrowth following nerve injury. In the present study, PGF-containing cylindrical scaffolds of 1.8 mm diameter and 3 mm length were developed and implanted into the gap between the proximal and distal stumps following complete transection of rat spinal cords at T9. The PGF-free collagen scaffolds were implanted into the transected spinal cords of the control group. The open-field Basso, Beattie and Bresnahan locomotor scale revealed that the locomotor function of the experimental group was better than in the control group from 8 to 12 weeks after implantation, and urodynamic analysis revealed additional improvements in the experimental group in some parameters. Twelve weeks after implantation, some axon growth from the proximal and distal stumps to the scaffold was observed in the experimental group but not in the control group. Macrophages surrounded the injured thoracic spinal cord at 1 and 4 weeks after implantation; however, 6 h after implantation, the pro-inflammatory cytokines did not differ between the control and experimental groups. Anterograde corticospinal tract (CST) tracing with biotinylated dextran amine showed that, in the experimental group, some CST outgrowths could reach the lumbar enlargement. By 12 weeks, the mRNA levels of brain-derived neurotrophic factor in the bladder had increased more in the experimental group than in the controls. We conclude that PGFs can have a beneficial effect on functional recovery following complete transection of the thoracic spinal cord in rats.

We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V–VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles. J. Comp. Neurol. 519:3470–3487, 2011. © 2011 Wiley-Liss, Inc.

Exercise provides a number of important benefits after spinal cord injury in clinical studies and animal models. However, the amount of functional improvement in overground locomotion obtained with exercise alone has been limited thus far, for reasons that are still poorly understood. One hypothesis is that the complex network of endogenous extracellular matrix components, including chondroitin sulfate proteoglycans (CSPGs), can inhibit exercise-induced remodeling and limit plasticity of spared circuitry in the adult central nervous system. Recent animal studies have shown that chondroitinase ABC (ChABC) can enhance plasticity in the adult nervous system by cleaving glycosaminoglycan sidechains from CSPGs. In this article we review the current literature on plasticity observed with locomotor training and following degradation of CSPGs with ChABC and then present a rationale for the use of exercise combined with ChABC to promote functional recovery after spinal cord injury. We also present results of a preliminary study that tested the simplest approach for combining these treatments; use of a single intraparenchymal injection of ChABC administered to the lumbar enlargement of mice with voluntary wheel running exercise after a mid-thoracic spinal contusion injury. The results are negative, yet serve to highlight limitations in our understanding of the most effective protocols for combining these approaches. Further work is directed to identify the timing, type, and quantity of exercise and pharmacological interventions that can be used to maximize functional improvements by strengthening appropriate synaptic connections.

The motor effects induced by pulsed magnetic fields (PMF) projected onto the lumbar and cervical spinal cord were studied in decerebrate cats. A magnetic coil (inductor) of diameter 8 cm was positioned 1–2 cm above the surface of the spinal cord. Stimulation of the spinal cord with PMF was performed in two regimes: with single impulses with an intensity of 0.5–1 T and with continuous rhythmic stimulation at a frequency of 1 Hz and an intensity of 0.5 T. Application of single stimuli to the lumbar enlargement evoked reflex responses in the proximal and distal hindlimb muscles. Rhythmic stimulation initiated locomotor activity of the limb on a running treadmill, i.e., activated the neural locomotor network of the spinal cord (stepping movement generator). Magnetic stimulation of the lumbar enlargement evoked coordinated stepping movements of the hindlimbs only. Application of PMF to the cervical enlargement induced coordinated stepping movements of all four limbs, hindlimb movements starting before forelimb movements. After cessation of magnetic stimulation, the limbs completed several further coordinated movement cycles. This is the first report of the triggering of limb stepping movement generators with PMF in decerebrate cats. The results obtained here demonstrate that the neural locomotor networks of the spinal cord can be activated noninvasively and open new perspectives for the clinical use of PMF.