RESUMO
We have recently documented that treatment with the alternative biofuel, acetyl-L-carnitine (ALC, 300 mg/kg), as late as 1 h after T10 contusion spinal cord injury (SCI), significantly maintained mitochondrial function 24 h after injury. Here we report that after more severe contusion SCI centered on the L1/L2 segments that are postulated to contain lamina X neurons critical for locomotion (the "central pattern generator"), ALC treatment resulted in significant improvements in acute mitochondrial bioenergetics and long-term hind limb function. Although control-injured rats were only able to achieve slight movements of hind limb joints, ALC-treated animals produced consistent weight-supported plantar steps 1 month after injury. Such landmark behavioral improvements were significantly correlated with increased tissue sparing of both gray and white matter proximal to the injury, as well as preservation of choline acetyltransferase (ChAT)-positive neurons in lamina X rostral to the injury site. These findings signify that functional improvements with ALC treatment are mediated, in part, by preserved locomotor circuitry rostral to upper lumbar contusion SCI. Based on beneficial effects of ALC on mitochondrial bioenergetics after injury, our collective evidence demonstrate that preventing mitochondrial dysfunction acutely "promotes" neuroprotection that may be associated with the milestone recovery of plantar, weight-supported stepping.
Assuntos
Acetilcarnitina/farmacologia , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Metabolismo Energético/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
While activity-based rehabilitation is one of the most promising therapeutic approaches for spinal cord injury, the necessary components for optimal locomotor retraining have not yet been determined. Currently, a number of different activity-based approaches are being investigated including body weight-supported treadmill training (with and without manual assistance), robotically-assisted treadmill training, bicycling and swimming, among others. We recently showed, in the adult rat, that intensive rehabilitation based on swimming brought about significant improvements in hindlimb performance during swimming but did not alter the normal course of recovery of over-ground walking (Smith et al., 2006a,b, 2009). However, swimming lacks the phasic limb-loading and plantar cutaneous feedback thought to be important for weight-supported step training. So, we are investigating an innovative approach based on walking in shallow water where buoyancy provides some body weight support and balance while still allowing for limb-loading and appropriate cutaneous afferent feedback during retraining. Thus, the aim of this study is to determine if spinal cord injured animals show improved overground locomotion following intensive body weight-supported locomotor training in shallow water. The results show that training in shallow water successfully improved stepping in shallow water, but was not able to bring about significant improvements in overground locomotion despite the fact that the shallow water provides sufficient body weight support to allow acutely injured rats to generate frequent plantar stepping. These observations support previous suggestions that incompletely injured animals retrain themselves while moving about in their cages and that daily training regimes are not able to improve upon this already substantial functional improvement due to a ceiling effect, rather than task-specificity, per se. These results also support the concept that moderately-severe thoracic contusion injuries decrease the capacity for body weight support, but do not decrease the capacity for pattern generation. In contrast, animals with severe contusion injuries could not support their body weight nor could they generate a locomotor pattern when provided with body weight support via buoyancy.
Assuntos
Terapia por Exercício/métodos , Marcha/fisiologia , Locomoção/fisiologia , Atividade Motora/fisiologia , Traumatismos da Medula Espinal/reabilitação , Análise de Variância , Animais , Feminino , Membro Posterior/fisiologia , Membro Posterior/fisiopatologia , Fibras Nervosas Mielinizadas/patologia , Condicionamento Físico Animal , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Vértebras Torácicas , Gravação em Vídeo , ÁguaRESUMO
In the mammalian spinal cord, the ventrolateral funiculus (VLF) has been identified as critical to postural control and locomotor function, in part due to the reticulospinal pathways it contains. The primary purpose of this descriptive study was to investigate the distribution of neurons in the medulla labeled retrogradely from the VLF and the intermediate gray matter of specific lumbar and cervical spinal cord segments in the adult rat. We made discrete injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5/6, 7/8 or lumbar (L) 2 segmental levels followed by a single injection of Fluoro-Gold (FG) into the right VLF at T9. Double-labeled medullary neurons were found primarily in the gigantocellular group of nuclei (Gi), distributed both ipsilaterally and contralaterally following cervical or lumbar FR injections. In addition, a substantial population of neurons contained within the vestibular group of nuclei was double labeled both ipsilaterally and contralaterally. We also identified a substantial population of Gi-related neurons located ipsilateral to the VLF injections that were double labeled following left unilateral FR injections at C5/6, C7/8 or L2. These results describe a substantial population of ipsilateral and commissural medullary neurons that project to both cervical and thoracolumbar segments. Two different populations of commissural neurons are described, one with axons that cross the midline rostral to T9, and one with axons that cross the midline caudal to T9. These observations provide strong additional evidence for a pattern of reticulo- and vestibulospinal projections that include substantial numbers of commissural neurons and project to multiple cervical and thoracolumbar levels.
Assuntos
Vias Neurais/fisiologia , Formação Reticular/fisiologia , Medula Espinal/fisiologia , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Contagem de Células , Dextranos , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Bulbo/citologia , Bulbo/fisiologia , Vias Neurais/citologia , Ratos , Ratos Sprague-Dawley , Formação Reticular/citologia , Rodaminas , Medula Espinal/citologia , Fixação de TecidosRESUMO
The ventrolateral funiculus (VLF) in the spinal cord contains important ascending and descending pathways related to locomotion and interlimb coordination. The primary purpose of this descriptive study was to investigate the distribution of inter-enlargement pathways in the adult rat spinal cord with an emphasis on the VLF. We made discrete unilateral injections of Fluoro-Gold (FG) into the right VLF at thoracic segment (T) 9, and either unilateral or bilateral injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5-6, C7-8, or lumbar (L) 2 segmental levels. Inter-enlargement neurons with ascending axons in the right VLF were found bilaterally in laminae VII and VIII throughout the rostral lumbar spinal cord (L1-L3) and predominantly contralaterally in the caudal lumbosacral (L4-S1) spinal cord. Following left unilateral FR injections at C5-6 or C7-8 and right unilateral VLF injections of FG at T9, very few double-labeled neurons could be found anywhere in the lumbar spinal cord. Similar injections of FR at L2 revealed an almost symmetrical bilateral distribution of double-labeled neurons throughout the cervical spinal cord (C1-8). These results describe ascending and descending pathways within the spinal cord that interconnect the two enlargements and involve both commissural and ipsilateral interneurons. The majority of inter-enlargement neurons had axons within the VLF at T9. These observations support the hypothesis that the VLF contains long ascending and descending axons with propriospinal inter-enlargement, commissural and ipsilateral connections that are anatomically well-suited to mediate interlimb coordination.
Assuntos
Axônios/ultraestrutura , Interneurônios/citologia , Vias Neurais/anatomia & histologia , Medula Espinal/anatomia & histologia , Animais , Axônios/fisiologia , Dextranos , Extremidades/inervação , Extremidades/fisiologia , Feminino , Lateralidade Funcional/fisiologia , Interneurônios/fisiologia , Locomoção/fisiologia , Movimento/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Vias Neurais/fisiologia , Ratos , Ratos Sprague-Dawley , Rodaminas , Medula Espinal/fisiologia , EstilbamidinasRESUMO
Mice overexpressing mutant alpha-synuclein develop a progressive loss of motor function associated with the accumulation of aggregated alpha-synuclein in neurons of the brainstem. Recent reports suggest that tau pathology might also be associated with Parkinson disease (PD) and aggregation of alpha-synuclein. We now report that mice overexpressing A30P alpha-synuclein develop abnormally phosphorylated tau in parallel with the accumulation of aggregated alpha-synuclein. Enhanced phosphorylation of tau occurs only in symptomatic mice that also harbor abundant aggregated alpha-synuclein. The increased phosphorylation of tau occurs at S396/404 and S202 as shown by immunoblotting and immunocytochemical studies with the antibodies PHF-1 and AT8. Neurons that accumulated alpha-synuclein occurred in the dorsal brainstem and did not show strong colocalization with neurons that showed abnormal tau phosphorylation, which largely occurred in the ventral brainstem. Aggregation of alpha-synuclein and phosphorylation of tau are associated with increased levels of phosphorylated c-jun kinase (JNK), which is a stress kinase known to phosphorylate tau protein. These results suggest that alpha-synuclein pathology can stimulate early pathological changes in tau.
Assuntos
Transtornos dos Movimentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas tau/metabolismo , Fatores Etários , Alanina/genética , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting/métodos , Encéfalo/citologia , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/imunologia , Humanos , Imuno-Histoquímica/métodos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Doença de Parkinson/complicações , Doença de Parkinson/genética , Fosforilação , Prolina/genética , Sinucleínas , alfa-SinucleínaRESUMO
BACKGROUND/PURPOSE: Some Health Maintenance Organizations (HMO) limit access of their members to specialists to lower costs. The purpose of this study is to determine whether this policy affects the outcome of children with appendicitis. METHODS: At a large academic medical center, children 17 years or younger with appendicitis were treated either by an HMO Adult General Surgical Service (group A) or a Pediatric Surgical Service (group B). Board certified pediatric surgeons were not available on the HMO surgical service. Anesthesia, surgical residents, nursing, and ancillary support services were identical in both groups. Study parameters included imaging tests performed, operation type, complications, readmissions, and length of stay. Results were analyzed using chi(2) and Fischer's Exact tests. RESULTS: One-hundred seventy-five consecutive children underwent appendectomy, 96 in group A and 79 in group B. In patients with simple acute appendicitis, there was no significant difference between group A and group B for complications, readmissions, second operation, or length of stay. In patients with gangrenous or perforated appendicitis there was a significant difference between group A and group B for type of operation (laparoscopic appendectomy, group A, 4 of 27 v. group B, 0 of 34; P =.04); complications (group A, 9 of 27 v. group B, 3 of 34; P =.025); readmissions (group A, 6 of 27 v. group B, 0 of 34; P =.001); second operation (group A, 6 of 27 v. group B, 2 of 34; P =.001); and mean total length of stay in days (group A, 8.6 of 27 v. group B, 5.4 of 34; P =.05). CONCLUSIONS: Children with significantly perforated appendicitis have lower complication rates and shorter lengths of hospital stay when treated by pediatric surgeons as compared with HMO adult general surgeons.
Assuntos
Apendicectomia , Apendicite/cirurgia , Cirurgia Geral , Adolescente , Criança , Pré-Escolar , Tratamento de Emergência , Feminino , Sistemas Pré-Pagos de Saúde , Humanos , Lactente , Perfuração Intestinal/cirurgia , Masculino , Ohio , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Embryonic day 14 rat cerebral cortex-derived precursors were expanded with FGF2 and labeled with BrdU prior to being transplanted into the kainic acid-lesioned adult rat spinal cord. While these precursors give rise to cells with neuronal, astrocytic and oligodendroglial phenotypes vitro, they remained largely undifferentiated up to 12 weeks in vivo. Numerous BrdU-labeled cells were found in injured gray matter, and also lining the dilated central canal that sometimes accompanies these lesions. BrdU-labeled cells never co-expressed Map2ab, rarely co-expressed GFAP but often co-expressed nestin, even after 12 weeks in vivo. These observations suggest that the environment of the kainic acid-injured spinal cord is not hostile to transplanted embryonic cerebral cortex-derived precursors, but also is not conducive to their neuronal differentation.
Assuntos
Transplante de Tecido Encefálico/fisiologia , Embrião de Mamíferos/citologia , Agonistas de Aminoácidos Excitatórios , Transplante de Tecido Fetal/fisiologia , Ácido Caínico , Doenças da Medula Espinal/patologia , Medula Espinal/fisiologia , Animais , Diferenciação Celular/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Membro Posterior/fisiologia , Locomoção/fisiologia , Masculino , Ratos , Ratos Endogâmicos F344 , Doenças da Medula Espinal/induzido quimicamenteRESUMO
Neurofibrillary tangles, which contain abnormally hyperphosphorylated forms of tau protein, are one of the neuropathological hallmarks of Alzheimer's disease (AD). This altered phosphorylation state of tau protein may be due to increased kinase activity or/and decreased phosphatase activity. In the present study, we characterized human calcineurin phosphatase activity in postmortem superior frontal cortex and sensorimotor cortex and measured calcineurin phosphatase activity in samples from individuals with moderate to severe AD (n = 7) and age-matched controls (n = 5). Basal phosphatase activity was reduced by 25% (P < 0.05) in AD frontal cortex. Nickel-stimulated calcineurin activity was decreased by 52% (P < 0.05) and 30% (P < 0.05) in P2 and total cell homogenate, respectively, compared to age-matched controls. No differences in phosphatase activities were detected in the sensorimotor cortex. The decrease in nickel-stimulated calcineurin phosphatase activity in frontal lobe correlated with the neurofibrillary tangle pathology (total cell homogenate, r = -0.77, P < 0.05; P2 fraction, r = -0.76, P < 0.02), but not with diffuse or neuritic plaques. Despite the changes in calcineurin phosphatase activity in the superior frontal cortex, calcineurin protein levels determined by immunoblot were similar in control and AD cases. In addition, no changes in calcineurin regulatory proteins (cyclophilin A and FKBP12) levels were observed. These studies suggest that decrease of calcineurin activity may play a role in paired-helical filament formation and/or stabilization, and the decrease of activity was not accompanied by a decrease of calcineurin protein expression.
Assuntos
Doença de Alzheimer/metabolismo , Calcineurina/metabolismo , Córtex Cerebral/metabolismo , Mudanças Depois da Morte , Idoso , Doença de Alzheimer/patologia , Química Encefálica/efeitos dos fármacos , Calcineurina/análise , Membrana Celular/química , Membrana Celular/metabolismo , Córtex Cerebral/patologia , Cobalto/farmacologia , Ciclofilina A/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Immunoblotting , Manganês/farmacologia , Emaranhados Neurofibrilares/patologia , Níquel/farmacologia , Placa Amiloide/patologia , Córtex Somatossensorial/metabolismo , Córtex Somatossensorial/patologia , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
OBJECT: The aims of this study were to investigate further the role played by lumbar spinal cord interneurons in the generation of locomotor activity and to develop a model of spinal cord injury suitable for testing neuron replacement strategies. METHODS: Adult rats received intraspinal injections of kainic acid (KA). Locomotion was assessed weekly for 4 weeks by using the Basso, Beattie, and Bresnahan (BBB) 21-point locomotor scale, and transcranial magnetic motor evoked potentials (MMEPs) were recorded in gastrocnemius and quadriceps muscles at 1 and 4 weeks. No changes in transcranial MMEP latency were noted following KA injection, indicating that the descending motor pathways responsible for these responses, including the alpha motor neurons, were not compromised. Rats in which KA injections included much of the L-2 segment (10 animals) showed severe locomotor deficits, with a mean BBB score of 4.5 +/- 3.6 (+/- standard deviation). Rats that received lesions rostral to the L-2 segment (four animals) were able to locomote and had a mean BBB score of 14.6 +/- 2.6. Three rats that received only one injection bilaterally centered at L-2 (three animals) had a mean BBB score of 3.2 +/- 2. Histological examination revealed variable loss of motor neurons limited to the injection site. There was no correlation between motor neuron loss and BBB score. CONCLUSIONS: Interneuron loss centered on the L-2 segment induces lasting paraplegia independent of motor neuron loss and white matter damage, supporting earlier suggestions that circuitry critical to the generator of locomotor activity (the central pattern generator) resides in this area. This injury model may prove ideal for studies of neuron replacement strategies.
Assuntos
Interneurônios/fisiologia , Paraplegia/etiologia , Doenças da Medula Espinal/complicações , Doenças da Medula Espinal/fisiopatologia , Animais , Contagem de Células , Morte Celular/fisiologia , Eletrofisiologia , Interneurônios/patologia , Vértebras Lombares , Masculino , Atividade Motora , Placa Motora/fisiopatologia , Neurônios Motores/patologia , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Doenças da Medula Espinal/patologiaRESUMO
The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.
Assuntos
Ventrículos Laterais/citologia , Mitógenos/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Eletrofisiologia , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibronectinas/farmacologia , Substâncias de Crescimento/farmacologia , Heparina/farmacologia , Técnicas In Vitro , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/fisiologia , Fenótipo , Ratos , Células-Tronco/fisiologiaRESUMO
Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.
Assuntos
Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Mitógenos/farmacologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Estimulação Elétrica/métodos , Fator de Crescimento Epidérmico/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Neuroglia/citologia , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Neurokinin B and its cognate neurokinin-3 receptor are expressed more in the forebrain than in brain stem structures but little is known about the primary function of this peptide system in the central processing of information. In general, few studies have specifically addressed age-related changes of tachykinins, notably the changes in number and/or distribution of the neurokinin B-expressing and neurokinin-3 receptor-bearing neurons. Data on functions and changes of neurokinins in physiological aging are limited and apply mainly to the substance P/neurokinin-1 receptor system. In the present study, we analyzed neurokinin B/neurokinin-3 receptor system in young (5 months) versus middle aged (15 months) and old rats (23-25 months) and also in aging human brains. For the majority of the immunohistochemically examined regions of the rat brain, there was no statistically significant change in neuronal number and size of the neurokinin B and neurokinin-3 receptor staining. In the adult human brain, there was no age-associated change of the number or size of neurokinin-B-positive neurons. However, we found a major decline in number of neurokinin-3 receptor-expressing neurons between young/middle aged (30 years to 69 years) versus old (70 years and older) adults. Interestingly, numbers of neurokinin-3 receptor-positive microglia increased whereas the neurokinin-3 receptor-positive astrocytes remained unchanged in both aging rat and human brains. Finally, in addition to assessing the morphological and quantitative changes of the neurokinin B/neurokinin-3 receptor system in the rat and human brain, we discuss functional implications of the observed interspecies differences.
Assuntos
Envelhecimento/fisiologia , Química Encefálica/fisiologia , Neurocinina B/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Encéfalo/citologia , Contagem de Células , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neuroglia/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Ratos , Ratos Endogâmicos F344 , Especificidade da EspécieRESUMO
Autoradiographic and immunohistochemical studies have shown that the neurokinin-3 receptor is widely distributed in the rodent CNS. Expression of the neurokinin-3 receptor in human brain, however, has been debated. These conflicting findings, as well as the poor resolution of autoradiographic images, prompted us to develop a polyclonal antibody against an oligopeptide derived from the carboxy-terminus consensus sequence of both the rat and human neurokinin-3 receptor ([C]ASTTSSFISSPYTSVDEYS, amino acids 434-452 of the rat neurokinin-3 receptor). Western blot analysis of both human and rat brain tissue revealed a major band in the molecular weight range 65,000-67,000, the proposed molecular weight of the neurokinin-3 receptor based on its amino acid sequence and presumed glycosylation state. The distribution of selective high affinity neurokinin-3 receptor agonist [3H]senktide binding and neurokinin-3 receptor immunoreactivity were virtually identical in the brains of male Fischer 344 rats. The highest concentrations of neurokinin-3 receptors were observed in cortical layers IV-V; the basolateral amygdaloid nucleus; the hypothalamic paraventricular, perifornical and supraoptic nuclei; the zona incerta; and the entopeduncular and interpeduncular nuclei. [3H]senktide binding and neurokinin-3 receptor immunoreactivity were compared in homologous cortical areas of the human and rat brain. In contrast to the rat, autoradiographic analysis of normal control human brains (35-75 years) revealed a distinct and predominant superficial cortical labeling in the glia limitans and the cortical layer I. However, neurokinin-3 receptor immunoreactivity could be found not only in the superficial cortical layers, but also on pyramidal neurons and astrocytes in the neuropil and white matter. These findings suggest species differences in both the cellular and anatomical distribution of the neurokinin-3 receptor.
Assuntos
Encéfalo/metabolismo , Receptores da Neurocinina-3/metabolismo , Medula Espinal/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Encéfalo/citologia , Sequência Consenso , Humanos , Imunoglobulina G , Imuno-Histoquímica/métodos , Masculino , Dados de Sequência Molecular , Fibras Nervosas/metabolismo , Fibras Nervosas/ultraestrutura , Neurônios/citologia , Neurônios/metabolismo , Especificidade de Órgãos , Fragmentos de Peptídeos/farmacocinética , Ratos , Receptores da Neurocinina-3/química , Receptores da Neurocinina-3/imunologia , Medula Espinal/citologia , Substância P/análogos & derivados , Substância P/farmacocinéticaRESUMO
The majority of human spinal cord injuries involve gray matter loss from the cervical or lumbar enlargements. However, the deficits that arise from gray matter damage are largely masked by the severe deficits due to associated white matter damage. We have developed a model to examine gray matter-specific deficits and therapeutic strategies that uses intraspinal injections of the excitotoxin kainic acid into the T9 and L2 regions of the spinal cord. The resulting deficits have been compared to those from standard contusion injuries at the same levels. Injuries were assessed histologically and functional deficits were determined using the Basso, Beattie, and Bresnahan (BBB) 21-point open field locomotor scale and transcranial magnetic motor evoked potentials (tcMMEPs). Kainic acid injections into T9 resulted in substantial gray matter damage; however, BBB scores and tcMMEP response latencies were not different from those of controls. In contrast, kainic acid injections into L2 resulted in paraplegia with BBB scores similar to those following contusion injuries at either T9 or L2, without affecting tcMMEP response latencies. These observations demonstrate that gray matter loss can result in significant functional deficits, including paraplegia, in the absence of a disruption of major descending pathways.
Assuntos
Contusões/fisiopatologia , Agonistas de Aminoácidos Excitatórios , Ácido Caínico , Doenças da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Barreira Hematoencefálica , Contusões/patologia , Campos Eletromagnéticos , Potencial Evocado Motor , Locomoção , Masculino , Vias Neurais/fisiopatologia , Estimulação Física , Ratos , Ratos Sprague-Dawley , Medula Espinal/patologia , Doenças da Medula Espinal/induzido quimicamente , Doenças da Medula Espinal/patologia , Traumatismos da Medula Espinal/patologiaRESUMO
Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.
Assuntos
Produtos do Gene tat/farmacologia , HIV-1/metabolismo , Neurônios/efeitos dos fármacos , Animais , Cálcio/fisiologia , Estimulação Elétrica , Eletrofisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Estimulação Química , Tetrodotoxina/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Spinal cords from 2- to 8-day-old rats, maintained in vitro, were used to investigate the effects of discrete electrical stimuli applied to the ventrolateral funiculus (VLF) on motor neuron activity recorded from the lumbar ventral roots. Short trains of stimuli (1-3 s) delivered to one VLF in the low cervical region elicited rhythmic activity that persisted for up to 30 s. Responses consisted of short periods of activity (1-5 s) occurring simultaneously in the ipsilateral L5 and contralateral L3 ventral roots that alternated with similar bursts of activity in the ipsilateral L3 ventral root, a pattern consistent with locomotion. The rhythmicity of the ventral root responses to VLF stimulation was not affected by midsagittal sectioning of the preparation rostral to T10 and/or caudal to L4. Midsagittal sectioning of the lower thoracic or upper lumbar segments, however, disrupted the rhythmicity of the ventral root responses, leaving only long-duration simultaneous activation of the ipsilateral roots following VLF stimulus trains. The minimum lesion that effectively abolished the rhythmicity was one that divided only the L2 and L3 segments. In preparations rendered arrhythmic to VLF stimulation by an L2/L3 midsagittal lesion, rhythmicity could still be induced by N-methyl-D-aspartate (NMDA; 2-5 microM) and serotonin (5-HT; 20-50 microM), a drug combination commonly used to induce locomotor-like rhythmicity and air-stepping in vitro. Field potentials recorded following single stimuli delivered to the VLF revealed short-latency, large-amplitude responses in the ventral horn and intermediate gray both ipsilateral and contralateral to the stimulus site at T12 and L2. These observations suggest that 1) the discrete pathway under study may be an important descending locomotor command pathway and 2) this pathway has a strong bilateral projection in the lower thoracic and upper lumbar segments that is crucial for the initiation of VLF-induced rhythmic motor output. The induction of rhythmicity by NMDA/5-HT in an L2/L3-lesioned preparation suggests that these two rhythmogenic mechanisms may represent different levels within the circuitry that comprises the central pattern generator for locomotion. The rhythmic activity resulting from VLF stimulation is dependent on a bilateral projection that can be bypassed by the generalized excitation and subsequent rhythmicity that results from bath application of the NMDA/5-HT combination.
Assuntos
Animais Recém-Nascidos/fisiologia , Locomoção/fisiologia , Medula Espinal/fisiologia , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/fisiologia , Animais , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Locomoção/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologia , Medula Espinal/efeitos dos fármacosRESUMO
Differences in outcome and cost of laparoscopic and open surgery are continuously being evaluated. Two-year-old monozygotic twin boys with a previous history of prematurity, severe gastroesophageal reflux disease, and intractable reactive airway disease were each scheduled to undergo a laparoscopic Nissen fundoplication (LNF) on the same day. Current medications for both patients included albuterol, cromolyn sodium, dexamethasone, ranitidine, and metoclopramide. In the first case, the laparoscopic procedure was converted to an open Nissen fundoplication (ONF) to gain expeditious control of bleeding from a short gastric vessel close to the spleen. The second patient underwent LNF without complication. Operative time for each patient was 3.5 h. The postoperative length of stay for each patient was 6 days (ONF) and 4 days (LNF). The total hospital charges were $21,931 (ONF) and $19,108 (LNF). The first patient (ONF) was readmitted later on the day of discharge (postoperative day 6) for vomiting and was discharged after 24 h with no further treatment. The subsequent course of each patient was similar. At a 6-week follow-up visit, both patients were tolerating a regular diet with weight gain and dramatic improvement in pulmonary symptoms.
Assuntos
Doenças em Gêmeos , Fundoplicatura/métodos , Refluxo Gastroesofágico/cirurgia , Laparoscopia/normas , Gêmeos Monozigóticos , Pré-Escolar , Preços Hospitalares , Humanos , Laparoscopia/efeitos adversos , Laparoscopia/economia , Laparotomia/economia , Laparotomia/normas , Tempo de Internação , Masculino , Readmissão do Paciente , Fatores de Tempo , Resultado do TratamentoRESUMO
Tat is an 86- to 104-amino-acid viral protein that activates human immunodeficiency virus type 1 expression, modifies several cellular functions, and causes neurotoxicity. Here, we determined the extent to which peptide fragments of human immunodeficiency virus type 1 BRU Tat1-86 produced neurotoxicity, increased levels of intracellular calcium ([Ca2+]i), and affected neuronal excitability. Tat31-61 but not Tat48-85 dose dependently increased cytotoxicity and levels of [Ca2+]i in cultured human fetal brain cells. Similarly, Tat31-61 but not Tat48-85 depolarized rat hippocampal CA1 neurons in slices of rat brain. The neurotoxicity and increases in [Ca2+]i could be significantly inhibited by non-N-methyl-D-aspartate excitatory amino acid receptor antagonists. Shorter 15-mer peptides which overlapped by 10 amino acids each and which represented the entire sequence of Tat1-86 failed to produce any measurable neurotoxicity. Although it remains to be determined if Tat acts directly on neurons and/or indirectly via glial cells, these findings do suggest that Tat neurotoxicity is conformationally dependent, that the active site resides within the first exon of Tat between residues 31 to 61, and that these effects are mediated at least in part by excitatory amino acid receptors.
Assuntos
Produtos do Gene tat/fisiologia , HIV-1/patogenicidade , Neurônios/virologia , Animais , Cálcio/metabolismo , Morte Celular , Células Cultivadas , Epitopos/imunologia , Epitopos/fisiologia , Feminino , Produtos do Gene tat/imunologia , HIV-1/imunologia , Humanos , Potenciais da Membrana , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
In Alzheimer's disease (AD), there are marked deficits in the function of both the cholinergic and serotonergic systems. We and others have shown altered muscarinic M1 receptor/G-protein coupling in the superior frontal cortex (Brodmann areas 8 and 9) from AD patients. In the present study, we investigated whether similar receptor/G-protein alterations would occur at the 5-HT1A receptor in the same brain area. Using [3H]8-OH-DPAT as a radioligand, two binding sites with high and low affinity were found. The high affinity site (Kd = 1 nM), which is consistent with the classical 5-HT1A receptor, was not affected in AD either in the absence or presence of the non-hydrolyzable guanine nucleotide analog, GppNHp (100 microM). On the other hand, although there was no change in the affinity of [3H]8-OH-DPAT at the low affinity site (Kd = 8-10 nM), agonist binding was less sensitive to inhibition by GppNHp in AD (-29%) compared to age-matched controls (-51%). Based on these findings, alterations in receptor/G-protein coupling of both cholinergic and serotonergic receptors may be one explanation why neurotransmitter replacement drug therapies have been unsuccessful thus far in the treatment of AD.