RESUMO
Fluid and enzyme secretion from exocrine glands is initiated by Ca2+ signalling in acinar cells and is activated by external neural or hormonal signals. A wealth of information has been derived from studies in acutely isolated exocrine cells but Ca2+ signalling has until recently not been studied in undisrupted intact tissue in live mice. Our in vivo observations using animals expressing genetically encoded Ca2+ indicators in specific cell types in exocrine glands revealed both similarities to and differences from the spatiotemporal characteristics previously reported in isolated cells. These in vivo studies facilitate further understanding of how both neuronal and hormonal input shapes Ca2+ signalling events in a physiological setting and how these signals are translated into the stimulation of fluid secretion and exocytosis.
Assuntos
Sinalização do Cálcio , Glândulas Exócrinas , Animais , Glândulas Exócrinas/metabolismo , Glândulas Exócrinas/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Camundongos , Hormônios/metabolismo , Hormônios/fisiologia , Cálcio/metabolismoRESUMO
Saliva is essential for oral health. The molecular mechanisms leading to physiological fluid secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of inflammatory immune cell infiltration within the salivary glands and glandular hypofunction. In this study, we investigated in a mouse model system, mechanisms of glandular hypofunction caused by the activation of the stimulator of interferon genes (STING) pathway. Glandular hypofunction and SS-like disease were induced by treatment with 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), a small molecule agonist of murine STING. Contrary to our expectations, despite a significant reduction in fluid secretion in DMXAA-treated mice, in vivo imaging demonstrated that neural stimulation resulted in greatly enhanced spatially averaged cytosolic Ca2+ levels. Notably, however, the spatiotemporal characteristics of the Ca2+ signals were altered to signals that propagated throughout the entire cytoplasm as opposed to largely apically confined Ca2+ rises observed without treatment. Despite the augmented Ca2+ signals, muscarinic stimulation resulted in reduced activation of TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. However, super-resolution microscopy revealed a disruption in the intimate colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels in relation to TMEM16a. TMEM16a channel activation was also reduced when intracellular Ca2+ buffering was increased. These data are consistent with altered local coupling between the channels contributing to the reduced activation of TMEM16a. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics and secretion is an energetically expensive process. Disrupted mitochondrial morphology, a depolarized mitochondrial membrane potential, and reduced oxygen consumption rate were observed in DMXAA-treated animals compared to control animals. We report that early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction and likely the progression of SS disease.
RESUMO
An increase in intracellular [Ca2+ ] in exocrine acinar cells resident in the salivary glands or pancreas is a fundamental event that drives fluid secretion and exocytosis of proteins. Stimulation with secretagogues initiates Ca2+ signals with precise spatiotemporal properties thought to be important for driving physiological output. Both in vitro, in acutely isolated acini, and in vivo, in animals expressing genetically encoded indicators, individual cells appear specialized to initiate Ca2+ signals upon stimulation. Furthermore, these signals appear to spread to neighbouring cells. These properties are present in the absence of a conventional pacemaker mechanism dependent on the cyclical activation of Ca2+ -dependent or Ca2+ -conducting plasma membrane ion channels. In this article, we propose a model for 'pacing' intracellular Ca2+ signals in acinar cells based on the enhanced sensitivity of a subpopulation of individual cells and the intercellular diffusion through gap junctions of inositol 1,4,5-trisphosphate and Ca2+ to neighbouring cells.
RESUMO
Currently, all salivary ducts (intercalated, striated and collecting) are assumed to function broadly in a similar manner, reclaiming ions that were secreted by the secretory acinar cells while preserving fluid volume and delivering saliva to the oral cavity. Nevertheless, there has been minimal investigation into the structural and functional differences between distinct types of salivary duct cells. Therefore, in this study, the expression profile of proteins involved in stimulus-secretion coupling, as well as the function of the intercalated duct (ID) and striated duct cells, was examined. Particular focus was placed on defining differences between distinct duct cell populations. To accomplish this, immunohistochemistry and in situ hybridization were utilized to examine the localization and expression of proteins involved in reabsorption and secretion of ions and fluid. Further, in vivo calcium imaging was employed to investigate cellular function. Based on the protein expression profile and functional data, marked differences between the IDs and striated ducts were observed. Specifically, the ID cells express proteins native to the secretory acinar cells while lacking proteins specifically expressed in the striated ducts. Further, the ID and striated duct cells display different calcium signalling characteristics, with the IDs responding to a neural stimulus in a manner similar to the acinar cells. Overall, our data suggest that the IDs have a distinct role in the secretory process, separate from the reabsorptive striated ducts. Instead, based on our evidence, the IDs express proteins found in secretory cells, generate calcium signals in a manner similar to acinar cells, and, therefore, are likely secretory cells. KEY POINTS: Current studies examining salivary intercalated duct cells are limited, with minimal documentation of the ion transport machinery and the overall role of the cells in fluid generation. Salivary intercalated duct cells are presumed to function in the same manner as other duct cells, reclaiming ions, maintaining fluid volume and delivering the final saliva to the oral cavity. Here we systematically examine the structure and function of the salivary intercalated duct cells using immunohistochemistry, in situ hybridization and by monitoring in vivo Ca2+ dynamics. Structural data revealed that the intercalated duct cells lack proteins vital for reabsorption and express proteins necessary for secretion. Ca2+ dynamics in the intercalated duct cells were consistent with those observed in secretory cells and resulted from GPCR-mediated IP3 production.
Assuntos
Cálcio , Células Epiteliais , Proteínas , ÍonsRESUMO
The exocrine pancreas secretes fluid and digestive enzymes in response to parasympathetic release of acetylcholine (ACh) via the vagus nerve and the gut hormone cholecystokinin (CCK). Both secretion of fluid and exocytosis of secretory granules containing enzymes and zymogens are dependent on an increase in the cytosolic [Ca2+ ] in acinar cells. It is thought that the specific spatiotemporal characteristics of the Ca2+ signals are fundamental for appropriate secretion and that these properties are disrupted in disease states in the pancreas. While extensive research has been performed to characterize Ca2+ signalling in acinar cells, this has exclusively been achieved in ex vivo preparations of exocrine cells, where it is difficult to mimic physiological conditions. Here we have developed a method to optically observe pancreatic acinar Ca2+ signals in vivo using a genetically expressed Ca2+ indicator and imaged with multi-photon microscopy in live animals. In vivo, acinar cells exhibited baseline activity in fasted animals, which was dependent on CCK1 receptors (CCK1Rs). Both stimulation of intrinsic nervous input and administration of systemic CCK induced oscillatory activity in a proportion of the cells, but the maximum frequencies were vastly different. Upon feeding, oscillatory activity was also observed, which was dependent on CCK1Rs. No evidence of a vago-vagal reflex mediating the effects of CCK was observed. Our in vivo method revealed the spatial and temporal profile of physiologically evoked Ca2+ signals, which will provide new insights into future studies of the mechanisms underlying exocrine physiology and that are disrupted in pathological conditions. KEY POINTS: In the exocrine pancreas, the spatiotemporal properties of Ca2+ signals are fundamentally important for the appropriate stimulation of secretion by the neurotransmitter acetylcholine and gut hormone cholecystokinin. These characteristics were previously defined in ex vivo studies. Here we report the spatiotemporal characteristics of Ca2+ signals in vivo in response to physiological stimulation in a mouse engineered to express a Ca2+ indicator in acinar cells. Specific Ca2+ 'signatures' probably important for stimulating secretion are evoked in vivo in fasted animals, by feeding, neural stimulation and cholecystokinin administration. The Ca2+ signals are probably the result of the direct action of ACh and CCK on acinar cells and not indirectly through a vago-vagal reflex.
Assuntos
Células Acinares , Pâncreas Exócrino , Camundongos , Animais , Acetilcolina/farmacologia , Pâncreas , Colecistocinina/farmacologia , Cálcio/farmacologiaRESUMO
OBJECTIVE: The synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, are crucial for lymphangiogenesis. However, their involvement in age-related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF-C/VEGFR-3 pathway contribute to the development and progression of age-related OA, using a murine model of naturally occurring joint disease. METHODS: SLS function was assessed in the knees of young (3-month-old) and aged (19-24-month-old) male and female C57BL/6J mice via a newly established in vivo IVIS-dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF-C treatment on SLS function and OA pathology in aged mice. RNA-sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints. RESULTS: Results showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P = 0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[µW/cm2 ]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA-sequencing of the synovial tissue showed that Vegf-c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF-C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF-C-treated aged mice versus 52 ± 15% in vehicle-treated aged mice; P = 0.012), increased total articular cartilage cross-sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF-C-treated aged mice versus 0.26 ± 0.07 mm2 in vehicle-treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13-positive staining area within total synovial area in 22-month-old VEGF-C-treated mice versus 22-month-old vehicle-treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P = 0.0004). CONCLUSION: SLS function is reduced in the knee joints of aged mice due to decreased VEGF-C/VEGFR-3 signaling. VEGF-C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF-C/VEGFR-3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age-related OA.
Assuntos
Osteoartrite , Fator C de Crescimento do Endotélio Vascular , Camundongos , Masculino , Feminino , Animais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Camundongos Endogâmicos C57BL , Osteoartrite/metabolismo , Membrana Sinovial/metabolismo , RNA/metabolismoRESUMO
Changes in intracellular calcium drive exocrine cell activity. In the salivary gland, acetylcholine released from parasympathetic neurons mobilizes endoplasmic reticulum calcium stores in acinar cells, which consequently initiates saliva secretion. However, our understanding of the signaling cascade is mainly based on ex vivo studies performed in enzymatically isolated cells. The dissociation process likely disrupts the extracellular matrix, removes neurons as the source of signal input, and disturbs the integrity of tight and gap junctional acinar connections. These alterations may affect the spatiotemporal properties of calcium signaling events. In vivo observations of calcium signals, where tissue organization is intact, are therefore important to establish the characteristics of physiological calcium signals that are crucial for the stimulation of fluid secretion. Here, we present a detailed protocol for in vivo imaging of calcium signaling events, following nervous stimulation by multi-photon microscopy in mouse salivary gland acinar cells, expressing the genetically encoded calcium indicator GCamp6F.
RESUMO
Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels, present on the apical and basolateral plasma membrane, respectively. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.
Assuntos
Sinalização do Cálcio/fisiologia , Saliva/metabolismo , Glândulas Salivares/metabolismo , Células Acinares/metabolismo , Animais , Cálcio/metabolismo , Biologia Computacional , Retículo Endoplasmático/metabolismo , Feminino , Canais Iônicos/metabolismo , Masculino , Camundongos , Glândulas Salivares/patologia , Glândula SubmandibularRESUMO
Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism triggered by depletion of Ca2+ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca2+ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca2+ entry, as well as sustained Ca2+ release and force generation during repetitive stimulation. Skeletal muscle from mice lacking calsequestrin-1 (CASQ1-null), the primary Ca2+-binding protein in the lumen of SR terminal cisternae, exhibits significantly reduced total Ca2+ store content and marked SR Ca2+ depletion during high-frequency stimulation. Here, we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of sedentary CASQ1-null mice. The higher density of CEUs in EDL (39.6 ± 2.1/100 µm2 versus 2.0 ± 0.3/100 µm2) and FDB (16.7 ± 1.0/100 µm2 versus 2.7 ± 0.5/100 µm2) muscles of CASQ1-null compared with WT mice correlated with enhanced constitutive- and store-operated Ca2+ entry and increased expression of STIM1, Orai1, and SERCA. The higher ability to recover Ca2+ ions via SOCE in CASQ1-null muscle served to promote enhanced maintenance of peak Ca2+ transient amplitude, increased dependence of luminal SR Ca2+ replenishment on BTP-2-sensitive SOCE, and increased maintenance of contractile force during repetitive, high-frequency stimulation. Together, these data suggest that muscles from CASQ1-null mice compensate for the lack of CASQ1 and reduction in total releasable SR Ca2+ content by assembling CEUs to promote constitutive and store-operated Ca2+ entry.
Assuntos
Cálcio , Calsequestrina , Músculo Esquelético , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Calsequestrina/fisiologia , Íons , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , Proteína ORAI1 , Molécula 1 de Interação EstromalRESUMO
Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca2+ entry (SOCE). Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.
Assuntos
Cálcio/metabolismo , Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Proteína ORAI1/metabolismo , Condicionamento Físico Animal , Animais , Cátions Bivalentes/metabolismo , Masculino , Camundongos Endogâmicos C57BLRESUMO
Supramolecular hydrogels are emerging as next-generation alternatives to synthetic polymers for drug delivery applications. Self-assembling peptides are a promising class of supramolecular gelator for in vivo drug delivery that have been slow to be adopted despite advantages in biocompatibility due to the relatively high cost of producing synthetic peptide hydrogels compared to synthetic polymer gels. Herein we describe the development and use of inexpensive low molecular weight cationic derivatives of phenylalanine (Phe) as injectable hydrogels for in vivo delivery of an anti-inflammatory drug, diclofenac, for pain mitigation in a mouse model. N-Fluorenylmethoxycarbonyl phenylalanine (Fmoc-Phe) derivatives were modified at the carboxylic acid with diaminopropane (DAP) to provide Fmoc-Phe-DAP molecules that spontaneously and rapidly self-assemble in aqueous solutions upon addition of physiologically relevant sodium chloride concentrations to give hydrogels. When self-assembly occurs in the presence of diclofenac, the drug molecule is efficiently encapsulated within the hydrogel network. These hydrogels exhibit robust shear-thinning behavior, mechanical stability, and drug release profiles to enable application as injectable hydrogels for in vivo drug delivery. Delivery of diclofenac in vivo was demonstrated by a localized injection of an Fmoc-F5-Phe-DAP/diclofenac hydrogel into the ankle joint of mice with induced ankle injury and associated inflammation-induced pain. Remediation of pain in the ankle joint was observed immediately after initial injection and was sustained for a period of nearly two weeks while diclofenac controls remediated pain for less than one day. This data demonstrates the promise of these supramolecular hydrogels as inexpensive next-generation materials for sustained and localized drug delivery in vivo.
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Acupuncture is an alternative treatment for wide spectrum chronic pain. However, its validity remains controversial due to the disputed efficacy assessed in various clinical studies. Moreover, variability amongst individuals complicates the predictability of outcome, which impedes the integration of acupuncture into mainstream pain management programs. In light of our previous finding that the analgesic effect of acupuncture is mediated by adenosine A1 receptor activation at the acupuncture point, we here report that in acute and chronic animal pain models, oral intake of caffeine, a potent adenosine receptor antagonist, interferes with acupuncture analgesia, even at a low dose. Local administration of caffeine at the acupuncture point was sufficient to eliminate the analgesic effect, dismissing the systemic action of caffeine. Such interference was reversible, as caffeine withdrawal fully restored the efficacy of acupuncture by the next day, and long-term exposure to caffeine did not alter A1 receptor expression at the acupuncture point. Combined, these data indicate that a trace amount of caffeine can reversibly block the analgesic effects of acupuncture, and controlling caffeine consumption during acupuncture may improve pain management outcomes.
Assuntos
Analgesia por Acupuntura/métodos , Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Modelos Animais de Doenças , Inflamação/complicações , Dor/tratamento farmacológico , Receptor A1 de Adenosina/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor/etiologia , Manejo da DorRESUMO
BACKGROUND: Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it's expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation. METHODS: We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student's t- test. RESULTS: We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2 > apoE3 > apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state. CONCLUSIONS: Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Privação do Sono/metabolismo , Animais , Apolipoproteínas E/líquido cefalorraquidiano , Aquaporina 4/metabolismo , Transporte Biológico , Masculino , Camundongos , Isoformas de Proteínas/metabolismo , Privação do Sono/líquido cefalorraquidiano , Fatores de TempoRESUMO
Microglia are integral functional elements of the central nervous system, but the contribution of these cells to the structural integrity of the neurovascular unit has not hitherto been assessed. We show here that following blood-brain barrier (BBB) breakdown, P2RY12 (purinergic receptor P2Y, G-protein coupled, 12)-mediated chemotaxis of microglia processes is required for the rapid closure of the BBB. Mice treated with the P2RY12 inhibitor clopidogrel, as well as those in which P2RY12 was genetically ablated, exhibited significantly diminished movement of juxtavascular microglial processes and failed to close laser-induced openings of the BBB. Thus, microglial cells play a previously unrecognized protective role in the maintenance of BBB integrity following cerebrovascular damage. Because clopidogrel antagonizes the platelet P2Y12 receptor, it is widely prescribed for patients with coronary artery and cerebrovascular disease. As such, these observations suggest the need for caution in the postincident continuation of P2RY12-targeted platelet inhibition.
Assuntos
Barreira Hematoencefálica , Microglia/fisiologia , Receptores Purinérgicos P2Y12/fisiologia , Animais , Movimento Celular , Clopidogrel , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Ticlopidina/análogos & derivados , Ticlopidina/farmacologiaRESUMO
Aneurysmal subarachnoid hemorrhage remains one of the more devastating forms of stroke due in large part to delayed cerebral ischemia that appears days to weeks following the initial hemorrhage. Therapies exclusively targeting large caliber arterial vasospasm have fallen short, and thus we asked whether capillary dysfunction contributes to delayed cerebral ischemia after subarachnoid hemorrhage. Using a mouse model of subarachnoid hemorrhage and two-photon microscopy we showed capillary dysfunction unrelated to upstream arterial constriction. Subarachnoid hemorrhage decreased RBC velocity by 30%, decreased capillary pulsatility by 50%, and increased length of non-perfusing capillaries by 15%. This was accompanied by severe brain hypoxia and neuronal loss. Hyaluronidase, an enzyme that alters capillary blood flow by removing the luminal glycocalyx, returned RBC velocity and pulsatility to normal. Hyaluronidase also reversed brain hypoxia and prevented neuron loss typically seen after subarachnoid hemorrhage. Thus, subarachnoid hemorrhage causes specific changes in capillary RBC flow independent of arterial spasm, and hyaluronidase treatment that normalizes capillary blood flow can prevent brain hypoxia and injury after subarachnoid hemorrhage. Prevention or treatment of capillary dysfunction after subarachnoid hemorrhage may reduce the incidence or severity of subarachnoid hemorrhage-induced delayed cerebral ischemia.
Assuntos
Circulação Cerebrovascular/efeitos dos fármacos , Hialuronoglucosaminidase/uso terapêutico , Microcirculação/efeitos dos fármacos , Hemorragia Subaracnóidea/fisiopatologia , Animais , Isquemia Encefálica/prevenção & controle , Capilares/efeitos dos fármacos , Capilares/fisiopatologia , Hipóxia/prevenção & controle , CamundongosRESUMO
The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, but a small percentage of infected cells switch from the latent state to the lytic cycle, leading to potent viral DNA replication and progeny viruses production. We here focused on a lytic gene BGLF3.5, and first established BGLF3.5 mutants by marker cassette insertion. Unexpectedly, this insertion mutant failed to produce BGLF4 protein and thus progeny production was severely inhibited. Then we carefully made two point mutant viruses (stop codon insertion or frame-shift mutation) and found that BGLF3.5 is not essential for EBV lytic replication processes, such as viral gene expression, DNA replication, or progeny production in the HEK293 cells although its homolog in murine gammaherpesvirus 68 (MHV-68) was reported to be essential. In addition, we examined the roles of two short, upstream open reading frames within the 5'UTR of BGLF3.5 gene in translation of BGLF4.
Assuntos
Células Epiteliais/virologia , Herpesvirus Humano 4/fisiologia , Proteínas Virais/genética , Replicação Viral , Códon sem Sentido , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Proteínas Mutantes/genética , Mutação PuntualRESUMO
Metabolically, the brain is a highly active organ that relies almost exclusively on glucose as its energy source. According to the astrocyte-to-neuron lactate shuttle hypothesis, glucose is taken up by astrocytes and converted to lactate, which is then oxidized by neurons. Here we show, using two-photon imaging of a near-infrared 2-deoxyglucose analogue (2DG-IR), that glucose is taken up preferentially by neurons in awake behaving mice. Anaesthesia suppressed neuronal 2DG-IR uptake and sensory stimulation was associated with a sharp increase in neuronal, but not astrocytic, 2DG-IR uptake. Moreover, hexokinase, which catalyses the first enzymatic steps in glycolysis, was highly enriched in neurons compared with astrocytes, in mouse as well as in human cortex. These observations suggest that brain activity and neuronal glucose metabolism are directly linked, and identify the neuron as the principal locus of glucose uptake as visualized by functional brain imaging.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Glucose/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Vigília , Anestésicos Dissociativos/farmacologia , Animais , Antimetabólitos , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Desoxiglucose , Neuroimagem Funcional/métodos , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Hipnóticos e Sedativos/farmacologia , Imuno-Histoquímica , Ketamina/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Estimulação Física , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectroscopia de Luz Próxima ao Infravermelho , Xilazina/farmacologiaRESUMO
Experimental advances in the study of neuroglia signaling have been greatly accelerated by the generation of transgenic mouse models. In particular, an elegant manipulation that interferes with astrocyte vesicular release of gliotransmitters via overexpression of a dominant-negative domain of vesicular SNARE (dnSNARE) has led to documented astrocytic involvement in processes that were traditionally considered strictly neuronal, including the sleep-wake cycle, LTP, cognition, cortical slow waves, depression, and pain. A key premise leading to these conclusions was that expression of the dnSNARE was specific to astrocytes. Inconsistent with this premise, we report here widespread expression of the dnSNARE transgene in cortical neurons. We further demonstrate that the activity of cortical neurons is reversibly suppressed in dnSNARE mice. These findings highlight the need for independent validation of astrocytic functions identified in dnSNARE mice and thus question critical evidence that astrocytes contribute to neurotransmission through SNARE-dependent vesicular release of gliotransmitters.
Assuntos
Regulação da Expressão Gênica , Neurônios/metabolismo , Proteínas SNARE/biossíntese , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletroencefalografia/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas SNARE/genética , Fases do Sono/fisiologiaRESUMO
A flurry of studies over the past decade has shown that astrocytes play a more active role in neural function than previously recognized. Hippocampal slices prepared from young rodent pups have served as a popular model for studying the pathways by which astrocytes participate in synaptic transmission. It is, however, not known how well astrocytes tolerate traumatic injury and hypoxia, which are unavoidable when preparing acute slices. We here showed that astrocytes exhibit striking changes in expression of several receptors and structural proteins, including re-expression of the developmental marker nestin within 90 min following preparation of live vibratome slices. Moreover, immunoelectron microscopy showed a 2.7-fold loss of astrocytic processes in acute hippocampal slices prepared from glial fibrillary acidic protein-green fluorescent protein reporter mice. A sharp decrease in the number of mitochondria was also noted in acute slices, concurrently with an increase in mitochondrial size. Glycogen content decreased 3-fold upon slice preparation and did not recover despite stable recordings of field excitatory postsynaptic current. Analysis of Ca(2+) signaling showed that astrocytic responses to purine receptor and mGluR5 agonists differed in slice versus in vivo. These observations suggest that the functional properties and the fine structure of astrocytes in slices may be reflective of early stages of reactive gliosis and should be confirmed in vivo when possible.