Thoracic 9 Spinal Transection-Induced Model of Muscle Spasticity in the Rat: A Systematic Electrophysiological and Histopathological Characterization.

The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABAB receptor agonist), tizanidine (α2-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity.

Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival.

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague-Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6 weeks to 3 months after cell grafting. In animals implanted with TAC pellets (3.4 mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1 ± 4.9 ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4-5.1 mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats.

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

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

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

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

Development of AMPA receptor and GABA B receptor-sensitive spinal hyper-reflexia after spinal air embolism in rat: a systematic neurological, electrophysiological and qualitative histopathological study.

Decompression sickness results from formation of bubbles in the arterial and venous system, resulting in spinal disseminated neurodegenerative changes and may clinically be presented by motor dysfunction, spinal segmental stretch hyper-reflexia (i.e., spasticity) and muscle rigidity. In our current study, we describe a rat model of spinal air embolism characterized by the development of similar spinal disseminated neurodegenerative changes and functional deficit. In addition, the anti-spastic potency of systemic AMPA receptor antagonist (NGX424) or GABA B receptor agonist (baclofen) treatment was studied. To induce spinal air embolism, animals received an intra-aortic injection of air (50-200 µl/kg). After embolism, the development of spasticity was measured using computer-controlled ankle rotation. Animals receiving 150 or 200 µl of intra-aortic air injections displayed motor dysfunction with developed spastic (50-60% of animals) or flaccid (25-35% of animals) paraplegia at 5-7 days. MRI and spinal histopathological analysis showed disseminated spinal cord infarcts in the lower thoracic to sacral spinal segments. Treatment with NGX424 or baclofen provided a potent anti-spasticity effect (i.e., stretch hyper-reflexia inhibition). This model appears to provide a valuable experimental tool to study the pathophysiology of air embolism-induced spinal injury and permits the assessment of new treatment efficacy targeted to modulate neurological symptoms resulting from spinal air embolism.

Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

BACKGROUND: Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B) receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. METHODS/PRINCIPAL FINDINGS: Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2-3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. CONCLUSIONS/SIGNIFICANCE: These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments.

Suppression of stretch reflex activity after spinal or systemic treatment with AMPA receptor antagonist NGX424 in rats with developed baclofen tolerance.

BACKGROUND AND PURPOSE: Baclofen (a GABA(B) receptor agonist) is the most commonly used anti-spasticity agent in clinical practice. While effective when administered spinally or systemically, the development of progressive tolerance represents a serious limitation for its long-term use. The goal of the present study was to characterize the treatment potency after intrathecal or systemic treatment with the selective AMPA receptor antagonist NGX424 on stretch reflex activity (SRA) and background muscle activity (BMA) in rats with developed baclofen tolerance. EXPERIMENTAL APPROACH: Animals were exposed to 10 min of spinal ischaemia to induce an increase in BMA and SRA. Selected animals were implanted with an intrathecal PE-5 catheter and infused intrathecally with baclofen (1 µg·h⁻¹ ) for 14 days. Before and after baclofen infusion, changes in BMA and SRA were measured at 2 day intervals. After development of baclofen tolerance, the animals were injected intrathecally (1 µg) or subcutaneously (3, 6 or 12 mg·kg⁻¹) with NGX424, and changes in BMA and SRA were measured. KEY RESULTS: Intrathecal or systemic delivery of NGX424 significantly suppressed the BMA and SRA in baclofen-tolerant animals. This effect was dose dependent. The magnitude of BMA and SRA suppression seen after 1 µg (intrathecal) or 12 mg·kg ⁻¹ (s.c.) of NGX424 injection was similar to that seen during the first 5 days of baclofen infusion. CONCLUSIONS AND IMPLICATIONS These data demonstrate that the use of NGX424 can represent an effective therapy to modulate chronic spasticity in patients who are refractory or tolerant to baclofen treatment.

Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs.

In recent studies using a rat aortic balloon occlusion model, we have demonstrated that spinal grafting of rat or human neuronal precursors or human postmitotic hNT neurons leads to progressive amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In the present study, we characterized the optimal dosing regimen and safety profile of human spinal stem cells (HSSC) when grafted into the lumbar spinal cord segments of naive immunosuppressed minipigs. Gottingen-Minnesota minipigs (18-23 kg) were anesthetized with halothane, mounted into a spine-immobilization apparatus, and received five bilateral injections of HSSC delivered in 2, 4, 6, 8, or 10 µl of media targeted into L2-L5 central gray matter (lamina VII). The total number of delivered cells ranged between 2,500 and 100,000 per injection. Animals were immunosuppressed with Prograf® for the duration of study. After cell grafting, ambulatory function was monitored daily using a Tarlov's score. Sensory functions were assessed by mechanically evoked skin twitch test. Animals survived for 6-7 weeks. Three days before sacrifice animals received daily injections of bromodeoxyuridine (100 mg/kg; IV) and were then transcardially perfused with 4% paraformaldehyde. Th12-L6 spinal column was then dissected; the spinal cord was removed and scanned with MRI. Lumbar transverse spinal cord sections were then cut and stained with a combination of human-specific (hNUMA, hMOC, hNSE, hSYN) or nonspecific (DCX, MAP2, GABA, CHAT) antibodies. The total number of surviving cells was estimated using stereological quantification. During the first 12-24 h after cell grafting, a modest motor weakness was observed in three of eight animals but was no longer present at 4 days to 7 weeks. No sensory dysfunction was seen at any time point. Postmortem MRI scans revealed the presence of the individual grafts in the targeted spinal cord areas. Histological examination of spinal cord sections revealed the presence of hNUMA-immunoreactive grafted cells distributed between the base of the dorsal horn and the ventral horn. In all grafts intense hMOC, DCX, and hSYN immunoreactivity in grafted cells was seen. In addition, a rich axodendritic network of DCX-positive processes was identified extending 300-700 µm from the grafts. On average, 45% of hNUMA-positive neurons were GABA immunoreactive. Stereological analysis of hNUMA-positive cells showed an average of 2.5- to 3-fold increase in number of surviving cells compared with the number of injected cells. Analysis of spinal structural morphology showed that in animals injected with more than 50,000 cells/injection or volumes of injectate higher than 6 µl/injection there was tissue expansion and disruption of the local axodendritic network. Based on these data the safe total number of injected cells and volume of injectate were determined to be 30,000 cells delivered in ≤6 µl of media. These data demonstrate that highly reproducible delivery of a potential cell therapeutic candidate into spinal parenchyma can be achieved across a wide range of cell doses by direct intraspinal injections. The resulting grafts uniformly showed robust cell survival and progressive neuronal maturation.

Mutant dynein (Loa) triggers proprioceptive axon loss that extends survival only in the SOD1 ALS model with highest motor neuron death.

Dominant mutations in cytoplasmic dynein (Loa or Cra) have been reported to provoke selective, age-dependent killing of motor neurons, while paradoxically slowing degeneration and death of motor neurons in one mouse model of an inherited form of ALS. Examination of Loa animals reveals no degeneration of large caliber alpha-motor neurons beyond an age-dependent loss (initiating only after 18 months) that was comparable in Loa and wild-type littermates. Absence of Loa-mediated alpha-motor neuron loss contrasted with dramatic, sustained, mutant dynein-mediated postnatal loss of lumbar proprioceptive sensory axons, accompanied by decreased excitatory glutamatergic inputs to motor neurons. In mouse models of inherited ALS caused by mutations in superoxide dismutase (SOD1), mutant dynein modestly prolonged survival in the one mouse model with the most extensive motor neuron loss (SOD(G93A)) while showing marginal (SOD(G85R)) or no (SOD(G37R)) benefit in models with higher numbers of surviving motor neurons at end stage. These findings support a noncell autonomous, excitotoxic contribution from proprioceptive sensory neurons that modestly accelerates disease onset in inherited ALS.

Spinal astrocyte glutamate receptor 1 overexpression after ischemic insult facilitates behavioral signs of spasticity and rigidity.

Using a rat model of ischemic paraplegia, we examined the expression of spinal AMPA receptors and their role in mediating spasticity and rigidity. Spinal ischemia was induced by transient occlusion of the descending aorta combined with systemic hypotension. Spasticity/rigidity were identified by simultaneous measurements of peripheral muscle resistance (PMR) and electromyography (EMG) before and during ankle flexion. In addition, Hoffman reflex (H-reflex) and motor evoked potentials (MEPs) were recorded from the gastrocnemius muscle. Animals were implanted with intrathecal catheters for drug delivery and injected with the AMPA receptor antagonist NGX424 (tezampanel), glutamate receptor 1 (GluR1) antisense, or vehicle. Where intrathecal vehicle had no effect, intrathecal NGX424 produced a dose-dependent suppression of PMR [ED50 of 0.44 microg (0.33-0.58)], as well as tonic and ankle flexion-evoked EMG activity. Similar suppression of MEP and H-reflex were also seen. Western blot analyses of lumbar spinal cord tissue from spastic animals showed a significant increase in GluR1 but decreased GluR2 and GluR4 proteins. Confocal and electron microscopic analyses of spinal cord sections from spastic animals revealed increased GluR1 immunoreactivity in reactive astrocytes. Selective GluR1 knockdown by intrathecal antisense treatment resulted in a potent reduction of spasticiy and rigidity and concurrent downregulation of neuronal/astrocytic GluR1 in the lumbar spinal cord. Treatment of rat astrocyte cultures with AMPA led to dose-dependent glutamate release, an effect blocked by NGX424. These data suggest that an AMPA/kainate receptor antagonist can represent a novel therapy in modulating spasticity/rigidity of spinal origin and that astrocytes may be a potential target for such treatment.

Measurement of peripheral muscle resistance in rats with chronic ischemia-induced paraplegia or morphine-induced rigidity using a semi-automated computer-controlled muscle resistance meter.

In experimental and clinical studies, an objective assessment of peripheral muscle resistance represents one of the key elements in determining the efficacy of therapeutic manipulations (e.g. pharmacological, surgical) aimed to ameliorate clinical signs of spasticity and/or rigidity. In the present study, we characterize a newly developed limb flexion resistance meter which permits a semi-automated, computer-controlled measurement of peripheral muscle resistance (PMR) in the lower extremities during a forced flexion of the ankle in the awake rat. Ischemic paraplegia was induced in Sprague-Dawley rats by transient aortic occlusion (10 min) in combination with systemic hypotension (40 mm Hg). After ischemia the presence of spasticity component was determined by the presence of an exaggerated EMG activity recorded from gastrocnemius muscle after nociceptive or proprioceptive afferent activation and by velocity-dependent increase in muscle resistance. Rigidity was induced by high dose (30 mg/kg, i.p.) of morphine. Animals with defined ischemic spasticity or morphine-induced rigidity were then placed into a plastic restrainer and a hind paw attached by a tape to a metal plate driven by a computer-controlled stepping motor equipped with a resistance transducer. The resistance of the ankle to rotation was measured under several testing paradigms: (i) variable degree of ankle flexion (40 degrees, 50 degrees, and 60 degrees), (ii) variable speed/rate of ankle flexion (2, 3, and 4 sec), (iii) the effect of inhalation anesthesia, (iv) the effect of intrathecal baclofen, (v) the effect of dorsal L2-L5 rhizotomy, or (vi) systemic naloxone treatment. In animals with ischemic paraplegia an increased EMG response after peripheral nociceptive or proprioceptive activation was measured. In control animals average muscle resistance was 78 mN and was significantly increased in animals with ischemic spasticity (981-7900 mN). In ischemic-spastic animals a significant increase in measured muscle resistance was seen after increased velocity (4 > 3 > 2 sec) and the angle (40 degrees > 50 degrees > 60 degrees) of the ankle rotation. In spastic animals, deep halothane anesthesia, intrathecal baclofen or dorsal rhizotomy decreased muscle resistance to 39-80% of pretreatment values. Systemic treatment with morphine induced muscle rigidity and corresponding increase in muscle resistance. Morphine-induced increase in muscle resistance was independent on the velocity of the ankle rotation and was reversed by naloxone. These data show that by using this system it is possible to objectively measure the degree of peripheral muscle resistance. The use of this system may represent a simple and effective experimental tool in screening new pharmacological compounds and/or surgical manipulations targeted to modulate spasticity and/or rigidity after a variety of neurological disorders such as spinal cord traumatic or ischemic injury, multiple sclerosis, cerebral palsy, or Parkinson's disease.

The activation of spinal N-methyl-D-aspartate receptors may contribute to degeneration of spinal motor neurons induced by neuraxial morphine after a noninjurious interval of spinal cord ischemia.

We investigated the relationship between the degeneration of spinal motor neurons and activation of N-methyl-d-aspartate (NMDA) receptors after neuraxial morphine following a noninjurious interval of aortic occlusion in rats. Spinal cord ischemia was induced by aortic occlusion for 6 min with a balloon catheter. In a microdialysis study, 10 muL of saline (group C; n = 8) or 30 mug of morphine (group M; n = 8) was injected intrathecally (IT) 0.5 h after reflow, and 30 mug of morphine (group SM; n = 8) or 10 muL of saline (group SC; n = 8) was injected IT 0.5 h after sham operation. Microdialysis samples were collected preischemia, before IT injection, and at 2, 4, 8, 24, and 48 h of reperfusion (after IT injection). Second, we investigated the effect of IT MK-801 (30 mug) on the histopathologic changes in the spinal cord after morphine-induced spastic paraparesis. After IT morphine, the cerebrospinal fluid (CSF) glutamate concentration was increased in group M relative to both baseline and group C (P < 0.05). This increase persisted for 8 hrs. IT MK-801 significantly reduced the number of dark-stained alpha-motoneurons after morphine-induced spastic paraparesis compared with the saline group. These data indicate that IT morphine induces spastic paraparesis with a concomitant increase in CSF glutamate, which is involved in NMDA receptor activation. We suggest that opioids may be neurotoxic in the setting of spinal cord ischemia via NMDA receptor activation.

Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.

In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague-Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1-4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3-4 weeks after plating. A sub-population of plated neurons (35-40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10 mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 +/- 950%) and glutamate release (130 +/- 42%) during first 10 min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.

Spinal implantation of hNT neurons and neuronal precursors: graft survival and functional effects in rats with ischemic spastic paraplegia.

Transient spinal ischemia, a complication associated with aortic cross-clamp may lead to spastic paraplegia. Once fully developed this deficit is permanent. Quantitative histopathological assessments and pharmacological studies show that the ischemic spasticity is secondary to the loss of lumbar GABA and glycinergic inhibitory interneurons. In the present study, we investigated whether human hNT neurons or committed Sprague-Dawley rat spinal neuronal precursors (SNPs) when grafted into previously ischemic spinal segments depleted of inhibitory neurons would restore local inhibitory tone and ameliorate spasticity. Rats with functionally and electrophysiologically defined spasticity that received spinal graft of hNT neurons or neuronal precursors and immunosuppressive treatment displayed a progressive recovery of motor function that correlated with the improvement of otherwise exacerbated peripheral motor response evoked by stimulation of motor cortex. In contrast, in control, medium-injected or oligodendrocyte-grafted animals no significant therapeutic effect was seen. Stereological quantification of grafted neurons revealed 1-2% survival at three months after transplantation. These surviving neurons displayed a robust axo-dendritic sprouting and expression of markers typical of mature neurons including NSE, NeuN and synaptophysin. In both treatment groups a subpopulation of grafted neurons developed GABA immunoreactivity. These data provide evidence that a region specific grafting of hNT neurons or other neuronally committed cells, which have a potential to develop inhibitory neurotransmitter phenotype, represent an effective treatment modality to modulate ischemia-induced spastic paraplegia.

Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study.

In the present study, we have characterized an atraumatic grafting technique which permits multiple, segmental, and lamina-specific injections into cervical or lumbar spinal cord. Cell injections were performed in spinally mounted rats of different ages and spinal cord size, using a micromanipulator and glass microcapillary connected to a digital microinjector. For grafting, we used human neuroteratoma (hNT) cells, BrdU-labeled rat spinal precursors or primary embryonic spinal cord neurons isolated from E14 spinal cord of the eGFP+ rat. Systematic quantification of grafted cells was performed using stereological principles of systematic random sampling and semi-automated optical Disector software. Volume reconstruction was performed using serial sections from grafted areas and custom-developed software (Ellipse) which permits "two reference points" semi-automated alignment of images, as well as volume reconstruction and calculation. By coupling these techniques, it is possible to achieve a relatively precise and atraumatic cell delivery into multiple spinal cord segments and specific spinal laminae. Consistency of the multiple grafts position in the targeted laminar areas was verified by a systematic volume reconstruction. Good survival of implanted cells for the three different cell lines used indicate that this grafting technique coupled with a systematic analysis of the individual grafting sites can represent a valuable implantation-analytical system.

Mediators of ischemic preconditioning identified by microarray analysis of rat spinal cord.

Spinal ischemia is a frequent cause of paralysis. Here we explore the biological basis of ischemic preconditioning (IPC), the phenomenon in which a brief period of ischemia can confer protection against subsequent longer and normally injurious ischemia, to identify mediators of endogenous neuroprotection. Using microarrays, we examined gene expression changes induced by brief spinal ischemia using a rat balloon occlusion model. Among the nearly 5000 genes assayed, relatively few showed two-fold changes, and three groups stood out prominently. The first group codes for heat shock protein 70, which is induced selectively and robustly at 30 min after brief ischemia, with increases up to 100-fold. A second group encodes metallothioneins 1 and 2. These mRNAs are increased at 6 and 12 h after ischemia, up to 12-fold. The third group codes for a group of immediate-early genes not previously associated with spinal ischemia: B-cell translocation gene 2 (BTG2), the transcription factors early growth response 1 (egr-1) and nerve growth factor inducible B (NGFI-B), and a mitogen-activated protein kinase phosphatase, ptpn16, an important cell signaling regulator. These mRNAs peak at 30 min and return to baseline or are decreased 6 h after ischemia. Several other potentially protective genes cluster with these induced mRNAs, including small heat shock proteins, and many have not been previously associated with IPC. These results provide both putative mediators of IPC and molecular targets for testing preconditioning therapies.

Characterization of spinal HSP72 induction and development of ischemic tolerance after spinal ischemia in rats.

Induction of heat shock protein (HSP72) has been implicated in the development of ischemic tolerance in several tissue organs including brain and spinal cord. In the present study, using an aortic balloon occlusion model in rats, we characterized the effect of transient noninjurious (3 or 6 min) or injurious intervals (10 min) of spinal ischemia followed by 4-72 h of reflow on spinal expression of HSP72 and GFAP protein. In a separate group of animals, the effect of ischemic preconditioning (3 or 6 min) on the recovery of function after injurious interval of spinal ischemia (10 min) was studied. After 3 min of ischemia, there was a modest increase in HSP72 protein immunoreactivity in the dorsal horn neurons at 12 h after reperfusion. After 6 min of ischemia, a more robust and wide spread HSP72 protein expression in both dorsal and ventral horn neurons was detected. The peak of the expression was seen at 24 h after ischemia. At the same time point, a significant increase in spinal tissue GFAP expression was measured with Western blots and corresponded morphologically with the presence of activated astrocytes in spinal segments that had been treated similarly. After 10 min of ischemia and 24 h of reflow, a significant increase in spinal neuronal HSP72 expression in perinecrotic regions was seen. Behaviorally, 3 min preconditioning ischemia led to the development of a biphasic ischemic tolerance (the first at 30 min and the second at 24 h after preconditioning) and was expressed as a significantly better recovery of motor function after exposure to a second 10-min interval of spinal ischemia. After 6 min ischemic preconditioning, a more robust ischemic tolerance at 24 h after preconditioning then seen after 3-min preconditioning was detected. These data indicate that 3 min of spinal ischemia represents a threshold for spinal neuronal HSP72 induction, however, a longer sublethal interval (6 min) of preconditioning ischemia is required for a potent neuronal HSP72 induction. More robust neurological protection, seen after 6 min of preconditioning ischemia, also indicates that HSP72 expression in spinal interneurons seen at 24 h after preconditioning may represent an important variable in modulating ischemic tolerance observed during this time frame.

Changes in spinal GDNF, BDNF, and NT-3 expression after transient spinal cord ischemia in the rat.

Previous studies have demonstrated that the expression of several growth factors including glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and neurotrophin-3 (NT-3) play an important role in defining neuronal survival after brain ischemia. In the present study, using a well-defined model of transient spinal ischemia in rat, we characterized the changes in spinal GDNF, BDNF, and NT-3 expression as defined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence coupled with deconvolution microscopy. In control animals, baseline levels of GDNF, BDNF, and NT-3 (74 +/- 22, 3,600 +/- 270, 593 +/- 176 pg/g tissue, respectively) were measured. In the ischemic group, 6 min of spinal ischemia resulted in a biphasic response with increases in tissue GDNF and BDNF concentrations at the 2-hr and 72-hr points after ischemia. No significant differences in NT-3 concentration were detected. Deconvolution analysis revealed that the initial increase in tissue GDNF concentration corresponded to a neuronal upregulation whereas the late peak seen at 72 hr corresponded with increased astrocyte-derived GDNF synthesis. Increased expression of BDNF was seen in neurons, astrocytes, and oligodendrocytes. These data suggest that the early increase in neuronal GDNF/BDNF expression likely modulates neuronal resistance/recovery during the initial period of postischemic reflow. Increased astrocyte-derived BDNF/GDNF expression corresponds with transient activation of astrocytes and may play an active role in neuronal plasticity after non-injurious intervals of spinal ischemia.

[Higher blood propofol concentrations upon awakening after total intravenous anesthesia in patients with panperitonitis compared with patients for elective abdominal surgery].

We examined the blood propofol concentrations when patients responded to vocal command in patients with panperitonitis (Group P:N = 11) and patients for elective laparotomy (Group E:N = 19). In both groups, general anesthesia was induced and maintained by intravenous administration of propofol, fentanyl and ketamine following insertion of an epidural catheter. Blood samples were taken from the left femoral artery at emergence from anesthesia, and blood propofol concentrations were measured by the HPLC. Although the demographic factors and the anesthetic conditions were similar in both groups, the blood propofol concentration at emergence from anesthesia in Group P was significantly higher than that in Group E (1.04 + 0.11 vs 0.84 + 0.14 microgram.ml-1, P = 0.03). Our data suggest that blood propofol levels should be kept higher during propofol anesthesia in patients with panperitonitis.