Spinal Cord Transection In Rats Research Articles

Spinal cord transection significantly influences nNOS-IR in neuronal circuitry that underlies the tail-flick reflex activity.

Spinal cord transection interrupts supraspinal input and leads to the development of prominent spasticity. In this study, we investigated the effect of rat spinal cord transection performed at low thoracic level on changes in (i) neuronal nitric oxide synthase immunoreactivity (nNOS-IR), and (ii) the level of neuronal nitric oxide synthase (nNOS) protein in the neuronal circuitry that underlies tail-flick reflex. nNOS-IR was detected by immunohistochemistry and the level of nNOS protein was determined by the Western blot analysis. The tail-flick reflex was tested by a noxious thermal stimulus delivered to the tail of experimental animals. After surgery, experimental animals survived for 7 days. A significant increase in the level of nNOS protein was found 1 week after thoracic transection in the L2-L6 segments. Immunohistochemical analysis discovered that this increase may be a result of (1) a high nNOS-IR in a large number of axons, located predominantly in the dorsal columns (DCs) of lower lumbosacral segments, and (2) a slight increase of density in nNOS-IR in motoneurons. On the other hand the number of nNOS-IR neurons in the superficial dorsal horn and in area surrounded the central canal (CC) was greatly reduced. The tail-flick response was immediate in animals after spinal transection, while control rats responded to thermal stimulus with a slight delay. However, the tail-flick latency in experimental animals was significantly higher than in control. These data indicate that transection of the spinal cord significantly influences nNOS-IR in neuronal circuitry that underlies the tail-flick reflex activity.

Proteomic Profiling of the Insoluble Pellets of the Transected Rat Spinal Cord

This study investigated the proteomic changes at different time points in the precipitated pellets of rat spinal cords after applying complete spinal cord transection. By two-dimensional electrophoresis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, MALDI-TOF/TOF and peptide mass fingerprinting analysis, 44 proteins were identified, most of which are membrane and/or organellar proteins. They are mainly involved in metabolic processes (75%), developmental processes (30%), or responses to stimuli (30%), playing negative or positive roles. In particular, decreases of pyruvate dehydrogenase beta, aconitase 2, fumarate hydratase 1, and ATP synthase subunit 6 can lead to ATP depletion by crippling tricarboxylic acid cycle and oxidative phosphorylation. Decreases of several antioxidant proteins such as catalase, peroxiredoxin 1, Parkinson disease 7, and stress-induced phosphoprotein 1 can contribute to the secondary injury of spinal cord. Decreases of development-related 3-phosphoglycerate dehydrogenase and stathmin 1 may be not propitious for spinal cord regeneration. On the other hand, increases of isocitrate dehydrogenase 3 alpha/gamma and glutamate dehydrogenase 1 can help compensate the impaired energy metabolism. Increases of sirtuin 2, crystallin alpha B (CRYAB), and heat shock 27-kDa protein 1 can help resist stresses induced by injury. Increases of adenylate cyclase-associated protein 1 and galactose binding lectin 3 can help regeneration by replaying their roles in neural development. To our knowledge, this is the first case of characterization of the proteomic changes seen in the precipitated fraction of injured spinal cord. Most of the identified proteins were found for the first time to be differentially expressed after spinal cord injury, which may provide new clues about the molecular mechanisms of spinal cord injury and repair.

Effect of Antiepileptic Agent, Levetiracetam, on Urodynamic Parameters and Neurogenic Bladder Overactivity in Chronically Paraplegic Rats

To investigate the effects of different levetiracetam (LEV) doses on urodynamic parameters in an animal model of neurogenic detrusor overactivity (NDO). A total of 54 female rats were studied. Of the 54 rats, 6 served as normal controls, and 48 underwent T10 spinal cord transection (SCT). Of the latter 48 rats, 12 were paraplegic controls, and the remaining 36 rats were divided into 3 equal subgroups that received LEV by way of a subcutaneous osmotic minipump at a dose of 17, 54, and 108 mg/kg daily, respectively. The paraplegic control and treatment groups were further subdivided (n = 6), and cystometry was performed at 3 and 4 weeks after SCT, respectively. All paraplegic controls developed NDO, with spontaneous contractions. At 3 and 4 weeks after SCT, the mean frequency of the contractions was 1.6 +/- 0.3/min and 1.7 +/- 0.2/min. The contraction amplitude and bladder capacity were not significantly different. After 1 week of LEV treatment, these urodynamic parameters improved significantly in a dose-dependent manner, and the changes were more striking at 2 weeks. At a LEV dosage of 17, 54, and 108 mg/kg, respectively, the NDO frequency increased from 1.7 +/- 0.3 to 0.7 +/- 0.2 contractions/min (P = .01), 0.48 +/- 0.16 contractions/min (P = .009), and 0.5 +/- 0.17 contractions/min (P = .01). The bladder capacity increased from 0.51 +/- 0.1 mL to 1.5 +/- 0.2 mL (P = .0001), 2.5 +/- 1.7 mL (P = .006), and 2.6 +/- 0.3 mL (P = .0003), and the micturition pressure improved from 105.8 +/- 6.9 to 73.8 +/- 6.8 cm H(2)O (P = .01), 58.6 +/- 8.9 cm H(2)O (P = .006), and 49.7 +/- 8.9 cm H(2)O (P = .002). The results of our study have shown that LEV is an effective treatment of NDO after SCT in rats. It might prove to be a novel, alternative therapeutic approach to NDO. The follow-up of these experimental results with a clinical trial is warranted.

Reduction of cystic cavity, promotion of axonal regeneration and sparing, and functional recovery with transplanted bone marrow stromal cell–derived Schwann cells after contusion injury to the adult rat spinal cord

The authors previously reported that Schwann cells (SCs) could be derived from bone marrow stromal cells (BMSCs) in vitro and that they promoted axonal regeneration of completely transected rat spinal cords in vivo. The aim of the present study is to evaluate the efficacy of transplanted BMSC-derived SCs (BMSC-SCs) in a rat model of spinal cord contusion, which is relevant to clinical spinal cord injury. Bone marrow stromal cells were cultured as plastic-adherent cells from the bone marrow of GFPtransgenic rats. The BMSC-SCs were derived from BMSCs in vitro with sequential treatment using beta-mercaptoethanol, all-trans-retinoic acid, forskolin, basic fibroblast growth factor, platelet derived-growth factor, and heregulin. Schwann cells were cultured from the sciatic nerve of neonatal, GFP-transgenic rats. Immunocytochemical analysis and the reverse transcriptase-polymerase chain reaction were performed to characterize the BMSC-SCs. For transplantation, contusions with the New York University impactor were delivered at T-9 in 10- to 11-week-old male Wistar rats. Four groups of rats received injections at the injury site 7 days postinjury: the first received BMSCSCs and matrigel, a second received peripheral SCs and matrigel, a third group received BMSCs and matrigel, and a fourth group received matrigel alone. Histological and immunohistochemical studies, electron microscopy, and functional assessments were performed to evaluate the therapeutic effects of BMSC-SC transplantation. Immunohistochemical analysis and reverse transcriptase-polymerase chain reaction revealed that BMSC-SCs have characteristics similar to SCs not only in their morphological characteristics but also in their immunocytochemical phenotype and genotype. Histological examination revealed that the area of the cystic cavity was significantly reduced in the BMSC-SC and SC groups compared with the control rats. Immunohistochemical analysis showed that transplanted BMSCs, BMSC-SCs, and SCs all maintained their original phenotypes. The BMSC-SC and SC groups had a larger number of tyrosine hydroxilase-positive fibers than the control group, and the BMSC-SC group had more serotonin-positive fibers than the BMSC or control group. The BMSC-SC group showed significantly better hindlimb functional recovery than in the BMSC and control group. Electron microscopy revealed that transplanted BMSC-SCs existed in association with the host axons. Based on their findings, the authors concluded that BMSC-SC transplantation reduces the size of the cystic cavity, promotes axonal regeneration and sparing, results in hindlimb functional recovery, and can be a useful tool for spinal cord injury as a substitute for SCs.

Rigid Fixation of the Spinal Column Improves Scaffold Alignment and Prevents Scoliosis in the Transected Rat Spinal Cord

A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging. To evaluate the stability of implanted scaffolds and the extent of kyphoscoliotic deformities after spinal fixation. Biodegradable scaffolds provide an excellent platform for the quantitative assessment of cellular and molecular factors that promote regeneration within the transected cord. Successful delivery of scaffolds to the damaged cord can be hampered by malalignment following transplantation, which in turn, hinders the assessment of neural regeneration. Radio-opaque barium sulfate-impregnated poly-lactic-co-glycolic acid scaffolds were implanted into spinal transection injuries in adult rats. Spinal fixation was performed in one group of animals using a metal rod fixed to the spinous processes above and below the site of injury, while the control group received no fixation. Radiographic morphometry was performed after 2 and 4 weeks, and 3-dimensional magnetic resonance microscopy analysis 4 weeks after surgery. Over the course of 4 weeks, progressive scoliosis was evident in the unfixed group, where a Cobb angle of 8.13 +/- 2.03 degrees was measured. The fixed group demonstrated significantly less scoliosis, with a Cobb angle measurement of 1.89 +/- 0.75 degrees (P = 0.0004). Similarly, a trend for less kyphosis was evident in the fixed group (7.33 +/- 1.68 degrees ) compared with the unfixed group (10.13 +/- 1.46 degrees ). Quantitative measurements of the degree of malalignment of the scaffolds were also significantly less in the fixed group (5 +/- 1.23 degrees ) compared with the unfixed group (11 +/- 2.82 degrees ) (P = 0.0143). Radio-opaque barium sulfate allows for visualization of scaffolds in vivo using radiographic analysis. Spinal fixation was shown to prevent scoliosis, reduce kyphosis, and reduce scaffold malalignment within the transected rat spinal cord. Using a highly optimized model will increase the potential for finding a therapy for restoring function to the injured cord.

Transplantation of Human Umbilical Mesenchymal Stem Cells from Wharton's Jelly after Complete Transection of the Rat Spinal Cord

BackgroundHuman umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton's jelly of the umbilical cord can be easily obtained and processed compared with embryonic or bone marrow stem cells. These cells may be a valuable source in the repair of spinal cord injury.Methodology/Principal FindingsWe examine the effects of HUMSC transplantation after complete spinal cord transection in rats. Approximately 5×105 HUMSCs were transplanted into the lesion site. Three groups of rats were implanted with either untreated HUMSCs (referred to as the stem cell group), or HUMSCs treated with neuronal conditioned medium (NCM) for either three days or six days (referred to as NCM-3 and NCM-6 days, respectively). The control group received no HUMSCs in the transected spinal cord. Three weeks after transplantation, significant improvements in locomotion were observed in all the three groups receiving HUMSCs (stem cell, NCM-3 and NCM-6 days groups). This recovery was accompanied by increased numbers of regenerated axons in the corticospinal tract and neurofilament-positive fibers around the lesion site. There were fewer microglia and reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the stem cell group than in the control group. Transplanted HUMSCs survived for 16 weeks and produced large amounts of human neutrophil-activating protein-2, neurotrophin-3, basic fibroblast growth factor, glucocorticoid induced tumor necrosis factor receptor, and vascular endothelial growth factor receptor 3 in the host spinal cord, which may help spinal cord repair.Conclusions/SignificanceTransplantation of HUMSCs is beneficial to wound healing after spinal cord injury in rats.

Intrathecal delivery of resiniferatoxin (RTX) reduces detrusor overactivity and spinal expression of TRPV1 in spinal cord injured animals

Recently, it has been demonstrated that intrathecal delivery of resiniferatoxin (RTX) produces strong analgesia, even in models of bone cancer pain. RTX has been investigated to treat bladder dysfunction of spinal origin, applied by intravesical instillation. However, RTX delivered by this route was not completely satisfactory in controlling urinary incontinence and high intravesical pressure. Thus, the present study assessed the effects of intrathecal injections of RTX in bladder dysfunction in rats with spinal cord transection (SCT). Bladder function was evaluated in SCT rats 24 h following intrathecal administration of RTX. Detrusor overactivity and intravesical pressure were reduced in a dose-dependent manner. This was accompanied by a decrease in spinal cord TRPV1 and CGRP, but not in IB4 binding sensory fibres. Also, intrathecal RTX induced a dose-dependent reduction in spinal cord activation of the ERK pathway. Overall, our results show that intrathecal administration of RTX effectively reduces detrusor overactivity and reduces intravesical pressure in models of complete chronic spinal cord transection by suppressing the activity of TRPV1 expressing afferent fibres. Also, intrathecal RTX decreases sensory input, as shown by reduced spinal ERK activation. These findings might be relevant for the management of patients with spinal cord injuries.

Involvement of Acidic Fibroblast Growth Factor in Spinal Cord Injury Repair Processes Revealed by a Proteomics Approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

Modulation of bladder myofibroblast activity: implications for bladder function

Bladder suburothelial myofibroblasts may modulate both sensory responses from the bladder wall and spontaneous activity. This study aimed to characterize further these cells in their response to exogenous agents implicated in mediating the above activity. Detrusor strips, with or without mucosa, and isolated suburothelial myofibroblasts were prepared from guinea pig bladders. Isometric tension, intracellular Ca2+, and membrane current were recorded. Cell pairs were formed by pushing two cells together. Tension, intracellular Ca2+, and membrane potential were also recorded from bladder sheets using normal or spinal cord-transected (SCT) rats. Spontaneous contractions were greater in detrusor strips with an intact mucosa and were augmented by 10 μM UTP. ATP, UTP, or reduced extracellular pH elicited Ca2+ transients and inward currents (Erev −30 mV) in isolated cells. Capsaicin (5–30 μM) reduced membrane current (37 ± 12% of control) with minor effects on Ca2+ transients: sodium nitroprusside reduced membrane currents (40 ± 21% of control). Cell pair formation, without an increase in cell capacitance, augmented ATP and pH responses (180 ± 58% of control) and reduced the threshold to ATP and acidosis. Glivec (20–50 μM) reversibly blocked the augmentation and also reduced spontaneous activity in bladder sheets from SCT, but not normal, rats. Glivec also disrupted the spread of Ca2+ waves in SCT sheets, generating patterns similar to normal bladders. Suburothelial myofibroblasts respond to exogenous agents implicated in modulating bladder sensory responses; responses augmented by physical intercellular contact. The action of glivec and its selective suppression of spontaneous activity in SCT rats identifies a possible pathway to attenuate bladder overactivity.

A new model of autonomic dysreflexia induced by gastric distension in the spinal cord-transected rat

Upper gastrointestinal (GI) motility inhibition after spinal cord injury has been classically considered to result from autonomic dysreflexia (AD). Animal models have been designed in rats to evaluate the presence of AD induced by colonic or bladder distension. However, there are no animal models of AD induced by gastric distension (GD). We examined whether controlled GD could induce AD and compared the pattern of hemodynamic responses induced by GD with colonic distensions (CD) and the interaction between them. Male Wistar rats underwent spinal cord transections performed at the level of C7–T1, T4–T5 and T9–T10 (control) vertebrae and the presence of AD was evaluated after 1 day. In animals with C7–T1 lesions, each CD in a series of 4 consecutive CDs triggered AD while GD only triggered AD after the 2 initial distensions in a series of 4 consecutive GDs. In animals with T4–T5 lesions, in a protocol of 4 consecutive CDs or GDs, AD was triggered only by the 2 initial distensions. In 2 other protocols, consisting of 2 consecutive CDs or GDs followed respectively by 2 GDs or CDs, the effect of 2 GDs was attenuated in animals with C7–T1 and T4–T5 lesions but the hemodynamic changes induced by CDs were not affected by prior GDs. In summary, this is a new model of AD triggered by GD in rats. AD is more intense in animals with C7–T1 lesions than after T4–T5 lesions and AD triggered by GD can be attenuated by prior CDs.

Urotheliogenic modulation of intrinsic activity in spinal cord-transected rat bladders: role of mucosal muscarinic receptors

We examined the modulation of intrinsic (i.e., spontaneous) detrusor contractions by the urothelium and the lamina propria through optical mapping approaches. Normal adult and spinal cord-transected (SCT) rat bladders were stained with Ca2+- and voltage-sensitive dyes, and optical activity generated from intrinsic contractions was mapped from the mucosal surface of whole bladder sheets. Both normal adult and SCT rat bladders displayed intrinsic contractions, where normal bladders showed low-amplitude, high-frequency contractions with disorganized patterns of activity. In contrast, in the SCT animals there were high-amplitude, low-frequency contractions that displayed an organized spread of membrane potential and intracellular Ca2+. The difference in contractile activity was mirrored in the Ca2+ and membrane potential maps of bladder sheets. Normal bladders showed multiple initiation sites across the mucosal surface, whereas SCT bladders showed only one or two fixed initiation sites localized to the dome. The magnitude of intrinsic contractions could be enhanced by stretch or low-dose arecaidine (50 nM), a muscarinic-specific agonist. Partial removal of the mucosa decreased the amplitude of the intrinsic contractions and decreased the response to stretch or arecaidine. Optical mapping of mucosa-denuded sheets, where enhanced spontaneous activity was abolished, or application of 1 microM nifedipine to remove smooth muscle signals, but not the mucosal signals, shows that intrinsic activity in pathological bladders is driven by the mucosal layer. In summary, we suggest an urotheliogenic origin for intrinsic activity, where structures within the mucosal layer organize and thereby enhance intrinsic detrusor contractions.

Epidural Stimulation Induced Modulation of Spinal Locomotor Networks in Adult Spinal Rats

The importance of the in vivo dynamic nature of the circuitries within the spinal cord that generate locomotion is becoming increasingly evident. We examined the characteristics of hindlimb EMG activity evoked in response to epidural stimulation at the S1 spinal cord segment in complete midthoracic spinal cord-transected rats at different stages of postlesion recovery. A progressive and phase-dependent modulation of monosynaptic (middle) and long-latency (late) stimulation-evoked EMG responses was observed throughout the step cycle. During the first 3 weeks after injury, the amplitude of the middle response was potentiated during the EMG bursts, whereas after 4 weeks, both the middle and late responses were phase-dependently modulated. The middle- and late-response magnitudes were closely linked to the amplitude and duration of the EMG bursts during locomotion facilitated by epidural stimulation. The optimum stimulation frequency that maintained consistent activity of the long-latency responses ranged from 40 to 60 Hz, whereas the short-latency responses were consistent from 5 to 130 Hz. These data demonstrate that both middle and late evoked potentials within a motor pool are strictly gated during in vivo bipedal stepping as a function of the general excitability of the motor pool and, thus, as a function of the phase of the step cycle. These data demonstrate that spinal cord epidural stimulation can facilitate locomotion in a time-dependent manner after lesion. The long-latency responses to epidural stimulation are correlated with the recovery of weight-bearing bipedal locomotion and may reflect activation of interneuronal central pattern-generating circuits.

Suppression of detrusor-sphincter dysynergia by GABA-receptor activation in the lumbosacral spinal cord in spinal cord-injured rats

We investigated the effects of intrathecal application of GABAA- or GABAB-receptor agonists on detrusor-sphincter dyssynergia (DSD) in spinal cord transection (SCT) rats. Adult female Sprague-Dawley rats were used. At 4 wk after Th9-10 SCT, simultaneous recordings of intravesical pressure and urethral pressure were performed under an awake condition to examine the effect of intrathecal application of GABAA and GABAB agonists (muscimol and baclofen, respectively) or GABAA and GABAB antagonists (bicuculline and saclofen, respectively) at the level of L6-S1 spinal cord. In spinal-intact rats, the effects of bicuculline and saclofen on bladder and urethral activity were also examined. During urethral pressure measurements, DSD characterized by urethral pressure increases during isovolumetric bladder contractions were observed in 95% of SCT rats. However, after intrathecal application of muscimol or baclofen, urethral pressure showed urethral relaxation during isovolumetric bladder contractions. The effective dose to induce inhibition of urethral activity was lower compared with the dose that inhibited bladder contractions. The effect of muscimol and baclofen was antagonized by intrathecal bicuculline and saclofen, respectively. In spinal-intact rats, intrathecal application of bicuculline induced DSD-like changes. These results indicate that GABAA- and GABAB-receptor activation in the spinal cord exerts the inhibitory effects on DSD after SCT. Decreased activation of GABAA receptors due to hypofunction of GABAergic mechanisms in the spinal cord might be responsible, at least in part, for the development of DSD after SCT.

Slow‐ and fast‐twitch rat hind limb skeletal muscle phenotypes 8 months after spinal cord transection and olfactory ensheathing glia transplantation

Paralysed skeletal muscle of rats with spinal cord injury (SCI) undergoes atrophy and a switch in gene expression pattern which leads to faster, more fatigable phenotypes. Olfactory ensheathing glia (OEG) transplants have been reported to promote axonal regeneration and to restore sensory-motor function in animals with SCI. We hypothesized that OEG transplants could attenuate skeletal muscle phenotypic deterioration and that this effect could underlie the functional recovery observed in behavioural tests. A variety of morphological, metabolic and molecular markers were assessed in soleus (SOL) and extensor digitorum longus (EDL) muscles of spinal cord transected (SCT), OEG-transplanted rats 8 months after the intervention and compared with non-transplanted SCT rats and sham-operated (without SCT) controls (C). A multivariate analysis encompassing all the parameters indicated that OEG-transplanted rats displayed skeletal muscle phenotypes intermediate between non-transplanted and sham-operated controls, but different from both. A high correlation was observed between behaviourally tested sensory-motor functional capacity and expression level of slow- and fast-twitch hind limb skeletal muscle phenotypic markers, particularly the histochemical glycerol-3-phosphate dehydrogenase activity (-0.843, P < 0.0001) and the fraction of variant 2s of the slow regulatory myosin light chain isoform (0.848, P < 0.0001) in SOL. Despite the mean overall effect of OEG transplants in patterning skeletal muscle protein expression towards normal, in 6 out of 9 animals they appeared insufficient to overcome fibre type switching and to support a consistent and generalized long-term maintenance of normal skeletal muscle characteristics. The interplay of OEG and exercise-mediated neurotrophic actions is a plausible mechanism underlying OEG transplantation effects on paralysed skeletal muscle.

Effects of intrathecal administration of pituitary adenylate cyclase activating polypeptide on lower urinary tract functions in rats with intact or transected spinal cords

Effects of intrathecally administered pituitary adenylate cyclase activating polypeptide-38 (PACAP-38, 0.1–30 μg) on lower urinary tract function were examined in unanesthetized, decerebrate rats with an intact spinal cord and after chronic spinal cord transection (SCT). PACAP-38 was also studied in rats with intact or bilaterally transected hypogastric nerves (HGNs), to determine if sympathetic pathways to the bladder influenced responses. In SCT rats with intact HGNs under isovolumetric conditions, 30 μg of PACAP-38 but not lower doses (0.1–10 μg) increased (mean 194%) bladder contraction amplitude (BCA). In SCT rats with sectioned HGNs, 10 μg and 30 μg of PACAP-38 increased BCA by 62% and 195%, respectively. On the other hand, during continuous infusion cystometrograms (CMGs) in SCT rats with intact or sectioned HGNs, PACAP-38 (10 μg and 30 μg) markedly reduced or completely suppressed BCA (60% and 90%, respectively) and reduced external urethral sphincter (EUS) EMG activity (58% and 91%, respectively). During CMGs in spinal cord intact rats, with intact HGNs PACAP-38 30 μg increased BCA (26%) but after HGN section PACAP-38 10 μg and 30 μg increased BCA by 21% and 35%. These results suggest that after SCT, PACAP-38 activates spinal circuitry to facilitate the parasympathetic outflow to the urinary bladder and that the elimination of sympathetic pathways enhances this effect. The decrease in BCA by PACAP-38 during CMGs in SCT rats is most reasonably attributed to a reduction in urethral outlet resistance due to suppression of excitatory EUS reflexes.

Repair of spinal cord transection and its effects on muscle mass and myosin heavy chain isoform phenotype

A number of significant advances have been developed for treating spinal cord injury during the past two decades. The combination of peripheral nerve grafts and acidic fibroblast growth factor (hereafter referred to as PNG) has been shown to partially restore hindlimb function. However, very little is known about the effects of such treatments in restoring normal muscle phenotype. The primary goal of the current study was to test the hypothesis that PNG would completely or partially restore 1) muscle mass and muscle fiber cross-sectional area and 2) the slow myosin heavy chain phenotype of the soleus muscle. To test this hypothesis, we assigned female Sprague-Dawley rats to three groups: 1) sham control, 2) spinal cord transection (Tx), and 3) spinal cord transection plus PNG (Tx+PNG). Six months following spinal cord transection, the open-field test was performed to assess locomotor function, and then the soleus muscles were harvested and analyzed. SDS-PAGE for single muscle fiber was used to evaluate the myosin heavy chain (MHC) isoform expression pattern following the injury and treatment. Immunohistochemistry was used to identify serotonin (5-HT) fibers in the spinal cord. Compared with the Tx group, the Tx+PNG group showed 1) significantly improved Basso, Beattie, and Bresnahan scores (hindlimb locomotion test), 2) less muscle atrophy, 3) a higher percentage of slow type I fibers, and 4) 5-HT fibers distal to the lesion site. We conclude that the combined treatment of PNG is partially effective in restoring the muscle mass and slow phenotype of the soleus muscle in a T-8 spinal cord-transected rat model.

Changes in unmyelinated axons in the sciatic nerve following complete spinal cord transection in rats

Changes in unmyelinated axons in the sciatic nerve following complete spinal cord transection in rats

Therapeutic Strategy for Acute Spinal Cord Contusion Injury: Cell Elimination Combined with Microsurgical Intervention

BackgroundNo cure is available for human spinal cord injury. Cell elimination by localized radiation therapy that is timed within 2–3 weeks postinjury can facilitate repair of structure and function in transected rat spinal cord. In pilot studies in contusion spinal cord injury, a model similar to crush/fracture injury in human, we did not observe the expected beneficial effects of radiation therapy. Long forgotten data show that in contusion/crush injury, fluid accumulation from hemorrhage is critical. Alfred Reginald Allen observed that the most devastating sequelae in contusive injury are secondary to fluid accumulation which could be alleviated by surgical intervention, midline slits (myelotomy) at the lesion site.Methods and FindingsHere, we tested whether release of fluid buildup by microsurgery (partial myelotomy) would affect the structural outcome of radiation therapy in the severely contused rat spinal cord. Surgical intervention alone significantly enhanced tissue and functional preservation in the contused cord, thus confirming Allen's observations. Combining partial myelotomy with radiation therapy that is specifically timed postinjury elicited substantial beneficial therapeutic outcome; it led to significant increase in tissue repair/preservation compared with the group that received surgical intervention only, as determined by histology and in vivo MRI. Altogether, the combined treatments led to a 1.8 fold increase in tissue repair/preservation as compared with the contused group.ConclusionsThe data suggest that a clinical protocol could be developed to treat acute human spinal cord injury through conventional clinical procedures, a combination of microsurgical manipulation and radiation therapy. These also suggest it is imperative to first prevent the secondary damage caused by fluid accumulation for a cure to be possible.

Role of gap junctions in spontaneous activity of the rat bladder

Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity, leading to incontinence. Immunohistochemistry was used to compare connexin (Cx) 26, 43, and 45 expression in the bladders of neonatal, adult, and spinal cord-transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed, and the spinal cord was transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 mo old) and neonates (7-21 day old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca(2+)-sensitive dyes. The expression of Cx26 was localized to the urothelium, Cx43 to the lamina propria myofibroblasts, and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites, resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18beta-glycyrrhetinic acid, 1 microM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.

VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cord

Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro-Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2-immunoreactive axons and synapses. CTB-immunoreactive SPN and PPN received many close appositions from VGLUT2-immunoreactive axons. VGLUT2-immunoreactive synapses occurred on Fluoro-Gold-labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1-immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1-immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1-immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarke's column. These data indicate that SPN and PPN are densely innervated by VGLUT2-immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1-immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN.