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1.
Neuroscience ; 194: 160-9, 2011 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-21871540

RESUMO

BACKGROUND: Spasticity and rigidity are serious complications associated with spinal traumatic or ischemic injury. Clinical studies show that tizanidine (Tiz) is an effective antispasticity agent; however, the mechanism of this effect is still not clear. Tiz binds not only to α2-adrenoreceptors (AR) but also to imidazoline (I) receptors. Both receptor systems (AR+I) are present in the spinal cord interneurons and α-motoneurons. The aim of the present study was to evaluate the therapeutic potency of systematically or spinally (intrathecally [IT]) delivered Tiz on stretch reflex activity (SRA) in animals with ischemic spasticity, and to delineate supraspinal or spinal sites of Tiz action. EXPERIMENTAL PROCEDURES: Animals were exposed to 10 min of spinal ischemia to induce an increase in SRA. Increase in SRA was identified by simultaneous increase in recorded electromyography (EMG) activity and ankle resistance measured during computer-controlled ankle dorsiflexion (40°/3 s) in fully awake animals. Animals with increased SRA were divided into several experimental subgroups and treated as follows: (i) Tiz administered systemically at the dose of 1 mg kg(-1), or IT at 10 µg or 50 µg delivered as a single dose; (ii) treatment with systemic Tiz was followed by the systemic injection of vehicle, or by nonselective AR antagonist without affinity for I receptors; yohimbine (Yoh), α2A AR antagonist; BRL44408 (BRL), α2B AR antagonist; ARC239 (ARC), nonselective AR and I(1) receptor antagonist; efaroxan (Efa), or nonselective AR and I(2) receptor antagonist; idazoxan (Ida); (iii) treatment with IT Tiz was followed by the IT injection of selective α2A AR antagonist; atipamezole (Ati). In a separate group of spastic animals the effect of systemic Tiz treatment (1 mg/kg) or isoflurane anesthesia on H-reflex activity was also studied. RESULTS: Systemic and/or IT treatment with Tiz significantly suppressed SRA. This Tiz-mediated anti-SRA effect was reversed by BRL (5 mg kg(-1)), Efa (1 mg kg(-1)), and Ida (1 mg kg(-1)). No reversal was seen after Yoh (3 mg kg(-1)) or ARC (5 mg kg(-1)) treatment. Anti-SRA induced by IT Tiz (50 µg) was reversed by IT injection of Ati (50 µg). Significant suppression of H-reflex was measured after systemic Tiz treatment (1 mg/kg) or isoflurane (2%) anesthesia, respectively. Immunofluorescence staining of spinal cord sections taken from animals with spasticity showed upregulation of α2A receptor in activated astrocytes. CONCLUSIONS: These data suggest that α2A AR and I receptors, but not α2B AR, primarily mediate the Tiz-induced antispasticity effect. This effect involves spinal and potentially supraspinal sites and likely targets α2A receptor present on spinal neurons, primary afferents, and activated astrocytes. Further studies using highly selective antagonists are needed to elucidate the involvement of specific subtypes of the AR and I receptors in the antispasticity effect seen after Tiz treatment.


Assuntos
Clonidina/análogos & derivados , Paraplegia/tratamento farmacológico , Paraplegia/fisiopatologia , Reflexo de Estiramento/efeitos dos fármacos , Isquemia do Cordão Espinal/fisiopatologia , Animais , Doença Crônica , Clonidina/farmacologia , Modelos Animais de Doenças , Masculino , Relaxantes Musculares Centrais/farmacologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Paraplegia/etiologia , Ratos , Ratos Sprague-Dawley , Reflexo de Estiramento/fisiologia , Isquemia do Cordão Espinal/complicações
2.
Cell Transplant ; 20(8): 1163-78, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21669049

RESUMO

A major limitation of neural transplantation studies is assessing the degree of host-graft interaction. In the present study, rat hippocampal/cortical embryonic neurons (E18) were infected with a lentivirus encoding enhanced green fluorescent protein (GFP) under control of the neuron-specific synapsin promoter, thus permitting robust identification of labeled neurons after in vivo grafting. Two weeks after transient forebrain ischemia or sham-surgery, GFP-expressing neurons were transplanted into CA1 hippocampal regions in immunosuppressed adult Wistar rats. The survival, distribution, phenotype, and axonal projections of GFP-immunoreactive (IR) positive transplanted neurons were evaluated in the sham-operated and ischemia- damaged CA1 hippocampal regions 4, 8, and 12 weeks after transplantation. In both experimental groups, we observed that the main phenotype of host-derived afferents projecting towards grafted GFP-IR neurons as well as transplant-derived GFP-IR efferents were glutamatergic in both animal groups. GFP axonal projections were seen in the nucleus accumbens, septal nuclei, and subiculum-known target areas of CA1 pyramidal neurons. Compared to sham-operated animals, GFP-IR neurons grafted into the ischemia-damaged CA1 migrated more extensively throughout a larger volume of host tissue, particularly in the stratum radiatum. Moreover, enhanced axonal sprouting and neuronal plasticity of grafted cells were evident in the hippocampus, subiculum, septal nuclei, and nucleus accumbens of the ischemia-damaged rats. Our study suggests that the adult rat brain retains some capacity to direct newly sprouting axons of transplanted embryonic neurons to the correct targets and that this capacity is enhanced in previously ischemia-injured forebrain.


Assuntos
Axônios/metabolismo , Região CA1 Hipocampal/patologia , Dendritos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Ataque Isquêmico Transitório/terapia , Neurônios/transplante , Sinapsinas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Ataque Isquêmico Transitório/patologia , Lentivirus/genética , Masculino , Neurônios Eferentes/citologia , Neurônios Eferentes/metabolismo , Fenótipo , Ratos , Ratos Wistar , Coloração e Rotulagem
3.
Neuroscience ; 154(3): 1077-87, 2008 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-18511207

RESUMO

Activation of the spinal phospholipase A(2) (PLA(2)) -cyclooxygenase (COX) -prostaglandin signaling pathway is widely implicated in nociceptive processing. Although the role of spinal COX isoforms in pain signal transmission has been extensively characterized, our knowledge of PLA(2) enzymes in this cascade is limited. Among all PLA(2) groups, cytosolic calcium-dependent PLA(2) group IVA (cPLA(2)IVA) appears to be the predominant PLA(2) enzyme in the spinal cord. In the present study we sought to (i) characterize anatomical and cellular distribution and localization of cPLA(2)IVA in dorsal horn of rat spinal cord, (ii) verify efficacy and selectivity of intrathecal (IT) delivery of an antisense oligonucleotide (AS) targeting rat cPLA(2)IVA mRNA on spinal expression of this enzyme, and (iii) examine the effect of down-regulation of spinal cPLA(2)IVA on peripheral tissue injury-induced pain behavior. Here we demonstrate that cPLA(2)IVA is constitutively expressed in rat spinal cord, predominantly in dorsal horn neurons and oligodendrocytes but not in astrocytes or microglia. Intrathecal injection of AS significantly down-regulated both protein and gene expression of cPLA(2)IVA in rat spinal cord, while control missense oligonucleotide (MS) had no effect. Immunocytochemistry confirmed that the reduction occurred in neurons and oligodendrocytes. cPLA(2)IVA AS did not alter expression of several other PLA(2) isoforms, such as secretory PLA(2) (groups IIA and V) and calcium-independent PLA(2) (group VI), indicating that the AS was specific for cPLA(2)IVA. This selective knockdown of spinal cPLA(2)IVA did not change acute nociception (i.e. paw withdrawal thresholds to acute thermal stimuli and intradermal formalin-induced first phase flinching), however, it significantly attenuated formalin-induced hyperalgesia (i.e. second phase flinching behavior), which reflects spinal sensitization. Thus the present findings suggest that cPLA(2)IVA may specifically participate in spinal nociceptive processing.


Assuntos
Citosol/enzimologia , Formaldeído , Hiperalgesia/prevenção & controle , Hiperalgesia/psicologia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Fosfolipases A2/biossíntese , Medula Espinal/enzimologia , Animais , Comportamento Animal/efeitos dos fármacos , Western Blotting , Citosol/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Temperatura Alta , Hiperalgesia/induzido quimicamente , Imuno-Histoquímica , Injeções Espinhais , Masculino , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/efeitos dos fármacos
4.
Neuroscience ; 147(2): 546-60, 2007 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-17524565

RESUMO

Transient spinal cord ischemia in humans can lead to the development of permanent paraplegia with prominent spasticity and rigidity. Histopathological analyses of spinal cords in animals with ischemic spastic paraplegia show a selective loss of small inhibitory interneurons in previously ischemic segments but with a continuing presence of ventral alpha-motoneurons and descending cortico-spinal and rubro-spinal projections. The aim of the present study was to examine the effect of human spinal stem cells (hSSCs) implanted spinally in rats with fully developed ischemic paraplegia on the recovery of motor function and corresponding changes in motor evoked potentials. In addition the optimal time frame for cell grafting after ischemia and the optimal dosing of grafted cells were also studied. Spinal cord ischemia was induced for 10 min using aortic occlusion and systemic hypotension. In the functional recovery study, hSSCs (10,000-30,000 cells/0.5 mul/injection) were grafted into spinal central gray matter of L2-L5 segments at 21 days after ischemia. Animals were immunosuppressed with Prograf (1 mg/kg or 3 mg/kg) for the duration of the study. After cell grafting the recovery of motor function was assessed periodically using the Basso, Beattie and Bresnahan (BBB) scoring system and correlated with the recovery of motor evoked potentials. At predetermined times after grafting (2-12 weeks), animals were perfusion-fixed and the survival, and maturation of implanted cells were analyzed using antibodies recognizing human-specific antigens: nuclear protein (hNUMA), neural cell adhesion molecule (hMOC), neuron-specific enolase (hNSE) and synapthophysin (hSYN) as well as the non-human specific antibodies TUJ1, GFAP, GABA, GAD65 and GLYT2. After cell grafting a time-dependent improvement in motor function and suppression of spasticity and rigidity was seen and this improvement correlated with the recovery of motor evoked potentials. Immunohistochemical analysis of grafted lumbar segments at 8 and 12 weeks after grafting revealed intense hNSE immunoreactivity, an extensive axo-dendritic outgrowth as well as rostrocaudal and dorsoventral migration of implanted hNUMA-positive cells. An intense hSYN immunoreactivity was identified within the grafts and in the vicinity of persisting alpha-motoneurons. On average, 64% of hSYN terminals were GAD65 immunoreactive which corresponded to GABA immunoreactivity identified in 40-45% of hNUMA-positive grafted cells. The most robust survival of grafted cells was seen when cells were grafted 21 days after ischemia. As defined by cell survival and laminar distribution, the optimal dose of injected cells was 10,000-30,000 cells per injection. These data indicate that spinal grafting of hSSCs can represent an effective therapy for patients with spinal ischemic paraplegia.


Assuntos
Paraplegia/terapia , Isquemia do Cordão Espinal/terapia , Medula Espinal/citologia , Transplante de Células-Tronco , Adulto , Animais , Astrócitos/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Potencial Evocado Motor/fisiologia , Feminino , Glutamato Descarboxilase/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Humanos , Imuno-Histoquímica , Interneurônios/fisiologia , Isoenzimas/metabolismo , Locomoção/fisiologia , Microscopia Confocal , Rigidez Muscular/fisiopatologia , Rigidez Muscular/terapia , Espasticidade Muscular/fisiopatologia , Espasticidade Muscular/terapia , Neurotransmissores/metabolismo , Gravidez , Ratos , Isquemia do Cordão Espinal/patologia , Isquemia do Cordão Espinal/fisiopatologia , Sinaptofisina/metabolismo , Sinaptofisina/fisiologia , Fixação de Tecidos , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/fisiologia
5.
Neuroscience ; 141(3): 1569-83, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16797137

RESUMO

Transient spinal cord ischemia may lead to a progressive degeneration of spinal interneurons and subsequently to increased hind limb motor tone. In the present work we sought to characterize the rigidity and spasticity components of this altered motor function by: i) tonic electromyographic activity measured in gastrocnemius muscle before and after ischemia, ii) measurement of muscle resistance during the period of ankle flexion and corresponding changes in electromyographic activity, iii) changes in Hoffmann reflex, and, iv) motor evoked potentials. In addition the effect of intrathecal treatment with baclofen (GABAB receptor agonist; 1 microg), nipecotic acid (GABA uptake inhibitor; 300 microg) and dorsal L2-L5 rhizotomy on spasticity and rigidity was studied. Finally, the changes in spinal choline acetyltransferase (ChAT) and vesicular glutamate transporter 2 and 1 (VGLUT2 and VGLUT1) expression were characterized using immunofluorescence and confocal microscopy. At 3-7 days after ischemia an increase in tonic electromyographic activity with a variable degree of rigidity was seen. In animals with modest rigidity a velocity-dependent increase in muscle resistance and corresponding appearance in electromyographic activity (consistent with the presence of spasticity) was measured during ankle rotation (4-612 degrees /s rotation). Measurement of the H-reflex revealed a significant increase in Hmax/Mmax ratio and a significant loss of rate-dependent inhibition. In the same animals a potent increase in motor evoked potential amplitudes was measured and this change correlated positively with the increased H-reflex responses. Spasticity and rigidity were consistently present for a minimum of 3 months after ischemia. Intrathecal treatment with baclofen (GABA B receptor agonist) and nipecotic acid (GABA uptake inhibitor) provided a significant suppression of spasticity, rigidity, H-reflex or motor evoked potentials. Dorsal L2-L5 rhizotomy significantly decreased muscle resistance but had no effect on increased amplitudes of motor evoked potentials. Confocal analysis of spinal cord sections at 8 weeks-12 months after ischemia revealed a continuing presence of ChAT positive alpha-motoneurons, Ia afferents and VGLUT2 and VGLUT1-positive terminals but a selective loss of small presumably inhibitory interneurons between laminae V-VII. These data demonstrate that brief transient spinal cord ischemia in rat leads to a consistent development of spasticity and rigidity. The lack of significant suppressive effect of dorsal L2-L5 rhizotomy on motor evoked potentials response indicates that descending motor input into alpha-motoneurons is independent on Ia afferent couplings and can independently contribute to increased alpha-motoneuronal excitability. The pharmacology of this effect emphasizes the potent role of GABAergic type B receptors in regulating both the spasticity and rigidity.


Assuntos
Potencial Evocado Motor/fisiologia , Reflexo H/fisiologia , Rigidez Muscular/etiologia , Espasticidade Muscular/etiologia , Isquemia do Cordão Espinal/complicações , Ácido gama-Aminobutírico/metabolismo , Acetiltransferases/metabolismo , Análise de Variância , Animais , Baclofeno/administração & dosagem , Relação Dose-Resposta à Radiação , Sistemas de Liberação de Medicamentos/métodos , Estimulação Elétrica , Eletromiografia/métodos , Agonistas GABAérgicos/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Reflexo H/efeitos dos fármacos , Imuno-Histoquímica/métodos , Masculino , Rigidez Muscular/tratamento farmacológico , Espasticidade Muscular/tratamento farmacológico , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Exame Neurológico/métodos , Ácidos Nipecóticos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Rizotomia/métodos , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
6.
Cell Mol Neurobiol ; 26(7-8): 1295-310, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16786431

RESUMO

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments. 2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [(3)H] l-arginine to [(3)H] l-citrulline. 3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult. 4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min). 5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.


Assuntos
Aorta Abdominal , Arteriopatias Oclusivas/complicações , Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Isquemia do Cordão Espinal/metabolismo , Animais , Plexo Lombossacral/metabolismo , Masculino , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Placebos , Coelhos , Traumatismo por Reperfusão/metabolismo , Isquemia do Cordão Espinal/etiologia
7.
J Pharmacol Exp Ther ; 296(3): 756-61, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11181903

RESUMO

Tactile allodynia can be modeled in experimental animals by acutely blocking spinal glycine or GABA(A) receptors with intrathecal (i.t.) strychnine (STR) or bicuculline (BIC), respectively. To test the hypothesis that glycine and GABA effect cooperative (supra-additive) inhibition of touch-evoked responses in the spinal cord, male Sprague-Dawley rats, fitted with chronic i.t. catheters, were used. Following i.t. STR, BIC, or STR + BIC, hair deflection evoked cardiovascular (increased blood pressure and heart rate), motor (scratching, kicking and rippling of the affected dermatomes), and cortical encephalographic responses. Hair deflection was without effect in i.t. saline-treated rats. Isobolographic analysis of STR (ED(50) = 25.1-36.9 microg), BIC (ED(50) = 0.5-0.6 microg), and BIC:STR combination (ED(50) = 0.026-0.034:2.6-3.4 microg) dose-response curves confirmed a supra-additive interaction between BIC and STR in this model. BIC-allodynia was reproduced by i.t. picrotoxin. Pretreatment with i.t. scopolamine, or i.t. muscarine had no effect. STR-allodynia was dose dependently inhibited by i.t. muscimol but not baclofen. The results of this study indicate that 1) glycine and GABA effect cooperative inhibition of low-threshold mechanical input in the spinal cord of the rat; and 2) BIC-allodynia arises from the blockade of GABA(A) receptors and is unrelated to any secondary anticholinesterase activity. The allodynic state induced by the blockade of glycine or GABA receptors is clearly exacerbated by the removal of both inhibitory systems. Their combined loss after neural injury may explain the exaggerated sensitivity to and subsequent miscoding of tactile information as pain.


Assuntos
Bicuculina/toxicidade , Dor/induzido quimicamente , Estricnina/toxicidade , Análise de Variância , Animais , Convulsivantes/toxicidade , Sinergismo Farmacológico , Injeções Espinhais , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/fisiologia
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