RESUMO
Imidazole-4-acetic acid-ribotide (IAARP) is a putative neurotransmitter/modulator and an endogenous regulator of sympathetic drive, notably systemic blood pressure, through binding to imidazoline receptors. IAARP is present in neurons and processes throughout the CNS, but is particularly prevalent in regions that are involved in blood pressure control. The goal of this study was to determine whether IAARP is present in neurons in the caudal vestibular nuclei that participate in the vestibulo-sympathetic reflex (VSR) pathway. This pathway is important in modulating blood pressure upon changes in head position with regard to gravity, as occurs when humans rise from a supine position and when quadrupeds climb or rear. Sinusoidal galvanic vestibular stimulation was used to activate the VSR and cfos gene expression in VSR pathway neurons of rats. These subjects had previously received a unilateral FluoroGold tracer injection in the rostral or caudal ventrolateral medullary region. The tracer was transported retrogradely and filled vestibular neuronal somata with direct projections to the injected region. Brainstem sections through the caudal vestibular nuclei were immunostained to visualize FluoroGold, cFos protein, IAARP and glutamate immunofluorescence. The results demonstrate that IAARP is present in vestibular neurons of the VSR pathway, where it often co-localizes with intense glutamate immunofluorescence. The co-localization of IAARP and intense glutamate immunofluorescence in VSR neurons may represent an efficient chemoanatomical configuration, allowing the vestibular system to rapidly up- and down-modulate the activity of presympathetic neurons in the ventrolateral medulla, thereby altering blood pressure.
Assuntos
Imidazóis/metabolismo , Bulbo/citologia , Neurônios/metabolismo , Ribosemonofosfatos/metabolismo , Sistema Nervoso Simpático/metabolismo , Núcleos Vestibulares/citologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Lateralidade Funcional , Ácido Glutâmico/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Long-Evans , Reflexo/fisiologia , Estilbamidinas/metabolismoRESUMO
The first 72 h after aneurysmal subarachnoid hemorrhage (SAH) is a critical period for the patient. Most of the deaths in the SAH patient population occur during this time, and a number of key events activate and trigger mechanisms that not only contribute to early brain injury but evolve over time and participate in the delayed complications. This review highlights the contribution of key events to the early brain injury and to overall outcome after SAH.
Assuntos
Química Encefálica/fisiologia , Isquemia Encefálica , Hiperemia , Hipertensão Intracraniana , Hemorragia Subaracnóidea , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Progressão da Doença , Humanos , Hiperemia/metabolismo , Hiperemia/patologia , Hiperemia/fisiopatologia , Hipertensão Intracraniana/metabolismo , Hipertensão Intracraniana/patologia , Hipertensão Intracraniana/fisiopatologia , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/patologia , Hemorragia Subaracnóidea/fisiopatologiaRESUMO
Most subarachnoid hemorrhage (SAH) patients exhibit clinical signs of cerebral ischemia at admission but no angiographic vasospasm. Consequently, the source of early cerebral ischemia is not understood. Parenchymal microvessels may contribute to early cerebral ischemia, but the low resolution of current imaging has prevented their analysis in SAH patients. Animal studies demonstrated that early after SAH structure and function of parenchymal vessels are compromised to the level that may very well contribute to early ischemia. We review these studies.
Assuntos
Córtex Cerebral/patologia , Microvasos/fisiopatologia , Hemorragia Subaracnóidea/patologia , Animais , Modelos Animais de Doenças , Encefalite/etiologia , Encefalite/patologia , Humanos , Microvasos/ultraestrutura , Doenças do Sistema Nervoso/etiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Hemorragia Subaracnóidea/complicaçõesRESUMO
Deletion of the human SHANK3 gene near the terminus of chromosome 22q is associated with Phelan-McDermid syndrome and autism spectrum disorders. Nearly all such deletions also span the tightly linked IB2 gene. We show here that IB2 protein is broadly expressed in the brain and is highly enriched within postsynaptic densities. Experimental disruption of the IB2 gene in mice reduces AMPA and enhances NMDA receptor-mediated glutamatergic transmission in cerebellum, changes the morphology of Purkinje cell dendritic arbors, and induces motor and cognitive deficits suggesting an autism phenotype. These findings support a role for human IB2 mutation as a contributing genetic factor in Chr22qter-associated cognitive disorders.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doenças Cerebelares/genética , Transtornos Globais do Desenvolvimento Infantil/genética , Transtornos Globais do Desenvolvimento Infantil/fisiopatologia , Transmissão Sináptica/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Doenças Cerebelares/metabolismo , Doenças Cerebelares/fisiopatologia , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Cromossomos Humanos Par 22/genética , Transtornos Cognitivos/genética , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença/genética , Humanos , Recém-Nascido , Masculino , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Transtornos Mentais/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
BACKGROUND: Subarachnoid haemorrhage (SAH) elicits rapid pathological changes in the structure and function of parenchymal vessels (≤ 100 µm). The role of neutrophils in these changes has not been determined. This study investigates the role of neutrophils in early microvascular changes after SAH METHOD: Rats were either untreated, treated with vinblastine or anti-polymorphonuclear (PMN) serum, which depletes neutrophils, or treated with pyrrolidine dithiocarbamate (PDTC), which limits neutrophil activity. SAH was induced by endovascular perforation. Neutrophil infiltration and the integrity of vascular endothelium and basement membrane were assessed immunohistochemically. Vascular collagenase activity was assessed by in situ zymography. RESULTS: Vinblastine and anti-PMN serum reduced post-SAH accumulation of neutrophils in cerebral vessels and in brain parenchyma. PDTC increased the neutrophil accumulation in cerebral vessels and decreased accumulation in brain parenchyma. In addition, each of the three agents decreased vascular collagenase activity and post-SAH loss of vascular endothelial and basement membrane immunostaining. CONCLUSIONS: Our results implicate neutrophils in early microvascular injury after SAH and indicate that treatments which reduce neutrophil activity can be beneficial in limiting microvascular injury and increasing survival after SAH.
Assuntos
Microcirculação , Ativação de Neutrófilo , Neutrófilos/fisiologia , Hemorragia Subaracnóidea/patologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Biomarcadores/metabolismo , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Permeabilidade Capilar , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Humanos , Masculino , Infiltração de Neutrófilos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Prolina/análogos & derivados , Prolina/metabolismo , Prolina/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/fisiopatologia , Tiocarbamatos/metabolismo , Tiocarbamatos/farmacologia , Moduladores de Tubulina/farmacologia , Vimblastina/farmacologiaRESUMO
During the last decade much effort has been invested in understanding the events that occur early after SAH. It is now widely accepted that these early events not only participate in the early ischemic injury but also set the stage for the pathogenesis of delayed vasospasm. That early cerebral ischemia occurs after SAH is documented in both experimental SAH and in human autopsy studies; however, angiographic evidence for vasoconstriction early after SAH is lacking and the source of early ischemic injury is therefore unclear. Recently, the cerebral microvasculature has been identified as an early target of SAH. Changes in the anatomical structure of cerebral microvessels, sufficient to cause functional deficits, are found early after experimental SAH. These changes may explain cerebral ischemia in human in the absence of angiographic evidence of large vessel vasoconstriction. This paper summarizes known alterations in cerebral microvasculature during the first 48 h after SAH.
Assuntos
Vasos Sanguíneos/fisiopatologia , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/patologia , Animais , Membrana Basal/fisiopatologia , Plaquetas/metabolismo , Endotélio/fisiopatologia , HumanosRESUMO
gamma-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify gamma-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for gamma-H2AX focus formation by immunofluorescence analysis. The gamma-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and gamma-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r(2) correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when gamma-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of gamma-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify gamma-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.
Assuntos
Histonas/metabolismo , Imuno-Histoquímica/métodos , Automação , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Estudos de Viabilidade , Humanos , Microscopia , Fosforilação , Doses de RadiaçãoRESUMO
The Cre-loxP system is widely used for making conditional alterations to the mouse genome. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Given the distinct advantages of fluorescent reporters, we developed a transgenic reporter line, termed IRG, in which DsRed-Express, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and an enhanced green fluorescent protein (EGFP) following recombination. Besides their utility for monitoring Cre-mediated recombination, we show that in IRG mice red and green native fluorescence can be imaged simultaneously in thick tissue sections by confocal microscopy allowing for complex reconstructions to be created that are suitable for analysis of neuronal morphologies as well as neurovascular interactions in brain. IRG mice should provide a versatile tool for analyzing complex cellular relationships in both neural and nonneural tissues.
Assuntos
Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Recombinação Genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Cruzamentos Genéticos , Diagnóstico por Imagem , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Genes Reporter , Marcadores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Integrases/genética , Proteínas de Filamentos Intermediários/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Nestina , Regiões Promotoras Genéticas , Distribuição Tecidual , Transgenes , Proteína Vermelha FluorescenteRESUMO
A substantial body of research has led to the recognition that the vestibular system participates in blood pressure modulation during active movements and changes in posture, and that this modulation is effected at least partly by the caudal vestibular nuclei. The I-4 isomer of imidazoleacetic acid-ribotide (IAA-RP) is a putative neurotransmitter/modulator that is thought to be an endogenous regulator of general sympathetic drive, particularly systemic blood pressure. The present study employed immunofluorescence and light and electron microscopic immunocytochemistry to visualize IAA-RP in the vestibular nuclei of adult male rats. The results demonstrate IAA-RP immunolabeling of subpopulations of vestibular neurons in the descending nucleus and the caudal half of the medial nucleus, with scattered immunostained vestibular neurons also present more rostrally. On the basis of double immunofluorescence staining for IAA-RP and calbindin, many of these ribotide-immunoreactive neurons appear to be innervated by cerebellar Purkinje cell afferents. Ultrastructural observations in the caudal vestibular nuclei confirm the IAA-RP immunolocalization in cell bodies and dendritic processes, and in some myelinated axons and presynaptic boutons. The regional distribution of IAA-RP immunoreactivity corresponds to the location of vestibular neurons involved in autonomic functions. The presence of IAA-RP in those neurons suggests that they participate specifically in vestibulo-autonomic regulation of blood pressure. The localization of immunostain in processes and terminals suggests that vestibulo-autonomic activity is subject to local feedback control. Overall, the observations offer a chemoanatomic basis for understanding the vestibular side effects commonly experienced by patients treated with clonidine and other imidazoline-related drugs.
Assuntos
Neurônios/metabolismo , Ribosemonofosfatos/fisiologia , Núcleos Vestibulares/citologia , Animais , Calbindinas , Citrulina/metabolismo , Imidazóis , Imuno-Histoquímica/métodos , Masculino , Microscopia Imunoeletrônica/métodos , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Imidazoleacetic acid-ribotide (IAA-RP) is a putative neurotransmitter/modulator recently discovered in mammalian brain. The present study examines the distribution of IAA-RP in the rat CNS using a highly specific antiserum raised in rabbit against IAA-RP with immunostaining of aldehyde-fixed rat CNS. IAA-RP-immunoreactive neurons were present throughout the neuraxis; neuroglia were not labeled. In each region, only a subset of the neuronal pool was immunostained. In the forebrain, ribotide-immunolabeled neurons were common in neocortex, in hippocampal formation, and in subcortical structures including basal ganglia, thalamus and hypothalamus. Labeling was prominent in limbic areas including olfactory bulb, basal forebrain, pyriform cortex and amygdala. In the mid- and hindbrain, immunolabeled neurons were concentrated in specific nuclei and, in some areas, in specific subregions of those nuclei. Structures of the motor system, including cranial nerve motor nuclei, precerebellar nuclei, the substantia nigra, and the red nucleus were clearly labeled. Staining was intense in cells and/or puncta in the rostral and caudal ventrolateral medullary reticular formation, nucleus tractus solitarius and the caudal vestibular nuclear complex. Within neurons, the ribotide was found predominantly in somata and dendrites; some myelinated axons and occasional synaptic terminals were also immunostained. These data indicate that IAA-RP contributes to the neurochemical phenotype of many neuronal populations and further supports our suggestion that, in autonomic structures, IAA-RP may serve as a chemical mediator in complex circuits involved in blood pressure regulation and, more generally, sympathetic drive.
Assuntos
Química Encefálica/fisiologia , Imidazóis/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores de Droga/metabolismo , Ribosemonofosfatos/metabolismo , Animais , Tronco Encefálico/metabolismo , Cerebelo/metabolismo , Ensaio de Imunoadsorção Enzimática , Hipocampo/metabolismo , Imidazóis/imunologia , Receptores de Imidazolinas , Imuno-Histoquímica , Prosencéfalo/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Ribosemonofosfatos/imunologia , Núcleos do Trigêmeo/metabolismo , Núcleos Vestibulares/metabolismoRESUMO
OBJECT: Structural changes in brain parenchymal vessels occur within minutes after subarachnoid hemorrhage (SAH). These changes include platelet aggregation, activation of vascular collagenases, and destruction of perivascular collagen IV. Because collagen IV is an important component of the basal lamina, the authors attempted to further define changes in vascular structure (length and luminal diameter) and their relationship to vascular permeability immediately after SAH. In addition, the authors explored whether such alterations were attenuated by administration of a nitric oxide (NO) donor. METHODS: Endovascular perforation was used to induce SAH in rats. Two sets of experiments were performed. The first established changes in vascular structure and permeability (collagen IV and endothelial barrier antigen [EBA] dual immunofluorescence) during the first 24 hours after SAH. In the second, the investigators examined the effects of an NO donor on vascular structure, permeability, and collagenase activity (in situ zymography). In this second study, animals received intravenous infusion of the NO donor S-nitrosoglutathione (GSNO, 1 microM/8 microl/min) 15 minutes after induction of SAH and were killed 3 hours after SAH onset. Controls were naive unoperated animals for the first study and saline-infused SAH animals for the second. The authors found a time-dependent decrease in area fraction, length, and luminal diameter of collagen IV- and EBA-immunofluorescent vessels after SAH. The greatest change occurred at 3 hours after onset of SAH. Administration of GSNO was associated with striking preservation of collagen IV and EBA immunofluorescence compared with saline treatment. Zymography indicated decreased collagenase activity in GSNO-treated SAH animals compared with saline-treated SAH animals. CONCLUSIONS: These results demonstrate changes in the structure and permeability of brain parenchymal microvessels after SAH and their reversal by treatment with an NO donor.
Assuntos
Encéfalo/irrigação sanguínea , Doadores de Óxido Nítrico/uso terapêutico , S-Nitrosoglutationa/uso terapêutico , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/prevenção & controle , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Permeabilidade Capilar/fisiologia , Masculino , Microcirculação/patologia , Microcirculação/fisiopatologia , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/patologia , Fatores de TempoRESUMO
The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first demonstration of two disparate chemoanatomic VSR pathways.
RESUMO
OBJECT: The mechanisms underlying acute cerebral ischemia after subarachnoid hemorrhage (SAH) are not well established. Platelets aggregate within major cerebral vessels hours after SAH, but this has not been studied in the microvasculature. Platelet aggregates within the microvasculature could mechanically obstruct the lumen and initiate events that injure vessel structure. In the present study the authors examined the hypothesis that platelets aggregate within the cerebral microvasculature acutely after SAH. METHODS: Subarachnoid hemorrhage was induced in the rat by using the endovascular perforation model. The animals were killed between 10 minutes and 48 hours after SAH. Immunostaining for the platelet surface receptor glycoprotein (GP)IIb/IIIa, which mediates platelet aggregation, was used to detect platelet aggregation. Sham-operated animals were used as controls. The GPIIb/IIIa immunoreactive platelet aggregates were abundant in the microvasculature of the basal and frontal cortex, striatum, and hippocampus 10 minutes after SAH. These aggregates decreased in number from 1 to 6 hours post-SAH and then increased to a peak at 24 hours. No immunoreactive aggregates were observed 48 hours after SAH. CONCLUSIONS: The data indicate that widespread platelet aggregation occurs very rapidly in response to SAH followed by a decrease within 6 hours and a subsequent increase 24 hours after SAH. Microvascular platelet aggregates may contribute to decreased cerebral blood flow and ischemic injury after SAH via a number of mechanisms.
Assuntos
Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular , Agregação Plaquetária , Hemorragia Subaracnóidea/fisiopatologia , Doença Aguda , Animais , Plaquetas/metabolismo , Pressão Sanguínea , Isquemia Encefálica/sangue , Pressão Intracraniana , Masculino , Microcirculação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/sangueRESUMO
BACKGROUND AND PURPOSE: Inflammation and compromise in structure and function of cerebral parenchymal microvasculature begins early after subarachnoid hemorrhage (SAH). We recently found greater inflammation and greater vascular compromise in male than in female rats following SAH. In this study, we investigated whether this cross-sexual difference in pathology is reflected in expression levels of genes related to vascular inflammation and structural compromise. METHOD: Age-matched male and female rats underwent sham surgery or SAH by endovascular perforation. Early physiology (intracranial pressure (ICP), blood pressure (BP), heart rate, and cerebral blood flow) was monitored. Cerebral RNA was extracted at sacrifice 3 h after surgery and assayed for expression of thrombomodulin (Thbd), endothelial nitric oxide synthase (eNos;Nos3), intracellular adhesion molecule-1 (Icam1), vascular endothelial growth factor (Vegf), interleukin-1beta (Il1ß) tumor necrosis factor-alpha (Tnf-α), and arginine vasopressin (Avp). RESULTS: Increases in ICP and BP at SAH appeared slightly greater in males but the difference did not reach statistical difference, indicating that SAH intensity did not differ significantly between the sexes. Of the seven genes studied two; Tnf-α and Vegf, did not change after injury, while the remainder showed significant responses to SAH. Response of Nos3 and Thbd was markedly different between the sexes, with expression greater in males. CONCLUSION: This study finds that sexual dimorphism is present in the response of some but not all genes to SAH. Since products of genes exhibiting sexual dimorphism have anti-inflammatory activities, our results indicate that previously found sex-based differences in vascular pathology are paralleled by sexually dimorphic changes in gene expression following SAH.
Assuntos
Regulação da Expressão Gênica/fisiologia , Caracteres Sexuais , Hemorragia Subaracnóidea , Animais , Arginina Vasopressina/genética , Arginina Vasopressina/metabolismo , Pressão Sanguínea/fisiologia , Circulação Cerebrovascular , Modelos Animais de Doenças , Feminino , Frequência Cardíaca/fisiologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pressão Intracraniana , Masculino , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/genética , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/fisiopatologia , Trombomodulina/genética , Trombomodulina/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Brain imaging, molecular genetic, and ultrastructural evidence indicate the existence of pathologic alterations in the cortical and subcortical white matter of schizophrenic patients. METHODS: We performed a stereologic analysis of numbers, densities, and spatial distribution of oligodendrocytes in layer III and in the gyral white matter of Brodmann's area 9 in the superior frontal gyrus to assess whether these cells are affected in schizophrenia. Counts were obtained on Nissl-stained materials and on sections immunolabeled for the oligodendrocyte marker 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) in seven schizophrenic and seven age-matched control cases. RESULTS: A 28% decrease in total numbers (or densities) of cortical layer III oligodendrocytes and a 27% decrease in the white matter were detected in schizophrenic compared with control cases based on CNPase immunostaining. Nissl and CNPase immunohistochemistry yielded comparable results. The spatial distribution of oligodendrocytes in area 9 white matter exhibited a less clustered arrangement in schizophrenic cases. CONCLUSIONS: These results suggest a severe pathology of oligodendrocytes in schizophrenia and provide a quantitative cellular correlate of the white matter changes observed by brain imaging in vivo, showing reduced fractional anisotropy in schizophrenia. The data support recent evidence that several genes encoding myelin-related proteins consistently exhibit reduced expression in schizophrenia.
Assuntos
Lobo Frontal/patologia , Oligodendroglia/patologia , Esquizofrenia/patologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/análise , Autopsia , Estudos de Casos e Controles , Contagem de Células , Humanos , Imuno-Histoquímica , Oligodendroglia/enzimologiaRESUMO
Although gamma-aminobutyric acid (GABA) and glutamate are known to be present in the vestibular sensory epithelia of a variety of species, the functional relationship between these two transmitters is not clear. The present study addresses the three-dimensional spatial distribution of GABA and glutamate immunoreactivity in the vestibular labyrinth of the oyster toadfish by using whole end organs labeled by immunofluorescence with monoclonal anti-GABA and/or antiglutamate antibodies and visualized as whole mounts by multiphoton confocal microscopy. We find glutamate-immunoreactive hair cells present throughout the sensory epithelium. In contrast, prominent GABA immunoreactivity is restricted to a small population of hair cells located in the central region of the crista. Double immunofluorescence reveals two distinct staining patterns in GABA-labeled hair cells. Most ( approximately 80%) GABA-labeled cells show trace levels of glutamate, appropriate for the metabolic/synthetic role of cytoplasmic glutamate. The remainder of the GABA-stained cells contain substantial levels of both GABA and glutamate, suggesting transmitter colocalization. In the toadfish utricle, glutamatergic hair cells are present throughout the macula. GABA-immunoreactive hair cells follow the arc of the striola, and most GABA-labeled receptor cells coexpress glutamate. The localization of GABA was explored in other species as well. In the pigeon, GABAergic hair cells are present throughout the crista ampullaris. Our findings demonstrate that multiple, neurochemically distinct types of hair cells are present in vestibular sensory epithelia. These observations, together with the excitatory activity generally associated with 8th nerve afferent fibers, strongly suggest that GABA serves an important, specific, and complex role in determining primary afferent response dynamics.
Assuntos
Batracoidiformes/metabolismo , Células Ciliadas Vestibulares/metabolismo , Vestíbulo do Labirinto/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Columbidae/metabolismo , Imunofluorescência , Ácido Glutâmico/metabolismo , Células Ciliadas Vestibulares/citologia , Neurônios Aferentes/metabolismo , Especificidade da Espécie , Distribuição Tecidual , Vestíbulo do Labirinto/citologiaRESUMO
The functions of the presenilin-1 (PS-1) protein remain largely unknown. In adult brain PS-1 is expressed principally in neurons. However during development PS-1 is expressed more widely including in embryonic neural progenitors. To determine if PS-1 is expressed in neural progenitors in adult hippocampus we used bromodeoxyuridine (BrdU) labeling combined with immunostaining for BrdU, PS-1 and markers of neuronal or glial differentiation. Most BrdU labeled cells also expressed PS-1 at a time when few BrdU labeled cells expressed the early neuronal markers beta-III tubulin or TOAD-64 and none expressed mature neuronal (NeuN or calbindin) or astrocytic (GFAP) markers. Cells expressing PS-1 and the neural progenitor marker nestin were also found. Thus PS-1 is expressed in neural progenitor cells in adult hippocampus implying its possible role in neurogenesis in adult brain.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Células-Tronco/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Bromodesoxiuridina , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/citologia , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nestina , Neurônios/citologia , Presenilina-1 , Células-Tronco/citologia , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: Subarachnoid hemorrhage (SAH) is associated with acute decreases and subsequent recovery of cerebral nitric oxide (NO) levels, but the mechanisms of these alterations are not known. In this study, we measured NO synthase (NOS) protein and kinetics to determine its involvement in the alterations of cerebral NO levels after SAH. METHODS: The endovascular rat model of SAH was used. The number of NOS-1 (neuronal) and NOS-2 (inducible)-positive cells (0-96 h) was determined by counting immunoreactive cells in 8-microm cryostat sections. The tissue content of active NOS and its kinetic parameters were studied with an enzymatic l-citrulline assay. RESULTS: The number of NOS-1-positive cells increased between 1 and 3 hours after SAH, decreased to and below control values at 6 and 72 hours after SAH, and increased to control values 96 hours after SAH. The number of NOS-2-positive cells increased 1 hour after SAH, decreased to control values at 24 hours, and increased above control values 96 hours after SAH. The Michaelis-Menten kinetic parameters (V(max), K(m), slope) of NOS remained unchanged at 10 and 90 minutes after SAH. CONCLUSION: NOS-1 and -2 proteins undergo a triphasic alteration after SAH, whereas the amount of active NOS and its kinetic parameters remain unchanged during the first 90 minutes after SAH. Depletion of NOS is not involved in the acute alterations of cerebral NO levels after SAH.
Assuntos
Encéfalo/enzimologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/metabolismo , Hemorragia Subaracnóidea/enzimologia , Animais , Encéfalo/patologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Contagem de Células , Técnicas Imunoenzimáticas , Cinética , Masculino , Neurônios/enzimologia , Neurônios/patologia , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Sprague-Dawley , Valores de Referência , Hemorragia Subaracnóidea/patologiaRESUMO
OBJECT: Aneurysmal subarachnoid hemorrhage (SAH) causes acute and delayed ischemic brain injuries. The mechanisms of acute ischemic injury following SAH are poorly understood, although an acute increase in microvascular permeability has been noted. The integrity of cerebral microvessels is maintained in part by components of basal lamina: collagen IV, elastin, lamina, and so forth. Destruction of basal lamina components by collagenases and matrix metalloproteinases (MMPs), especially MMP-9, has been known to occur in other ischemic models. The authors assessed the integrity of cerebral microvasculature after acute SAH by examining collagen IV and MMP-9 levels and collagenase activity in the microvessels. METHODS: Subarachnoid hemorrhage was induced in rats through endovascular perforation of the intracranial bifurcation of the internal carotid artery. Animals were killed 10 minutes to 48 hours after SAH or sham operation (time-matched controls). Levels of collagen IV and MMP-9 were studied in the microvasculature by performing immunoperoxidase and immunofluorescence staining, and collagenase activity was assessed by in situ zymography. Little change occurred in collagen IV and MMP-9 immunostaining or collagenase activity at 10 minutes or 1 hour after SAH. Starting 3 hours after SAH, collagen IV immunostaining was reduced or eliminated along segments of microvessels whereas MMP-9 staining was segmentally increased. These effects reached a maximum at 6 hours and returned toward those values in sham-operated controls at 48 hours. CONCLUSIONS: Results of this study demonstrated an acute loss of collagen IV from the cerebral microvasculature after SAH and indicated that MMP-9 contributes to this event. The loss of collagen IV might contribute to the known failure of the blood-brain barrier after SAH.
Assuntos
Membrana Basal/irrigação sanguínea , Membrana Basal/patologia , Isquemia Encefálica/etiologia , Aneurisma Intracraniano/complicações , Hemorragia Subaracnóidea/complicações , Doença Aguda , Animais , Colágeno Tipo IV/análise , Modelos Animais de Doenças , Masculino , Metaloproteinase 9 da Matriz/análise , Microcirculação , Ratos , Ratos Sprague-DawleyRESUMO
Changes in head position and posture are detected by the vestibular system and are normally followed by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by integration of vestibular system pathways with blood pressure control centers in the ventrolateral medulla. Orthostatic hypotension can reflect altered activity of this neural circuitry. Vestibular sensory input to the vestibulo-sympathetic pathway terminates on cells in the vestibular nuclear complex, which in turn project to brainstem sites involved in the regulation of cardiovascular activity, including the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). In the present study, sinusoidal galvanic vestibular stimulation was used to activate this pathway, and activated neurons were identified through detection of c-Fos protein. The retrograde tracer Fluoro-Gold was injected into the RVLM or CVLM of these animals, and immunofluorescence studies of vestibular neurons were conducted to visualize c-Fos protein and Fluoro-Gold concomitantly. We observed activated projection neurons of the vestibulo-sympathetic reflex pathway in the caudal half of the spinal, medial, and parvocellular medial vestibular nuclei. Approximately two-thirds of the cells were ipsilateral to Fluoro-Gold injection sites in both the RVLM and CVLM, and the remainder were contralateral. As a group, cells projecting to the RVLM were located slightly rostral to those with terminals in the CVLM. Individual activated projection neurons were multipolar, globular, or fusiform in shape. This study provides the first direct demonstration of the central vestibular neurons that mediate the vestibulo-sympathetic reflex.