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Neuroimage ; 192: 135-144, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30669007


The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics' potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed ∼40% larger LFP responses (p = 0.05) and twice as large cerebrovascular responses (p = 0.002) under focused vs. diffuse photostimulation (focused: 1.64 ±â€¯0.84 mV LFP amplitude and 75 ±â€¯48% increase in vRBC; diffuse: 1.14 ±â€¯0.75 mV LFP amplitude and 35 ±â€¯23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a ∼65% increase in the yield of cerebrovascular responses (73 ±â€¯10% for focused and 42 ±â€¯29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9 ±â€¯14.7 for focused and 10.4 ±â€¯1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.

Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Channelrhodopsins/metabolismo , Optogenética/métodos , Animais , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
Theranostics ; 8(17): 4824-4836, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30279740


Traumatic brain injury (TBI) research has focused on moderate to severe injuries as their outcomes are significantly worse than those of a mild TBI (mTBI). However, recent epidemiological evidence has indicated that a series of even mild TBIs greatly increases the risk of neurodegenerative and psychiatric disorders. Neuropathological studies of repeated TBI have identified changes in neuronal ionic concentrations, axonal injury, and cytoskeletal damage as important determinants of later life neurological and mood compromise; yet, there is a paucity of data on the contribution of neurogliovascular dysfunction to the progression of repeated TBI and alterations of brain function in the intervening period. Methods: Here, we established a mouse model of repeated TBI induced via three electromagnetically actuated impacts delivered to the intact skull at three-day intervals and determined the long-term deficits in neurogliovascular functioning in Thy1-ChR2 mice. Two weeks post the third impact, cerebral blood flow and cerebrovascular reactivity were measured with arterial spin labelling magnetic resonance imaging. Neuronal function was investigated through bilateral intracranial electrophysiological responses to optogenetic photostimulation. Vascular density of the site of impacts was measured with in vivo two photon fluorescence microscopy. Pathological analysis of neuronal survival and astrogliosis was performed via NeuN and GFAP immunofluorescence. Results: Cerebral blood flow and cerebrovascular reactivity were decreased by 50±16% and 70±20%, respectively, in the TBI cohort relative to sham-treated animals. Concomitantly, electrophysiological recordings revealed a 97±1% attenuation in peri-contusional neuronal reactivity relative to sham. Peri-contusional vascular volume was increased by 33±2% relative to sham-treated mice. Pathological analysis of the peri-contusional cortex demonstrated astrogliosis, but no changes in neuronal survival. Conclusion: This work provides the first in-situ characterization of the long-term deficits of the neurogliovascular unit following repeated TBI. The findings will help guide the development of diagnostic markers as well as therapeutics targeting neurogliovascular dysfunction.

Lesões Encefálicas Traumáticas/patologia , Transtornos Cerebrovasculares/patologia , Modelos Animais de Doenças , Neuroglia/patologia , Neurônios/patologia , Animais , Lesões Encefálicas Traumáticas/diagnóstico por imagem , Transtornos Cerebrovasculares/diagnóstico por imagem , Imagem por Ressonância Magnética , Camundongos , Microscopia de Fluorescência , Optogenética , Recidiva
Stroke ; 49(9): 2173-2181, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30354983


Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body ß-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Ácido 3-Hidroxibutírico/farmacologia , Astrócitos/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Encéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ácido 3-Hidroxibutírico/metabolismo , Acetoacetatos/metabolismo , Animais , Astrócitos/patologia , Glicemia/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Circulação Cerebrovascular , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Endotelina-1 , Hemodinâmica , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Microinjeções , Neurônios/patologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Córtex Sensório-Motor
J Magn Reson Imaging ; 46(2): 505-517, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28703413


PURPOSE: Stroke is the leading cause of adult disability worldwide. The absence of more effective interventions in the chronic stage-that most patients stand to benefit from-reflects uncertainty surrounding mechanisms that govern recovery. The present work investigated the effects of a novel treatment (selective cyclooxygenase-1, COX-1, inhibition) in a model of focal ischemia. MATERIALS AND METHODS: FR122047 (COX-1 inhibitor) was given beginning 7 days following stroke (cortical microinjection of endothelin-1) in 23 adult male rats. Longitudinal continuous-arterial-spin-labeling was performed prior to treatment (7 days), and repeated following treatment (21 days) on a 7T magnetic resonance imaging (MRI) system to estimate resting perfusion and reactivity to hypercapnia. These in vivo measurements were buttressed by immunohistochemistry. RESULTS: Stroke caused an increase in perilesional resting perfusion (peri-/contralesional perfusion ratio of 170 ± 10%) and perfusion responses to hypercapnia (180 ± 10%) at 7 days. At 21 days, placebo-administered rats showed normalized perilesional perfusion (100 ± 20%) but persistent hyperreactivity (190 ± 20%). Treated animals exhibited sustained perilesional hyperperfusion (180 ± 10%). Further, reactivity lateralization did not persist following treatment (peri- vs. contralesional reactivity: P = 0.002 at 7 vs. P = 0.2 at 21 days). Hemodynamic changes were accompanied by neuronal loss, increased endothelial density, and widespread microglial and astrocytic activation. Moreover, relative to controls, treated rats showed increased perilesional neuronal survival (22 ± 1% vs. 14.9 ± 0.8%, P = 0.02) and decreased microglia/macrophage recruitment (17 ± 1% vs. 20 ± 1%, P = 0.05). Finally, perilesional perfusion was correlated with neuronal survival (slope = 0.14 ± 0.05; R2 = 0.7, P = 0.03). CONCLUSION: These findings shed light on the role of COX-1 in chronic ischemic injury and suggest that delayed selective COX-1 inhibition exerts multiple beneficial effects on the neurogliovascular unit. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 4 J. MAGN. RESON. IMAGING 2017;46:505-517.

Inibidores de Ciclo-Oxigenase/farmacologia , Isquemia/diagnóstico por imagem , Imagem por Ressonância Magnética , Proteínas de Membrana/antagonistas & inibidores , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/fisiopatologia , Animais , Ciclo-Oxigenase 1 , Modelos Animais de Doenças , Endotelina-1/química , Macrófagos/patologia , Masculino , Microglia/patologia , Neuroglia/patologia , Neurônios/patologia , Perfusão , Piperazinas/química , Ratos , Ratos Sprague-Dawley , Marcadores de Spin , Tiazóis/química
Neuroimage ; 146: 869-882, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27664828


Brain plasticity following focal cerebral ischaemia has been observed in both stroke survivors and in preclinical models of stroke. Endogenous neurovascular adaptation is at present incompletely understood yet its potentiation may improve long-term functional outcome. We employed longitudinal MRI, intracranial array electrophysiology, Montoya Staircase testing, and immunofluorescence to examine function of brain vessels, neurons, and glia in addition to forelimb skilled reaching during the subacute stage of ischemic injury progression. Focal ischemic stroke (~100mm3 or ~20% of the total brain volume) was induced in adult Sprague-Dawley rats via direct injection of endothelin-1 (ET-1) into the right sensori-motor cortex, producing sustained impairment in left forelimb reaching ability. Resting perfusion and vascular reactivity to hypercapnia in the peri-lesional cortex were elevated by approximately 60% and 80% respectively seven days following stroke. At the same time, the normal topological pattern of local field potential (LFP) responses to peripheral somatosensory stimulation was abolished and the average power of spontaneous LFP activity attenuated by approximately 50% relative to the contra-lesional cortex, suggesting initial response attenuation within the peri-infarct zone. By 21 days after stroke, perilesional blood flow resolved, but peri-lesional vascular reactivity remained elevated. Concomitantly, the LFP response amplitudes increased with distance from the site of ET-1 injection, suggesting functional remodelling from the core of the lesion to its periphery. This notion was further buttressed by the lateralization of spontaneous neuronal activity: by day 21, the average ipsi-lesional power of spontaneous LFP activity was almost twice that of the contra-lesional cortex. Over the observation period, the peri-lesional cortex exhibited increased vascular density, along with neuronal loss, astrocytic activation, and recruitment and activation of microglia and macrophages, with neuronal loss and inflammation extending beyond the peri-lesional cortex. These findings highlight the complex relationship between neurophysiological state and behaviour and provide evidence of highly dynamic functional changes in the peri-infarct zone weeks following the ischemic insult, suggesting an extended temporal window for therapeutic interventions.

Isquemia Encefálica/fisiopatologia , Encéfalo/irrigação sanguínea , Encéfalo/fisiopatologia , Córtex Somatossensorial/irrigação sanguínea , Córtex Somatossensorial/fisiopatologia , Acidente Vascular Cerebral/fisiopatologia , Remodelação Vascular , Animais , Encéfalo/metabolismo , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/complicações , Ondas Encefálicas , Encefalite/complicações , Encefalite/metabolismo , Endotelina-1/administração & dosagem , Hipercapnia/fisiopatologia , Imagem por Ressonância Magnética , Masculino , Destreza Motora , Neuroglia/metabolismo , Neurônios/metabolismo , Estimulação Física , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Córtex Sensório-Motor/efeitos dos fármacos , Acidente Vascular Cerebral/induzido quimicamente , Acidente Vascular Cerebral/complicações , Percepção do Tato/fisiologia
Opt Express ; 19(25): 25057-65, 2011 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-22273897


A low-noise transducer based on a fiber Fabry-Perot (FFP) cavity was used as a pickup for an acoustic guitar. A distributed feedback (DFB) laser was locked to a 25 MHz-wide resonance of the FFP cavity using the Pound-Drever-Hall method. The correction signal was used as the audio output and was preamplified and sampled at up to 96 kHz. The pickup system is largely immune against optical noise sources, exhibits a flat frequency response from the infrasound region to about 25 kHz, and has a distortion-free audio output range of about 50 dB.

Acústica/instrumentação , Lasers , Música , Fibras Ópticas , Transdutores , Desenho de Equipamento , Análise de Falha de Equipamento