RESUMO
Microbubble-enhanced ultrasound provides a noninvasive physical method to locally overcome major obstacles to the accumulation of blood-borne therapeutics in the brain, posed by the blood-brain barrier (BBB). However, due to the highly nonlinear and coupled behavior of microbubble dynamics in brain vessels, the impact of microbubble resonant effects on BBB signaling and function remains undefined. Here, combined theoretical and prospective experimental investigations reveal that microbubble resonant effects in brain capillaries can control the enrichment of inflammatory pathways that are sensitive to wall shear stress and promote differential expression of a range of transcripts in the BBB, supporting the notion that microbubble dynamics exerted mechanical stress can be used to establish molecular, in addition to spatial, therapeutic windows to target brain diseases. Consistent with these findings, a robust increase in cytotoxic T-cell accumulation in brain tumors was observed, demonstrating the functional relevance and potential clinical significance of the observed immuno-mechano-biological responses.
Assuntos
Barreira Hematoencefálica , Encéfalo , Microbolhas , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos da radiação , Animais , Encéfalo/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Inflamação/metabolismo , Camundongos , Humanos , Estresse Mecânico , Ondas Ultrassônicas , Masculino , Capilares/metabolismo , FemininoRESUMO
The diffusion of oxygen through capillary to surrounding tissues through multiple points along the length has been addressed in many clinical studies, largely motivated by disorders including hypoxia. However relatively few analytical or numerical studies have been communicated. In this paper, as a compliment to physiological investigations, a novel mathematical model is developed which incorporates the multiple point diffusion of oxygen from different locations in the capillary to tissues, in the form of a fractional dynamical system of equations using the concept of system of balance equations with memory. Stability analysis of the model has been conducted using the well known Routh-Hurwitz stability criterion. Comprehensive analytical solutions for the differntial equation problem in the new proposed model are obtained using Henkel transformations. Both spatial and temporal variation of concentration of oxygen is visualized graphically for different control parameters. Close correlation with simpler models is achieved. Diffusion is shown to arise from different points of the capillary in decreasing order along the length of the capillary i.e. for the different values of z. The concentration magnitudes at low capillary length far exceed those further along the capillary. Furthermore with progrssive distance along the capillary, the radial distance of diffusion decreases, such that oxygen diffuses only effectively in very close proximity to tissues. The simulations provide a useful benchmark for more generalized mass diffusion computations with commercial finite element and finite volume software including ANSYS FLUENT.
Assuntos
Capilares , Hipóxia , Oxigênio , Oxigênio/metabolismo , Humanos , Difusão , Capilares/metabolismo , Capilares/fisiologia , Hipóxia/fisiopatologia , Hipóxia/metabolismo , Modelos Biológicos , Simulação por Computador , AnimaisRESUMO
Capillary plexus cultivation is crucial in tissue engineering and regenerative medicine. Theoretical simulations have been conducted to supplement the expensive experimental works. However, the mechanisms connecting mechanical and chemical stimuli remained undefined, and the functions of the different VEGF forms in the culture environment were still unclear. In this paper, we developed a hybrid model for simulating short-term in vitro capillary incubations. We used the Cellular Potts model to predict individual cell migration, morphology change, and continuum mechanics to quantify biogel deformation and VEGF transport dynamics. By bridging the mechanical regulation and chemical stimulation in the model, the results showed good agreement between the predicted network topology and experiments, in which elongated cells connected, forming the network cords and round cells gathered, creating cobblestone-like aggregates. The results revealed that the capillary-like networks could develop in high integrity only when the mechanical and chemical couplings worked adequately, with the cell morphology and haptotaxis driven by the soluble and bound forms of VEGF, respectively, functioning simultaneously.
Assuntos
Capilares , Simulação por Computador , Fator A de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular/metabolismo , Capilares/metabolismo , Humanos , Movimento Celular/fisiologia , Modelos Biológicos , Biologia Computacional , Neovascularização Fisiológica/fisiologia , Engenharia Tecidual/métodosRESUMO
Stroke is not only associated with muscle weakness, but also associated with reduced muscle fatigue resistance and reduced desaturation during exercise that may be caused by a reduced oxidative capacity and/or microvasculature. Therefore, the objective of the present study was to determine the effects of stroke on muscle mass, fiber size and shape, capillarization and oxidative capacity of the rat m. extensor carpi radialis (ECR) and m. flexor carpi ulnaris (FCU) after a photothrombotic stroke in the forelimb region of the primary sensorimotor cortex. The main observation of the present study was that 4 weeks after induction of stroke there were no significant changes in muscle fiber size and shape. Although there was no significant capillary rarefaction, there was some evidence for remodeling of the capillary bed as reflected by a reduced heterogeneity of capillary spacing (p = 0.006) that may result in improved muscle oxygenation. In the ECR, but not in the FCU, this was accompanied by reduction in muscle fiber oxidative capacity as reflected by reduced optical density of sections stained for succinate dehydrogenase (p = 0.013). The reduced oxidative capacity and absence of significant capillary rarefaction resulted in a capillary to fiber ratio per unit of oxidative capacity that was higher after stroke in the ECR (p = 0.01), but not in the FCU. This suggests that at least during the early stages, stroke is not necessarily accompanied by muscle fiber atrophy, and that stroke-induced reductions in oxidative capacity resulting in relative excess of capillarization are muscle specific.
Assuntos
Capilares , Músculo Esquelético , Animais , Masculino , Ratos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/irrigação sanguínea , Capilares/metabolismo , Capilares/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Oxirredução , Ratos Wistar , Ratos Sprague-DawleyRESUMO
Decreased myocardial capillary density has been reported as an important histopathological feature associated with various heart disorders. Quantitative assessment of cardiac capillarization typically involves double immunostaining of cardiomyocytes (CMs) and capillaries in myocardial slices. In contrast, single immunostaining of basement membrane protein is a straightforward approach to simultaneously label CMs and capillaries, presenting fewer challenges in background staining. However, subsequent image analysis always requires expertise and laborious manual work to identify and segment CMs/capillaries. Here, we developed an image analysis tool, AutoQC, for automatic identification and segmentation of CMs and capillaries in immunofluorescence images of basement membrane. Commonly used capillarization-related measurements can be derived from segmentation results. By leveraging the power of a pre-trained segmentation model (Segment Anything Model, SAM) via prompt engineering, the training of AutoQC required only a small dataset with bounding box annotations instead of pixel-wise annotations. AutoQC outperformed SAM (without prompt engineering) and YOLOv8-Seg, a state-of-the-art instance segmentation model, in both instance segmentation and capillarization assessment. Thus, AutoQC, featuring a weakly supervised algorithm, enables automatic segmentation and high-throughput, high-accuracy capillarization assessment in basement-membrane-immunostained myocardial slices. This approach reduces the training workload and eliminates the need for manual image analysis once AutoQC is trained.
Assuntos
Membrana Basal , Processamento de Imagem Assistida por Computador , Miocárdio , Miócitos Cardíacos , Membrana Basal/metabolismo , Animais , Miócitos Cardíacos/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Processamento de Imagem Assistida por Computador/métodos , Capilares/metabolismo , Algoritmos , Camundongos , Vasos Coronários/metabolismo , Vasos Coronários/patologiaAssuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Endoteliais , Hipertensão Pulmonar , Receptor Notch4 , Transdução de Sinais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Animais , Humanos , Receptor Notch4/metabolismo , Receptor Notch4/genética , Reprogramação Celular , Capilares/metabolismo , Capilares/patologia , Camundongos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/citologiaRESUMO
Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5's function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5-/- mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5-/- mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5-/- and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.
Assuntos
Apolipoproteína A-V , Hipertrigliceridemia , Lipase Lipoproteica , Animais , Lipase Lipoproteica/metabolismo , Lipase Lipoproteica/genética , Humanos , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/genética , Apolipoproteína A-V/genética , Apolipoproteína A-V/metabolismo , Capilares/metabolismo , Camundongos , Triglicerídeos/metabolismo , Triglicerídeos/sangueRESUMO
We sought to determine the physiological relevance of pannexin/purinergic-dependent signaling in mediating conducted vasodilation elicited by capillary stimulation through skeletal muscle contraction. Using hamster cremaster muscle and intravital microscopy we stimulated capillaries through local muscle contraction while observing the associated upstream arteriole. Capillaries were stimulated with muscle contraction at low and high contraction (6 and 60CPM) and stimulus frequencies (4 and 40 Hz) in the absence and presence of pannexin blocker mefloquine (MEF; 10-5 M), purinergic receptor antagonist suramin (SUR 10-5 M) and gap-junction uncoupler halothane (HALO, 0.07%) applied between the capillary stimulation site and the upstream arteriolar observation site. Conducted vasodilations elicited at 6CPM were inhibited by HALO while vasodilations at 60CPM were inhibited by MEF and SUR. The conducted response elicited at 4 Hz was inhibited by MEF while the vasodilation at 40 Hz was unaffected by any blocker. Therefore, upstream vasodilations resulting from capillary stimulation via muscle contraction are dependent upon a pannexin/purinergic-dependent pathway that appears to be stimulation parameter-dependent. Our data highlight a physiological importance of the pannexin/purinergic pathway in facilitating communication between capillaries and upstream arteriolar microvasculature and, consequently, indicating that this pathway may play a crucial role in regulating blood flow in response to skeletal muscle contraction.
Assuntos
Capilares , Conexinas , Mesocricetus , Contração Muscular , Músculo Esquelético , Vasodilatação , Animais , Masculino , Conexinas/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Contração Muscular/fisiologia , Capilares/fisiologia , Capilares/metabolismo , Vasodilatação/fisiologia , Transdução de Sinais/fisiologia , Cricetinae , Receptores Purinérgicos/metabolismo , Arteríolas/fisiologia , Arteríolas/metabolismoRESUMO
Capillaries are the smallest blood vessels (<10 µm in diameter) in the body and their walls are lined by endothelial cells. These microvessels play a crucial role in nutrient and gas exchange between blood and tissues. Capillary endothelial cells also produce vasoactive molecules and initiate the electrical signals that underlie functional hyperemia and neurovascular coupling. Accordingly, capillary function and density are critical for all cell types to match blood flow to cellular activity. This begins with the process of angiogenesis, when new capillary blood vessels emerge from pre-existing vessels, and ends with rarefaction, the loss of these microvascular structures. This review explores the mechanisms behind these processes, emphasizing their roles in various microvascular diseases and their impact on surrounding cells in health and disease. We discuss recent work on the mechanisms controlling endothelial cell proliferation, migration, and tube formation that underlie angiogenesis under physiological and pathological conditions. The mechanisms underlying functional and anatomical rarefaction and the role of pericytes in this process are also discussed. Based on this work, a model is proposed in which the balance of angiogenic and rarefaction signaling pathways in a particular tissue match microvascular density to the metabolic demands of the surrounding cells. This negative feedback loop becomes disrupted during microvascular rarefaction: angiogenic mechanisms are blunted, reactive oxygen species accumulate, capillary function declines and eventually, capillaries disappear. This, we propose, forms the foundation of the reciprocal relationship between vascular density, blood flow, and metabolic needs and functionality of nearby cells.
Assuntos
Capilares , Células Endoteliais , Rarefação Microvascular , Neovascularização Patológica , Neovascularização Fisiológica , Transdução de Sinais , Humanos , Animais , Capilares/metabolismo , Capilares/fisiopatologia , Capilares/patologia , Neovascularização Patológica/fisiopatologia , Neovascularização Patológica/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Rarefação Microvascular/fisiopatologia , Rarefação Microvascular/metabolismo , Pericitos/metabolismo , Pericitos/patologia , Proliferação de Células , Movimento Celular , Densidade Microvascular , AngiogêneseRESUMO
OBJECTIVE: This study aimed to clarify the effect of Type I diabetes (DIA) on transcapillary PO2 gradients, which are oxygen-driving factors between the blood and the interstitium, in the contracting muscle of rats. METHODS: Wistar male rats were divided into the diabetic (streptozocin i.p.) and sham groups. Microvascular and interstitial PO2 were measured in the extensor digitorum longus muscle during electrical stimulation-induced muscle contraction, using the phosphorescence quenching method. Transcapillary PO2 gradient, ΔPO2, was calculated as microvascular minus interstitial PO2. RESULTS: Resting microvascular PO2 was higher in the diabetic group than in the sham group (6.3 ± 1.7 vs. 4.7 ± 0.9 mmHg, p < 0.05) and remained for 180 s. Interstitial PO2 from rest to muscle contraction did not differ between the groups. The ΔPO2 was higher in the diabetic group than in the sham group at rest and during muscle contraction (4.03 ± 1.42 vs. 2.46 ± 0.90 mmHg at rest; 3.67 ± 1.51 vs. 2.22 ± 0.65 mmHg during muscle contraction, p < 0.05). Marked muscle atrophy was observed in the diabetic group. CONCLUSION: DIA increased microvascular and transcapillary PO2 gradients in the skeletal muscle. The enhanced PO2 gradients were maintained from rest to muscle contraction in diabetic muscle.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Contração Muscular , Músculo Esquelético , Oxigênio , Ratos Wistar , Animais , Masculino , Ratos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Oxigênio/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/metabolismo , Capilares/metabolismo , Capilares/fisiopatologia , Capilares/patologia , Microcirculação , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Atrofia Muscular/patologiaRESUMO
AIMS/HYPOTHESIS: Almost all beta cells contact one capillary and insulin granule fusion is targeted to this region. However, there are reports of beta cells contacting more than one capillary. We therefore set out to determine the proportion of beta cells with multiple contacts and the impact of this on cell structure and function. METHODS: We used pancreatic slices in mice and humans to better maintain cell and islet structure than in isolated islets. Cell structure was assayed using immunofluorescence and 3D confocal microscopy. Live-cell two-photon microscopy was used to map granule fusion events in response to glucose stimulation. RESULTS: We found that 36% and 22% of beta cells in islets from mice and humans, respectively, have separate contact with two capillaries. These contacts establish a distinct form of cell polarity with multiple basal regions. Both capillary contact points are enriched in presynaptic scaffold proteins, and both are a target for insulin granule fusion. Cells with two capillary contact points have a greater capillary contact area and secrete more, with analysis showing that, independent of the number of contact points, increased contact area is correlated with increased granule fusion. Using db/db mice as a model for type 2 diabetes, we observed changes in islet capillary organisation that significantly reduced total islet capillary surface area, and reduced area of capillary contact in single beta cells. CONCLUSIONS/INTERPRETATION: Beta cells that contact two capillaries are a significant subpopulation of beta cells within the islet. They have a distinct form of cell polarity and both contact points are specialised for secretion. The larger capillary contact area of cells with two contact points is correlated with increased secretion. In the db/db mouse, changes in capillary structure impact beta cell capillary contact, implying that this is a new factor contributing to disease progression.
Assuntos
Capilares , Polaridade Celular , Células Secretoras de Insulina , Insulina , Animais , Camundongos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Capilares/metabolismo , Capilares/patologia , Insulina/metabolismo , Humanos , Polaridade Celular/fisiologia , Secreção de Insulina/fisiologia , Camundongos Endogâmicos C57BL , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/irrigação sanguínea , Masculino , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de DoençasRESUMO
Based on Mader's mathematical model, the rate of capillary blood lactate concentration (νLamax) following intense exercise is thought to reflect the maximal glycolytic rate. We aimed to investigate the reliability of important variables of Mader's model (i.e. power output, lactate accumulation, predominant phosphagen contribution time frames (tP Cr)) and resulting νLamax values derived during and after a 15-s cycling sprint. Fifty cyclists performed a 15-s all-out sprint test on a Cyclus2 ergometer three times. The first sprint test was considered a familiarization trial. Capillary blood was sampled before and every minute (for 8 min) after the sprint to determine νLamax. Test-retest analysis between T2 and T3 revealed excellent reliability for power output (Pmean and Ppeak; ICC = 0.99, 0.99), ∆La and νLamax with tPCr of 3.5 s (ICC = 0.91, 0.91). νLamax calculated with tPCr = tP peak (ICC = 0.87) and tP Cr = tPpeak-3.5% (ICC = 0.79) revealed good reliability. tPpeak and tPpeak-3.5% revealed only poor and moderate reliability (ICC = 0.41, 0.52). Power output and ∆La are reliable parameters in the context of this test. Depending on tPCr, reliability of νLamax varies considerably with tP Cr of 3.5 s showing excellent reliability. We recommend standardization of this type of testing especially tP Cr.
Assuntos
Ciclismo , Ácido Láctico , Humanos , Ciclismo/fisiologia , Masculino , Adulto , Ácido Láctico/sangue , Capilares/fisiologia , Capilares/metabolismo , Reprodutibilidade dos Testes , Teste de Esforço/métodos , Adulto Jovem , FemininoRESUMO
We recently showed that the ratio of capillaries to myofibers in skeletal muscle, which accounts for 80% of insulin-directed glucose uptake and metabolism, was reduced in baboon fetuses in which estrogen was suppressed by maternal letrozole administration. Since vascular endothelial growth factor (VEGF) promotes angiogenesis, the present study determined the impact of estrogen deprivation on fetal skeletal muscle VEGF expression, capillary development, and long-term vascular and metabolic function in 4- to 8-year-old adult offspring. Maternal baboons were untreated or treated with letrozole or letrozole plus estradiol on days 100-164 of gestation (term = 184 days). Skeletal muscle VEGF protein expression was suppressed by 45% (P < 0.05) and correlated (P = 0.01) with a 47% reduction (P < 0.05) in the number of capillaries per myofiber area in fetuses of baboons in which serum estradiol levels were suppressed 95% (P < 0.01) by letrozole administration. The reduction in fetal skeletal muscle microvascularization was associated with a 52% decline (P = 0.02) in acetylcholine-induced brachial artery dilation and a 23% increase (P = 0.01) in mean arterial blood pressure in adult progeny of letrozole-treated baboons, which was restored to normal by letrozole plus estradiol. The present study indicates that estrogen upregulates skeletal muscle VEGF expression and systemic microvessel development within the fetus as an essential programming event critical for ontogenesis of systemic vascular function and insulin sensitivity/glucose homeostasis after birth in primate offspring.
Assuntos
Estradiol , Estrogênios , Letrozol , Músculo Esquelético , Nitrilas , Triazóis , Fator A de Crescimento do Endotélio Vascular , Animais , Feminino , Letrozol/farmacologia , Músculo Esquelético/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Gravidez , Nitrilas/farmacologia , Estrogênios/farmacologia , Estradiol/farmacologia , Triazóis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Papio , Masculino , Feto/metabolismo , Feto/irrigação sanguínea , Feto/efeitos dos fármacos , Capilares/metabolismo , Capilares/efeitos dos fármacos , Inibidores da Aromatase/farmacologiaRESUMO
Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium.
Assuntos
Capilares , Mitocôndrias Musculares , Músculo Esquelético , Sarcolema , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Sarcolema/fisiologia , Animais , Capilares/fisiologia , Capilares/metabolismo , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/ultraestrutura , Músculo Esquelético/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/irrigação sanguínea , Camundongos , Metabolismo Energético/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Potencial da Membrana Mitocondrial/fisiologiaAssuntos
Capilares , Mitocôndrias Musculares , Músculo Esquelético , Oxigênio , Músculo Esquelético/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/irrigação sanguínea , Humanos , Capilares/fisiologia , Capilares/metabolismo , Oxigênio/metabolismo , Mitocôndrias Musculares/metabolismo , AnimaisRESUMO
Overcoming the blood-brain barrier (BBB) is essential to enhance brain therapy. Here, we utilized nanobubbles with focused ultrasound for targeted and improved BBB opening in mice. A microscopy technique method assessed BBB opening at a single blood vessel resolution employing a dual-dye labeling technique using green fluorescent molecules to label blood vessels and Evans blue brain-impermeable dye for quantifying BBB extravasation. A deep learning architecture enabled blood vessels segmentation, delivering comparable accuracy to manual segmentation with a significant time reduction. Segmentation outcomes were applied to the Evans blue channel to quantify extravasation of each blood vessel. Results were compared to microbubble-mediated BBB opening, where reduced extravasation was observed in capillaries with a diameter of 2-6 µm. In comparison, nanobubbles yield an improved opening in these capillaries, and equivalent efficacy to that of microbubbles in larger vessels. These results indicate the potential of nanobubbles to serve as enhanced agents for BBB opening, amplifying bioeffects in capillaries while preserving comparable opening in larger vessels.
Assuntos
Barreira Hematoencefálica , Capilares , Microbolhas , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Capilares/metabolismo , Capilares/efeitos dos fármacos , Camundongos , Azul Evans/administração & dosagem , Sistemas de Liberação de Medicamentos , Aprendizado Profundo , Camundongos Endogâmicos C57BL , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Masculino , Feminino , NanopartículasRESUMO
The integrity of the blood-brain barrier (BBB) is essential for the normal functioning of the central nervous system (CNS). Isolated brain capillaries are essential for analyzing changes in protein and gene expression at the BBB under physiological and pathological conditions. The standard methods for isolating brain capillaries require the use of at least one or more mouse brains in order to obtain sufficient quantity and purity of brain capillaries. Here, we describe an optimized protocol for isolating and purifying capillaries from tiny amounts of mouse cerebral cortex using manual homogenization, density gradient centrifugation, and filtration while preserving the structural integrity and functional activity of microvessel fragments. Western blotting showed that proteins expressed at the BBB were enriched in mouse brain capillaries isolated by the optimized method compared to cerebral cortex protein homogenates. This approach can be used for the analysis of a variety of rare mouse genetic models and can also help the investigators to understand regional differences in susceptibility to pathological phenomena such as ischemia and traumatic brain injury. This will allow the investigators to better understand the physiology and pathology of the BBB.
Assuntos
Encéfalo , Capilares , Camundongos , Animais , Capilares/metabolismo , Encéfalo/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas/metabolismo , Transporte BiológicoRESUMO
Capillaries, composed of electrically coupled endothelial cells and overlying pericytes, constitute the vast majority of blood vessels in the brain. The most arteriole-proximate three to four branches of the capillary bed are covered by α-actin-expressing, contractile pericytes. These mural cells have a distinctive morphology and express different markers compared with their smooth muscle cell (SMC) cousins but share similar excitation-coupling contraction machinery. Despite this similarity, pericytes are considerably more depolarized than SMCs at low intravascular pressures. We have recently shown that pericytes, such as SMCs, possess functional voltage-dependent Ca2+ channels and ATP-sensitive K+ channels. Here, we further investigate the complement of pericyte ion channels, focusing on members of the K+ channel superfamily. Using NG2-DsRed-transgenic mice and diverse configurations of the patch-clamp technique, we demonstrate that pericytes display robust inward-rectifier K+ currents that are primarily mediated by the Kir2 family, based on their unique biophysical characteristics and sensitivity to micromolar concentrations of Ba2+. Moreover, multiple lines of evidence, including characteristic kinetics, sensitivity to specific blockers, biophysical attributes, and distinctive single-channel properties, established the functional expression of two voltage-dependent K+ channels: KV1 and BKCa. Although these three types of channels are also present in SMCs, they exhibit distinctive current density and kinetics profiles in pericytes. Collectively, these findings underscore differences in the operation of shared molecular features between pericytes and SMCs and highlight the potential contribution of these three K+ ion channels in setting pericyte membrane potential, modulating capillary hemodynamics, and regulating cerebral blood flow.
Assuntos
Encéfalo , Capilares , Pericitos , Pericitos/metabolismo , Pericitos/citologia , Animais , Capilares/metabolismo , Capilares/citologia , Camundongos , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/metabolismo , Canais de Potássio/metabolismo , Camundongos Transgênicos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Camundongos Endogâmicos C57BLRESUMO
The capillarization of hepatic sinusoids resulting from the activation of hepatic stellate cells poses a significant challenge, impeding the effective delivery of therapeutic agents to the Disse space for liver fibrosis treatment. Therefore, overcoming these barriers and achieving efficient drug delivery to activated hepatic stellate cells (aHSCs) are pressing challenge. In this study, we developed a synergistic sequential drug delivery approach utilizing neutrophil membrane hybrid liposome@atorvastatin/amlisentan (NCM@AtAm) and vitamin A-neutrophil membrane hybrid liposome @albumin (VNCM@Bai) nanoparticles (NPs) to breach the capillary barrier for targeted HSC cell delivery. Initially, NCM@AtAm NPs were successfully directed to the site of hepatic fibrosis through neutrophil-mediated inflammatory targeting, resulting in the normalization of liver sinusoidal endothelial cells (LSECs) and restoration of fenestrations under the combined influence of At and Am. Elevated tissue levels of the p-Akt protein and endothelial nitric oxide synthase (eNOS) indicated the normalization of LSECs following treatment with At and Am. Subsequently, VNCM@Bai NPs traversed the restored LSEC fenestrations to access the Disse space, facilitating the delivery of Bai into aHSCs under vitamin A guidance. Lastly, both in vitro and in vivo results demonstrated the efficacy of Bai in inhibiting HSC cell activation by modulating the PPAR γ/TGF-ß1 and STAT1/Smad7 signaling pathways, thereby effectively treating liver fibrosis. Overall, our designed synergistic sequential delivery system effectively overcomes the barrier imposed by LSECs, offering a promising therapeutic strategy for liver fibrosis treatment in clinical settings.