RESUMO
Brain function is substantially linked to the highly organized modular structure of neuronal networks. However, the structure of in vitro assembled neuronal circuits often exhibits variability, complicating the consistent recording of network functional output and its correlation to network structure. Therefore, engineering neuronal structures with predefined geometry and reproducible functional features is essential to precisely model in vivo neuronal circuits. Here, we engineered microchannel devices to assemble 2D and 3D modular networks. The microchannel devices were coupled with a multi-electrode array (MEA) electrophysiology system to enable recordings from circuits. Each network consisted of 64 modules connected to their adjacent modules by micron-sized channels. Modular circuits within microchannel devices showed enhanced activity and functional connectivity traits. This includes metrics such as connection weights, clustering coefficient, global efficiency, and the number of hub neurons with higher betweenness centrality. In addition, modular networks demonstrated an increased functional modularity score compared to the randomly formed circuits. Neurons within individual modules displayed uniform network characteristics and predominantly participated in their respective functional communities within the same or neighboring physical modules. These observations highlight that the modular network structure promotes the development of segregated functional connectivity traits while simultaneously enhancing the efficiency of overall network connectivity. Our findings emphasize the significant impact of physical constraints on the activity patterns and functional organization within engineered modular networks. These circuits, characterized by stable modular architecture and intricate functional dynamics-key features of the brain networks-offer a robust in vitro model for advancing neuroscience research.
Assuntos
Técnicas Biossensoriais , Dispositivos Lab-On-A-Chip , Rede Nervosa , Neurônios , Neurônios/fisiologia , Rede Nervosa/fisiologia , Animais , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Células Cultivadas , Encéfalo/fisiologiaRESUMO
During spaceflight, humans experience a variety of physiological changes due to deviations from familiar earth conditions. Specifically, the lack of gravity is responsible for many effects observed in returning astronauts. These impairments can include structural as well as functional changes of the brain and a decline in cognitive performance. However, the underlying physiological mechanisms remain elusive. Alterations in neuronal activity play a central role in mental disorders and altered neuronal transmission may also lead to diminished human performance in space. Thus, understanding the influence of altered gravity at the cellular and network level is of high importance. Previous electrophysiological experiments using patch clamp techniques and calcium indicators have shown that neuronal activity is influenced by altered gravity. By using multi-electrode array (MEA) technology, we advanced the electrophysiological investigation covering single-cell to network level responses during exposure to decreased (micro-) or increased (hyper-) gravity conditions. We continuously recorded in real-time the spontaneous activity of human induced pluripotent stem cell (hiPSC)-derived neural networks in vitro. The MEA device was integrated into a custom-built environmental chamber to expose the system with neuronal cultures to up to 6 g of hypergravity on the Short-Arm Human Centrifuge at the DLR Cologne, Germany. The flexibility of the experimental hardware set-up facilitated additional MEA electrophysiology experiments under 4.7 s of high-quality microgravity (10-6 to 10-5 g) in the Bremen drop tower, Germany. Hypergravity led to significant changes in activity. During the microgravity phase, the mean action potential frequency across the neural networks was significantly enhanced, whereas different subgroups of neurons showed distinct behaviors, such as increased or decreased firing activity. Our data clearly demonstrate that gravity as an environmental stimulus triggers changes in neuronal activity. Neuronal networks especially reacted to acute changes in mechanical loading (hypergravity) or de-loading (microgravity). The current study clearly shows the gravity-dependent response of neuronal networks endorsing the importance of further investigations of neuronal activity and its adaptive responses to micro- and hypergravity. Our approach provided the basis for the identification of responsible mechanisms and the development of countermeasures with potential implications on manned space missions.
RESUMO
Advances in primary and stem cell derived neuronal cell culture techniques and abundance of available neuronal cell types have enabledin vitroneuroscience as a substantial approach to modelin vivoneuronal networks. Survival of the cultured neurons is inevitably dependent on the cell culture incubators to provide stable temperature and humidity and to supply required CO2levels for controlling the pH of culture medium. Therefore, imaging and electrophysiology recordings outside of the incubator are often limited to the short-term experimental sessions. This restricts our understanding of physiological events to the short snapshots of recorded data while the major part of temporal data is neglected. Multiple custom-made and commercially available platforms like integrated on-stage incubators have been designed to enable long-term microscopy. Nevertheless, long-term high-spatiotemporal electrophysiology recordings from developing neuronal networks needs to be addressed. In the present work an incubator-independent polydimethylsiloxane-based double-wall perfusion chamber was designed and integrated with multi-electrode arrays (MEAs) electrophysiology and compartmentalized microfluidic device to continuously record from engineered neuronal networks at sub-cellular resolution. Cell culture media underwent iterations of conditioning to the ambient CO2and adjusting its pH to physiological ranges to retain a stable pH for weeks outside of the incubator. Double-wall perfusion chamber and an integrated air bubble trapper reduced media evaporation and osmolality drifts of the conditioned media for two weeks. Aligned microchannel-microfluidic device on MEA electrodes allowed neurite growth on top of the planar electrodes and amplified their extracellular activity. This enabled continuous sub-cellular resolution imaging and electrophysiology recordings from developing networks and their growing neurites. The on-chip versatile and self-contained system provides long-term, continuous and high spatiotemporal access to the network data and offers a robustin vitroplatform with many potentials to be applied on advanced cell culture systems including organ-on-chip and organoid models.
Assuntos
Incubadoras , Microscopia , Perfusão , Microeletrodos , Eletrofisiologia , Dispositivos Lab-On-A-ChipRESUMO
Comprehensive electrophysiological characterizations of human induced pluripotent stem cell (hiPSC)-derived neuronal networks are essential to determine to what extent these in vitro models recapitulate the functional features of in vivo neuronal circuits. High-density micro-electrode arrays (HD-MEAs) offer non-invasive recording with the best spatial and temporal resolution possible to date. For 3 months, we tracked the morphology and activity features of developing networks derived from a transgenic hiPSC line in which neurogenesis is inducible by neurogenic transcription factor overexpression. Our morphological data revealed large-scale structural changes from homogeneously distributed neurons in the first month to the formation of neuronal clusters over time. This led to a constant shift in position of neuronal cells and clusters on HD-MEAs and corresponding changes in spatial distribution of the network activity maps. Network activity appeared as scarce action potentials (APs), evolved as local bursts with longer duration and changed to network-wide synchronized bursts with higher frequencies but shorter duration over time, resembling the emerging burst features found in the developing human brain. Instantaneous firing rate data indicated that the fraction of fast spiking neurons (150-600 Hz) increases sharply after 63 days post induction (dpi). Inhibition of glutamatergic synapses erased burst features from network activity profiles and confirmed the presence of mature excitatory neurotransmission. The application of GABAergic receptor antagonists profoundly changed the bursting profile of the network at 120 dpi. This indicated a GABAergic switch from excitatory to inhibitory neurotransmission during circuit development and maturation. Our results suggested that an emerging GABAergic system at older culture ages is involved in regulating spontaneous network bursts. In conclusion, our data showed that long-term and continuous microscopy and electrophysiology readouts are crucial for a meaningful characterization of morphological and functional maturation in stem cell-derived human networks. Most importantly, assessing the level and duration of functional maturation is key to subject these human neuronal circuits on HD-MEAs for basic and biomedical applications.
RESUMO
The widespread adoption of microfluidic devices among the neuroscience and neurobiology communities has enabled addressing a broad range of questions at the molecular, cellular, circuit, and system levels. Here, we review biomedical engineering approaches that harness the power of microfluidics for bottom-up generation of neuronal cell types and for the assembly and analysis of neural circuits. Microfluidics-based approaches are instrumental to generate the knowledge necessary for the derivation of diverse neuronal cell types from human pluripotent stem cells, as they enable the isolation and subsequent examination of individual neurons of interest. Moreover, microfluidic devices allow to engineer neural circuits with specific orientations and directionality by providing control over neuronal cell polarity and permitting the isolation of axons in individual microchannels. Similarly, the use of microfluidic chips enables the construction not only of 2D but also of 3D brain, retinal, and peripheral nervous system model circuits. Such brain-on-a-chip and organoid-on-a-chip technologies are promising platforms for studying these organs as they closely recapitulate some aspects of in vivo biological processes. Microfluidic 3D neuronal models, together with 2D in vitro systems, are widely used in many applications ranging from drug development and toxicology studies to neurological disease modeling and personalized medicine. Altogether, microfluidics provide researchers with powerful systems that complement and partially replace animal models.
Assuntos
Microfluídica , Engenharia Tecidual , Animais , Encéfalo , Humanos , Dispositivos Lab-On-A-Chip , NeurôniosRESUMO
Spontaneous and optogenetically evoked activities of human induced pluripotent stem cell (hiPSC)-derived neurons can be assessed by patch clamp and multi-electrode array (MEA) electrophysiology. Optogenetic activation of these human neurons facilitates the characterization of their functional properties at the single neuron and circuit level. Here we showcase the preparation of hiPSC-derived neurons expressing optogenetic actuators, in vitro optogenetic stimulation and simultaneous functional recordings using patch clamp and MEA electrophysiology.
Assuntos
Células-Tronco Pluripotentes Induzidas , Optogenética , Potenciais de Ação/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Humanos , NeurôniosRESUMO
Neuronal networks derived from human induced pluripotent stem cells have been exploited widely for modeling neuronal circuits, neurological diseases, and drug screening. As these networks require extended culturing periods to functionally mature in vitro, most studies are based on immature networks. To obtain insights on long-term functional features, we improved a glia-neuron co-culture protocol within multi-electrode arrays, facilitating continuous assessment of electrical features in weekly intervals. By full-field optogenetic stimulation, we detected an earlier onset of neuronal firing and burst activity compared with spontaneous activity. Full-field stimulation enhanced the number of active neurons and their firing rates. Compared with full-field stimulation, which evoked synchronized activity across all neurons, holographic stimulation of individual neurons resulted in local activity. Single-cell holographic stimulation facilitated to trace propagating evoked activities of 400 individually stimulated neurons per multi-electrode array. Thereby, we revealed precise functional neuronal connectivity motifs. Holographic stimulation data over time showed increasing connection numbers and strength with culture age. This holographic stimulation setup has the potential to establish a profound functional testbed for in-depth analysis of human-induced pluripotent stem cell-derived neuronal networks.
Assuntos
Células-Tronco Pluripotentes Induzidas , Optogenética , Técnicas de Cocultura , Humanos , NeurôniosRESUMO
Despite substantial recent progress in network neuroscience, the impact of stroke on the distinct features of reorganizing neuronal networks during recovery has not been defined. Using a functional connections-based approach through 2-photon in vivo calcium imaging at the level of single neurons, we demonstrate for the first time the functional connectivity maps during motion and nonmotion states, connection length distribution in functional connectome maps and a pattern of high clustering in motor and premotor cortical networks that is disturbed in stroke and reconstitutes partially in recovery. Stroke disrupts the network topology of connected inhibitory and excitatory neurons with distinct patterns in these 2 cell types and in different cortical areas. These data indicate that premotor cortex displays a distinguished neuron-specific recovery profile after stroke.
Assuntos
Atividade Motora , Córtex Motor/fisiopatologia , Neurônios/fisiologia , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/fisiopatologia , Animais , Sinalização do Cálcio , Masculino , Camundongos Transgênicos , Imagem ÓpticaRESUMO
Transplantation of neural stem cells (NSCs) or NSC-derived neurons into the brain is a promising therapeutic approach to restore neuronal function. Rapid progress in the NSCs research field, particularly due to the exploitation of induced pluripotent stem cells (iPSCs), offers great potential and an unlimited source of stem cell-derived neural grafts. Studying the functional integration of these grafts into host brain tissues and their effects on each other have been boosted by the implementation of optogenetic technologies. Optogenetics provides high spatiotemporal functional manipulations of grafted or host neurons in parallel. This review aims to highlight the impact of optogenetics in neural stem cell transplantations.
Assuntos
Células-Tronco Neurais/transplante , Neurônios/transplante , Optogenética/métodos , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Medula Espinal/citologia , Medula Espinal/fisiologia , Transplante de Células-Tronco/métodosRESUMO
Chronic morphine (CM) treatment increases the phosphorylation of the mammalian target of rapamycin (mTOR), which confers neuroprotection against ischemia/reperfusion (I/R) injury. Besides its important regulatory role in the proliferation, metabolism, and survival of cells, the mTOR is critically involved in intracellular signaling events during I/R injury. In the present study, we investigated the interaction between the expressions of the mTOR and inducible nitric oxide synthase (iNOS) and their possible protective effects on hippocampal neurons against I/R injury in morphine-dependent mice. Additive doses of morphine were administered for 5 days to BALB/c mice so as to induce CM preconditioning before I/R injury. Global brain ischemia was induced via the occlusion of bilateral common carotid arteries for 30 min. CM attenuated iNOS expression, NO production, and malondialdehyde activity in the hippocampal tissue. Pretreatment with rapamycin, the inhibitor of mTOR, abolished all the above mentioned effects of CM. These findings suggested that CM acted through the mTOR signaling pathways to regulate iNOS expression and oxidative state in the hippocampal tissue after I/R injury.
RESUMO
Ischemic cerebral stroke is a major cause of death and morbidity. Currently, no neuroprotective agents have been shown to impact the clinical outcomes in cerebral stroke cases. Here, we report therapeutic effects of Se nanoparticles on ischemic stroke in a murine model. Anti-transferrin receptor monoclonal antibody (OX26)-PEGylated Se nanoparticles (OX26-PEG-Se NPs) were designed and synthesized and their neuroprotective effects were measured using in vitro and in vivo approaches. We demonstrate that administration of the biodegradable nanoparticles leads to resolution of brain edema, protection of axons in hippocampus region, and myelination of hippocampal area after cerebral ischemic stroke. Our nanoparticle design ensures efficient targeting and minimal side effects. Hematological and biochemical analyses revealed no undesired NP-induced changes. To gain mechanistic insights into the therapeutic effects of these particles, we characterized the changes to the relevant inflammatory and metabolic signaling pathways. We assessed metabolic regulator mTOR and related signaling pathways such as hippo, Ubiquitin-proteasome system (ERK5), Tsc1/Tsc2 complex, FoxO1, wnt/ß-catenine signaling pathway. Moreover, we examined the activity of jak2/stat3 signaling pathways and Adamts1, which are critically involved in inflammation. Together, our study provides a promising treatment strategy for cerebral stroke based on Se NP induced suppression of excessive inflammation and oxidative metabolism.
Assuntos
Inflamação/terapia , Nanopartículas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Selênio/uso terapêutico , Acidente Vascular Cerebral/terapia , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/terapia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
Curcumin (diferuloylmethane) is a natural bioactive compound with many health-promoting benefits. However, its poor water solubility and bioavailability has limited curcumin’s biomedical application. In the present study, we encapsulated curcumin into liposomes, formed from natural sources (salmon lecithin), and characterized its encapsulation efficiency and release profile. The proposed natural carriers increased the solubility and the bioavailability of curcumin. In addition, various physico-chemical properties of the developed soft nanocarriers with and without curcumin were studied. Nanoliposome-encapsulated curcumin increased the viability and network formation in the culture of primary cortical neurons and decreased the rate of apoptosis.
Assuntos
Curcumina/química , Curcumina/metabolismo , Lipossomos/química , Nanopartículas/química , Neurônios/efeitos dos fármacos , Salmão/metabolismo , Animais , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Portadores de Fármacos/química , Lecitinas/química , Solubilidade/efeitos dos fármacosRESUMO
The signaling pathway of chronic morphine treatment to prevent neuronal damage following transient cerebral ischemia is not clear. In this study, we examined the role of mammalian target of rapamycin (mTOR) to identify the neuroprotective effects of chronic morphine preconditioning on the hippocampus following ischemia-reperfusion (I/R) injury. Morphine was administered for 5 days, twice a day, before inducing I/R injury. The possible role of mTOR was evaluated by the injection of rapamycin (5 mg/kg body weight, by intraperitoneal injection) before I/R was induced. The passive avoidance test was used to evaluate memory performance. Neuronal density and apoptosis were measured in the CA1 region, 72 h after I/R injury. The expressions of mTOR and phosphorylated mTOR (p-mTOR), as well as superoxide dismutase (SOD) activity were determined 24 h after I/R injury. Chronic morphine treatment attenuated apoptosis and neuronal loss in the hippocampus after I/R injury, which led to improvement in memory (P < 0.05 vs. untreated I/R) and increase in the expression of p-mTOR (P < 0.05 vs. untreated I/R) and SOD activity (P < 0.05 vs. untreated I/R) in the hippocampus. Pretreatment with rapamycin abolished all the above-mentioned protective effects. These results describe novel findings whereby chronic morphine preconditioning in hippocampal CA1 neurons is mediated by the mTOR pathway, and through increased phosphorylation of mTOR can alleviate oxidative stress and apoptosis, and eventually protect the hippocampus from I/R injury.
Assuntos
Região CA1 Hipocampal/patologia , Morfina/farmacologia , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aprendizagem da Esquiva/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacos , Superóxido Dismutase/metabolismoRESUMO
PURPOSE: Pharmacologic preconditioning, through activating several mechanisms and mediators, can increase the tolerance of different tissues against ischemia/reperfusion (I/R) injury. Recent studies have shown that morphine preconditioning has protective effects in different organs, especially in the heart. Nevertheless, its mechanisms are not well elucidated in the brain. The present study aimed to clarify whether the activation of mitochondrial KATP (mKATP) channels in chronic morphine (CM) preconditioning could decrease hippocampus damage following I/R injury. MATERIALS AND METHODS: CM preconditioning was performed by the administration of additive doses of morphine for 5days before I/R injury induction. I/R injury was induced by the occlusion of bilateral common carotid arteries. The possible role of mKATP channels was evaluated by the injection of 5-hydroxydecanoate (5-HD) before I/R injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to detect apoptosis in hippocampal neurons. The expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (BAX) and levels of malondialdehyde (MDA) and catalase (CAT) enzymes were assessed. RESULTS: CM attenuated apoptosis in the hippocampal CA1 neurons (P<0.001 vs I/R), and mKATP channel blocking with 5-HD significantly increased apoptosis (P<0.001 vs CM+I/R). CM increased CAT activity (P<0.05 vs I/R) and Bcl-2 protein expression (P<0.01 vs I/R), while it decreased MDA level (P<0.05 vs I/R) and BAX protein expression (P<0.05 vs I/R). Pretreatment with 5-HD abolished all the above-mentioned effects of CM. CONCLUSIONS: These findings describe novel evidence whereby CM preconditioning in hippocampal CA1 neurons can improve oxidative stress and apoptosis through the activation of mKATP channels and eventually protect the hippocampal tissue against I/R injury.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Região CA1 Hipocampal/patologia , Morfina/farmacologia , Neurônios/patologia , Neuroproteção/efeitos dos fármacos , Canais de Potássio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Neurônios/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Due to their small dimensions, electrophysiology on thin and intricate axonal branches in support of understanding their role in normal and diseased brain function poses experimental challenges. To reduce experimental complexity, we coupled microelectrode arrays (MEAs) to bi-level microchannel devices for the long-term in vitro tracking of axonal morphology and activity with high spatiotemporal resolution. Our model allowed the long-term multisite recording from pure axonal branches in a microscopy-compatible environment. Compartmentalizing the network structure into interconnected subpopulations simplified access to the locations of interest. Electrophysiological data over 95 days in vitro (DIV) showed an age-dependent increase of axonal conduction velocity, which was positively correlated with, but independent of evolving burst activity over time. Conduction velocity remained constant at chemically increased network activity levels. In contrast, low frequency (1 Hz, 180 repetitions) electrical stimulation of axons or network subpopulations evoked amplitude-dependent direct (5-35 ms peri-stimulus) and polysynaptic (35-1,000 ms peri-stimulus) activity with temporarily (<35 ms) elevated propagation velocities along the perisomatic branches. Furthermore, effective stimulation amplitudes were found to be significantly lower (>250 mV) in microchannels when compared with those reported for unconfined cultures (>800 mV). The experimental paradigm may lead to new insights into stimulation-induced axonal plasticity.
Assuntos
Potenciais de Ação/fisiologia , Axônios/fisiologia , Microeletrodos , Condução Nervosa/fisiologia , Algoritmos , Animais , Células Cultivadas , Córtex Cerebral/citologia , Estimulação Elétrica , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Modelos NeurológicosRESUMO
Organ ischemia with inadequate oxygen supply followed by reperfusion (which initiates a complex of inflammatory responses and oxidative stress) occurs in different clinical conditions and surgical procedures including stroke, myocardial infarction, limb ischemia, renal failure, organ transplantation, free-tissue-transfer, cardiopulmonary bypass, and vascular surgery. Even though pharmacological treatments protect against experimental ischemia reperfusion (I/R) injury, there has not been enough success in their application for patient benefits. The main hurdles in the treatment of I/R injury are the lack of diagnosis tools for understanding the complicated chains of I/R-induced signaling events, especially in the acute phase after ischemia, determining the affected regions of the tissue over time, and then, targeting and safe delivery of antioxidants, drugs, peptides, genes and cells to the areas requiring treatment. Besides the innate antioxidant and free radical scavenging properties, some nanoparticles also show higher flexibility in drug delivery and imaging. This review highlights three main approaches in nanoparticle-mediated targeting of I/R injury: nanoparticles (1) as antioxidants for reducing tissue oxidative stress, (2) for targeted delivery of therapeutic agents to the ischemic regions or cells, and (3) for imaging I/R injury at the molecular, cellular or tissue level and monitoring its evolution using contrasts induced by nanoparticles. These approaches can also be combined to realize so called theranostics for providing simultaneous diagnosis of ischemic regions and treatments by targeted delivery.
RESUMO
Phospholipids in the brain cell membranes contain different polyunsaturated fatty acids (PUFAs), which are critical to nervous system function and structure. In particular, brain function critically depends on the uptake of the so-called "essential" fatty acids such as omega-3 (n-3) and omega-6 (n-6) PUFAs that cannot be readily synthesized by the human body. We extracted natural lecithin rich in various PUFAs from a marine source and transformed it into nanoliposomes. These nanoliposomes increased neurite outgrowth, network complexity and neural activity of cortical rat neurons in vitro. We also observed an upregulation of synapsin I (SYN1), which supports the positive role of lecithin in synaptogenesis, synaptic development and maturation. These findings suggest that lecithin nanoliposomes enhance neuronal development, which may have an impact on devising new lecithin delivery strategies for therapeutic applications.
Assuntos
Lecitinas/farmacologia , Rede Nervosa/fisiologia , Animais , Lipossomos , Microeletrodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanopartículas/química , Nanopartículas/ultraestrutura , Rede Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sinapsinas/genética , Sinapsinas/metabolismoRESUMO
Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.
RESUMO
We designed a miniaturized and thin polydimethylsiloxane (PDMS) microchannel device compatible with commercial microelectrode array (MEA) chips. It was optimized for selective axonal ablation by laser microdissection (LMD) to investigate the electrophysiological and morphological responses to a focal injury in distinct network compartments over 45 days in vitro (45 DIV). Low-density cortical or hippocampal networks (<3500 neurons per device) were cultured in quasi-closed somal chambers. Their axons were selectively filtered through neurite cavities and guided into the PDMS microchannels aligned over the recording electrodes. The device geometries amplified extracellularly recorded signals in the somal reservoir and the axonal microchannels to detectable levels. Locally extended areas along the microchannel, so-called working stations, forced axonal bundles to branch out and thereby allowed for their repeatable and controllable local, partial or complete dissections. Proximal and distal changes in the activity and morphology of the dissected axons were monitored and compared to those of their parent networks and of intact axons in the control microchannels. Microscopy images confirmed progressive anterograde degeneration of distal axonal segments over four weeks after surgery. Dissection on cortical and hippocampal axons revealed different cell type- and age-dependent network responses. At 17 DIV, network activity increased in both the somal and proximal microchannel compartments of the dissected hippocampal or cortical axons. At later days (24 DIV), the hippocampal networks were more susceptible to axonal injury. While their activity decreased, that in the cortical cultures actually increased. Subsequent partial dissections of the same axonal bundles led to a stepwise activity reduction in the distal hippocampal or cortical axonal fragments. We anticipate that the MEA-PDMS microchannel device for the combined morphological and electrophysiological study of axonal de- and regeneration can be easily merged with other experimental paradigms like molecular or pharmacological screening studies.
Assuntos
Axônios/fisiologia , Dispositivos Lab-On-A-Chip , Regeneração Nervosa , Animais , Axônios/ultraestrutura , Axotomia/instrumentação , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Fenômenos Eletrofisiológicos , Desenho de Equipamento , Hipocampo/citologia , Hipocampo/fisiologia , Microdissecção e Captura a Laser/instrumentação , Microeletrodos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Ratos , Análise Serial de Tecidos/instrumentaçãoRESUMO
Remote ischemic preconditioning (RIPC), which consists of several brief ischemia/reperfusion applied at the remote site of lethal ischemia reperfusion, can, through activating different mechanisms, increase the ability of the body's endogenous protection against prolonged ischemia/reperfusion. Recent studies have shown that RIPC has neuroprotective effects, but its mechanisms are not well elucidated. The present study aimed to determine whether activation of KATP channels in remote renal preconditioning decreases hippocampus damage induced by global cerebral ischemia. RIPC was induced by ischemia of the left renal artery (IPC); 24 h later, global cerebral ischemia reperfusion (IR) was induced by common carotid arteries occlusion. 5hydroxydecanoate (5HD) and glibenclamide (Gli) were injected before of IPC. The levels of malondialdehyde (MDA) and catalase (CAT) activity were assessed in hippocampus. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was assessed to detect apoptotic cells in hippocampus. RIPC inhibited apoptosis by decreasing positive TUNEL cells (P < 0.05). KATP channels blocking with 5HD and Gli markedly increased apoptosis in hippocampal cells in RIPC group (P < 0.001). RIPC decreased MDA level and increased CAT activity in ischemic hippocampus (P < 0.01). Also, 5HD and Gli inhibited the effect of RIPC on MDA level and CAT activity (P < 0.05). The present study shows that RIPC can effectively attenuate programmed cell death, increase activity of CAT, and reduce MDA levels. Blocking of KATP channels inhibited the protective effects of RIPC.