Ultrastructural examination of mouse kidney glomerular capillary loop by sandwich freezing and freeze-substitution.

Sandwich freezing is a method of rapid freezing by sandwiching specimens between two metal disks and has been used for observing exquisite the close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed animal and human tissues. In the present study, this method was applied to observe the fine structure of mouse glomerular capillary loops. Morphometry was then performed, and the results were compared with the data obtained by an in vivo cryotechnique, which may provide the closest ultrastructure to the native state of living tissue. The results show that the ultrastructure of glomerular capillary loops obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation was close to that of the ultrastructure obtained by in vivo cryotechnique not only in the quality of cell image but also in quantitative morphometry. They indicate that the ultrastructure obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation may more closely reflect the living state of cells and tissues than conventional chemical fixation and dehydration at room temperature and conventional rapid freezing-freeze-substitution of excised tissues without glutaraldehyde fixation. Sandwich freezing-freeze-substitution techniques should be used routinely as a standard method for observing the close-to-native ultrastructure of biological specimens.

Capillary pathology with prominent basement membrane reduplication is the hallmark histopathological feature of scleromyositis.

AIMS: We aim to perform ultrastructural and histopathological analysis of muscle biopsies from a large group of systemic sclerosis (SSc) patients, including some with early/mild SSc features, and examine whether capillary pathology differentiates 'scleromyositis' (SM) from other auto-immune myositis (AIM) subsets. METHODS: Muscle biopsies from a total of 60 SM patients and 43 AIM controls from two independent cohorts were examined by electron microscopy, collagen-4 immunofluorescence (Col4IF) and routine light microscopy. RESULTS: Ultrastructural examination revealed prominent capillary basement membrane (BM) reduplication (4+ layers in >50% of capillaries) in 65% of SM vs 0% of AIM controls (p < 0.001). In SM cases without prominent BM reduplication, capillary dilation was the most distinctive feature, present in 8% of capillaries in SM vs 2% in controls (p = 0.001). Accumulation of ensheathed pericyte processes was another characteristic feature of SM and closely correlated with the degree of BM reduplication (r = 0.833, p < 0.001). On light microscopy, BM marker Col4IF revealed more frequent capillary enlargement in SM than in controls (84% vs 21%, p < 0.001). SM cases were classified as non-inflammatory myopathy (36%), non-specific myositis (33%) or immune-mediated necrotizing myopathy (31%), but despite this histopathological heterogeneity, prominent BM reduplication remained a constant finding. In the 16 SM patients with early/mild SSc features, 63% showed prominent BM reduplication. CONCLUSIONS: These results show that capillary pathology, and in particular prominent capillary BM reduplication, is the hallmark histopathological feature of SM even in patients with early/mild SSc and support the concept of SM as an organ manifestation of SSc and a distinct subset of AIM.

Plasmalemmal Vesicle-Associated Protein Is Associated with Endothelial Cells Sprouting from the Peribiliary Capillary Plexus in Human Cirrhotic Liver.

INTRODUCTION: Plasmalemmal vesicle-associated protein (PLVAP) is an endothelial-specific integral membrane glycoprotein that localizes to caveolae and fenestrae in animal models; however, little is known about PLVAP in endothelial cells (ECs) in hepatic sinusoids during liver cirrhosis (LC). Here, we aimed to elucidate PLVAP localization and expression in the human liver during LC progression. METHODS: PLVAP protein expression was detected in specimens from normal control livers and hepatitis C-related cirrhotic livers using immunohistochemistry, Western blotting, and immunoelectron microscopy. RESULTS: PLVAP mainly localized to the peribiliary capillary plexus (PCP) and was rarely observed in hepatic artery branches and portal venules in control tissue, but was aberrantly expressed in capillarized sinusoids and proliferated capillaries in fibrotic septa within cirrhotic liver tissue. Ultrastructural analysis indicated that PLVAP localized to thin ECs in some caveolae, whereas PLVAP localized primarily to caveolae-like structures and proliferative sinusoid capillary EC vesicles in cirrhotic liver tissue. Western blot analysis confirmed that PLVAP was overexpressed at the protein level in advanced cirrhotic liver tissue. CONCLUSION: PLVAP was strongly expressed in the caveolae of proliferated capillaries directly connected with sinusoids linked with the PCP, suggesting that it plays a role in angiogenesis and sinusoidal remodeling in LC.

Ultrastructure of Endothelial Cells of Myocardial Capillaries in Burn Septicotoxemia.

We studied ultrastructure and vesicular structures in endothelial cells of myocardial micro-vessels in burn patients. Electron microscopy revealed a significant decrease in volume density of vesicular structures in the endotheliocytes of myocardial capillaries in patients with burn septicotoxemia. The observed structural signs of endothelial dysfunction revealed in this category of patients can be a promising area for further research and for the development of methods of pathogenetic correction of myocardial disorders in the case of burn injury.

VDAC1 regulates mitophagy in NLRP3 inflammasome activation in retinal capillary endothelial cells under high-glucose conditions.

Diabetic retinopathy (DR) has been considered to involve mitochondrial alterations and be related to the nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome activation. The voltage-dependent anion channel 1 (VDAC1) protein is one of the key proteins that regulates the metabolic and energetic functions of the mitochondria. To explore the involvement of VDAC1 in mitophagy regulation of NLRP3 inflammasome activation under high-glucose (HG) conditions, this study examined expressions of VDAC1, mitochondrial function and mitophagy-related proteins, and NLRP3 inflammasome-related proteins in human retinal capillary endothelial cells (HRCECs) cultured with 30 mM of glucose in the presence or absence of mitophagy inhibitor (Mdivi-1) using Western blot. Mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were detected using flow cytometry. GFP-tagged pAdTrack-VDAC1 adenovirus was used to overexpress VDAC1. Cell biological behaviors, including proliferation, migration, tubule formation, and apoptosis, were also observed. Our results showed that when compared to the normal glucose and high mannitol groups, increased amounts of mitochondrial fragments, reduced mitochondrial membrane potential, increased expression of mitochondrial fission protein Drp 1, decreased expression of mitochondrial fusion protein Mfn 2, accumulation of mtROS, and activation of the NLRP3 inflammasome were observed in the HG group. Meanwhile, HG markedly reduced the protein expressions of PINK1, Parkin and VDAC1. Inhibition of mitophagy reduced PINK1 expression, enhanced NLRP3 expression, but failed to alter VDAC1. VDAC1 overexpression promoted PINK1 expression, inhibited NLRP3 activation and changed the cell biological behaviors under HG conditions. These findings demonstrate that VDAC1-mediated mitophagy plays a crucial role in regulating NLRP3 inflammasome activation in retinal capillary endothelial cells under HG conditions, suggesting that VDAC1 may be a potential target for preventing or treating DR.

Age-related ultrastructural neurovascular changes in the female mouse cortex and hippocampus.

Blood-brain barrier (BBB) breakdown occurs in aging and neurodegenerative diseases. Although age-associated alterations have previously been described, most studies focused in male brains; hence, little is known about BBB breakdown in females. This study measured ultrastructural features in the aging female BBB using transmission electron microscopy and 3-dimensional reconstruction of cortical and hippocampal capillaries from 6- and 24-month-old female C57BL/6J mice. Aged cortical capillaries showed more changes than hippocampal capillaries. Specifically, the aged cortex showed thicker basement membrane, higher number and volume of endothelial pseudopods, decreased endothelial mitochondrial number, larger pericyte mitochondria, higher pericyte-endothelial cell contact, and increased tight junction tortuosity compared with young animals. Only increased basement membrane thickness and pericyte mitochondrial volume were observed in the aged hippocampus. Regional comparison revealed significant differences in endothelial pseudopods and tight junctions between the cortex and hippocampus of 24-month-old mice. Therefore, the aging female BBB shows region-specific ultrastructural alterations that may lead to oxidative stress and abnormal capillary blood flow and barrier stability, potentially contributing to cerebrovascular diseases, particularly in postmenopausal women.

Inhibiting Th1/2 cells influences hepatic capillarization by adjusting sinusoidal endothelial fenestrae through Rho-ROCK-myosin pathway.

CD4+ T cells are considered to be vital in chronic liver diseases, but their exact roles in hepatic capillarization, the typical characteristic of liver fibrosis, are poorly understood. This study aimed to assess the roles of typical subtype of CD4+ T cells, named T helper 1 (Th1) and Th2 cells in liver fibrosis. Taking advantage of well established fibrotic rat model, we conducted in vitro and in vivo experiments to explore the interactions between liver sinusoidal endothelial cells (LSECs) and Th1/2 cells; meanwhile we evaluated the degree of hepatic capillarization when inhibiting these interactions with inhibitory antibodies. Our results showed that prohibiting interactions between Th2 cells and LSECs caused the restoration of fenestrae, increased cytokine level of Th1 cells and reduction of hepatic capillarization; inhibiting the interaction between Th1 cells and LSECs produced the opposite effects. Moreover, increased Rho and myosin light chain phosphorylation were observed when Th1 cells were inhibited with the corresponding inhibitory antibody; Th2 cell inhibition yielded the opposite results. This study indicated that Th1/2 cells steer the capillarization process in different directions and this effect is probably mediated by the Rho-Rho kinase (ROCK)-myosin signaling pathway.

Clear Cell Change in Reactive Angiogenesis: A Potential Diagnostic Pitfall.

Reactive angiogenesis is commonplace, occurs in many circumstances, and is important in the repair of injured tissue. Histologically, it is characterized by newly formed capillaries arranged in a lobular architecture and lined by plump endothelial cells. We have encountered a form of reactive angiogenesis not well described; composed of large endothelial cells with abundant clear cytoplasm that causes diagnostic challenges. The cohort includes 10 patients, aged 4 to 61, mean 40 years; 7 males, 3 females. One case involved bone (ilium), and 9 involved soft tissue: fingers (n=2), toes (n=2), hip joint (n=1), shoulder (n=1), thigh (n=2), and anal mucosa (n=1). Clinically, the patients had chronic ulcers, osteomyelitis, or localized infection. All cases exhibited a lobular proliferation of capillaries lined by large polyhedral endothelial cells that obscured the vessel lumens and were admixed with acute and chronic inflammation. The endothelial nuclei were vesicular with small nucleoli and the cytoplasm was abundant and clear or palely eosinophilic. The endothelial cells were stained with CD31 and ERG (7/7 cases), CD34 (6/6), FLI1 (4/4), and were negative for keratin and CD68 (6/6). Periodic acid-Schiff stain and periodic acid-Schiff stain-diastase on 3 cases did not demonstrate glycogen. Using a polymerase chain reaction, no Bartonella henselae was found in all 6 cases tested. Reactive angiogenesis with clear cell change unassociated with Bartonella spp. has not been described. It causes diagnostic challenges and the differential diagnosis includes benign and malignant tumors, as well as unusual infections. It is important to distinguish between these possibilities because of the significant impact on treatment and prognosis.

The architecture of alveolar capillaries in the lungs of bactrian camels (Camelus bactrianus) / Arquitectura de los capilares alveolares de pulmones de camellos bactrianos (Camelus bactrianus)

SUMMARY: The Bactrian camel, which is native to China and Mongolia, is large in size and is an even-toed ungulate species. The double humps on the Bactrian camel back differentiate it from the dromedary camel, which has a single hump. This species has adapted to unsuitable conditions (lack of food and water) in the Gobi Desert and is advanced in unique anatomical and physiological characteristics during a prolonged evolution period. Several studies have been conducted on the anatomical features of the Bactrian camel, but none have given attention to the alveolar capillaries of the Bactrian camel lung. Therefore, the current study aims to explore the architecture of the alveolar capillary in the Bactrian camel lung and further explain the mechanism of blood flow in its lung. The current study extracted and examined the architecture of the alveolar capillary in the lung of the Bactrian camel (Camelus bactrianus) and further explained the mechanism of blood flow by performing lung casting and replica scanning electron microscopy methods. The reports showed that the resources of the alveolar-capillary originated from the capillaries of the subpleural space or interlobular septulum, sometimes originating from the precapillary arterioles or directly from the terminal arterioles. The alveolar capillaries anastomosed and formed a single layer of dense, basket-like network surrounding the alveolus. The mash diameter of the alveolar-capillary network was larger than that of the capillary, and the appearance of the mash was oval and elliptical. Many of the collapsed alveolar-capillary networks were found in the alveolar microvascular architecture in the lung of the Bactrian camel. The study found that, due to many collapsed alveoli in the Bactrian camel lung, the disproportional pressure between the pulmonary alveoli induced less imbalance of blood flow in the alveolar capillary, which affected the gas exchange efficiency. Therefore, the function of the anastomosing capillary branch was likely to regulate the blood flow between the alveolar-capillary network.

RESUMEN: El camello bactriano, es originario de China y Mongolia, es de gran tamaño y es una especie de ungulado de dedos pares. Las dobles jorobas del lomo del camello bactriano lo diferencian del dromedario, que tiene una sola joroba. Esta especie se ha adaptado a condiciones inadecuadas (falta de alimento y agua) en el desierto de Gobi y ha avanzado en características anatómicas y fisiológicas únicas durante un período de evolución prolongado. Se han realizado varios estudios sobre las características anatómicas del camello bactriano, pero ninguno ha prestado atención a los capilares alveolares del pulmón de este animal. Por lo tanto, el presente estudio tuvo como objetivo principal explorar la arquitectura del capilar alveolar en el pulmón del camello bactriano y explicar el mecanismo del flujo sanguíneo. A partir de nuestro trabajo se examinó la arquitectura del capilar alveolar en el pulmón del camello bactriano (Camelus bactrianus) mediante la realización de métodos de microscopía electrónica de barrido y escaneo pulmonar. Los informes mostraron que los recursos del alvéolo-capilar se originaban en los capilares del espacio subpleural o del tabique interlobulillar y a veces se originaban en las arteriolas precapilares o directamente en las arteriolas terminales. Los capilares alveolares se anastomosaban y formaban una densa red de capa única en forma de cesta que rodeaba el alvéolo. El diámetro del macerado de la red alveolar-capilar era mayor que el del capilar y el aspecto del macerado era ovalado y elíptico. Muchas de las redes alvéolo-capilares colapsadas se encontraron en la arquitectura microvascular alveolar en el pulmón del camello bactriano. El estudio encontró que, muchos alvéolos colapsados en el pulmón del camello bactriano, la presión desproporcionada entre los alvéolos pulmonares inducía un menor desequilibrio del flujo sanguíneo en el capilar alveolar, lo que afectaba la eficiencia del intercambio de gases. Por lo tanto, la función de la rama capilar anastomosante probablemente regularía el flujo sanguíneo entre la red alveolar-capilar.

Selective induction of sprouting and intussusception is associated with the concentration distributions of oxygen and hypoxia-induced VEGF.

Sprouting and intussusception are two important modes of capillary angiogenesis, the mechanisms of selective induction of which remain unclear. In this study, we focus on the two developing tissues of yolk sac and skeletal muscle of 2-week-old rat and try to explain the mechanisms to induce selectively sprouting and intussusception in a new way to combine numerical calculation, experimental observations and schematic simulation. We propose the concept of capillary network unit and show that the concentration and gradient of oxygen/hypoxia-induced VEGF around straight segments are lower/higher than that around vascular bifurcations; sprouting mainly occurs at straight segments and intussusception at vascular bifurcations. The results indicate that the locations susceptible to sprouting and intussusception are determined by the distribution characteristics of oxygen/hypoxia-induced VEGF in the capillary network unit. Furthermore, it is considered that the flow dynamics at these locations also play important roles, namely laminar flow at straight segments promotes sprouting, and flow disruption at bifurcations promotes intussusception. Our work suggests the presence of the location preference for sprouting and intussusception, and provides a new research perspective to reveal its core mechanisms.

Role of ultrastructural determinants of glomerular permeability in ultrafiltration function loss.

The epithelial filtration slit is a crucial component of the glomerular capillary membrane, which is essential for maintaining glomerular filtration function. Though chronic kidney diseases are an immense clinical problem, the mechanisms through which structural alterations reduce glomerular water filtration have not yet been understood completely. To investigate the mechanisms underlying filtration function loss, we studied rats with spontaneously occurring progressive kidney disease, either treated with angiotensin II antagonist or untreated, combining high-resolution electron microscopy of the glomerular capillary wall with theoretical water filtration modeling. Under pathological conditions, epithelial filtration pores and the extension of the subpodocyte space were larger than in normal controls. Numerical analyses indicated that these ultrastructural changes increased hydraulic resistance of the glomerular capillary wall by extending coverage of the filtration barrier by the subpodocyte space, with the changes in hydrodynamic forces acting on podocytes likely being responsible for their detachment. Angiotensin II inhibition normalized the subpodocyte space's hydraulic resistance, restored mechanical podocyte load, and preserved CD151-α3 integrin complex assembly, improving podocyte adherence and survival. Our results show that ultrastructural changes in podocytes are major determinants of the hydraulic resistance of the glomerular capillary wall and highlight the mechanism of podocyte loss in kidney disease progression, as well as the mechanisms underlying angiotensin II inhibition.

A Developmental Analysis of Juxtavascular Microglia Dynamics and Interactions with the Vasculature.

Microglia, a resident CNS macrophage, are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared with older ages and that the timing of these associations is dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages associate with vascular areas void of astrocyte endfeet, and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted on the arrival of astrocyte endfeet such that juxtavascular microglia become highly stationary and stable in the mature cortex.SIGNIFICANCE STATEMENT We report the first extensive analysis of juxtavascular microglia in the healthy, developing, and adult brain. Live imaging revealed that juxtavascular microglia within the cortex are highly motile and migrate along vessels as they are colonizing cortical regions. Using confocal, expansion, super-resolution, and electron microscopy, we determined that microglia associate with the vasculature at all ages in areas lacking full astrocyte endfoot coverage and motility of juxtavascular microglia ceases as astrocyte endfeet more fully ensheath the vasculature. Our data lay the fundamental groundwork to investigate microglia-astrocyte cross talk and juxtavascular microglial function in the healthy and diseased brain. They further provide a potential mechanism by which vascular interactions facilitate microglial colonization of the brain to later regulate neural circuit development.

[MICRO- AND ULTRASTRUCTURAL RECONSTRUCTION OF THE RAT SUBMANDIBULAR GLAND IN THE LATE STAGES OF EXPERIMENTAL TYPE 2 DIABETES MELLITUS].

Electron microscopic investigations of the animals' submandibular gland, conducted in 6 weeks of the experiment, established that ultrastructural changes increase in glandular cells of terminal secretory units in comparison with early period of the experiment. Serocytes have osmiophilic, rather small or picnotic nuclei. Perinuclear spaces of karyolemma are uneven, external nuclear membrane forms local protrusions. Electron density of the karyoplasm is significant, appearing homogenous, nuclei are not observed. Evident submicroscopic changes in blood capillaries of the submandibular gland in experimental diabetes mellitus indicate the impairment of blood-tissue barrier and transcapillary exchange. Deep destructive modifications of all branches of microcirculatory blood flow of the submandibular gland are observed in 8-week course of experimental diabetes mellitus. As compared with the 6th week of investigation, a reliable slight dilation of organ artery diameter, dilation of the diameter of interlobular arterioles, dilation of the diameter of intralobular (precapillary) arteriole and dilation of the capillary diameter were observed. Dilation, as compared to 6th weeks of the experiment, of postcapillary venules was observed. Compared to the indices of the 6th weeks of the experiment, an index of trophic activity of the submandibular gland tissuereaches its maximum meaning and an index of packing density of the capillaries reaches its minimum meaning. Capillary network loses delicate, tortuous pattern and often breaks due to destruction of the capillary component. Arteriovenous anastomoses dilate and blood from the arterioles flows into the venous bed avoiding destructed capillaries. Venules are dilated; thin-walled, retained fragments of the capillaries are significantly dilated in some areas. Swelling of connective tissue stroma and significant swelling of the interstitium are observed. Walls of the capillaries and venules are deformed. The walls of the arterioles are thickened due to plasmorrhagia, sclerosis and hyalinosis.

Sustained Ultrastructural Changes in Rat Hippocampal Formation After Repeated Electroconvulsive Seizures.

BACKGROUND: Electroconvulsive therapy (ECT) is a highly effective and fast-acting treatment for depression used in the clinic. Its mechanism of therapeutic action remains uncertain. Previous studies have focused on documenting neuroplasticity in the early phase following electroconvulsive seizures (ECS), an animal model of ECT. Here, we investigate whether changes in synaptic plasticity and nonneuronal plasticity (vascular and mitochondria) are sustained 3 months after repeated ECS trials. METHODS: ECS or sham treatment was given daily for 1 day or 10 days to a genetic animal model of depression: the Flinders Sensitive and Resistant Line rats. Stereological principles were employed to quantify numbers of synapses and mitochondria as well as length of microvessels in the hippocampus 24 hours after a single ECS. Three months after 10 ECS treatments (1 per day for 10 days) and sham-treatment, brain-derived neurotrophic factor and vascular endothelial growth factor protein levels were quantified with immunohistochemistry. RESULTS: A single ECS treatment significantly increased the volume of hippocampal CA1-stratum radiatum, the total length of microvessels, mitochondria number, and synapse number. Observed changes were sustained as shown in the multiple ECS treatment group analyzed 3 months after the last of 10 ECS treatments. CONCLUSION: A single ECS caused rapid effects of synaptic plasticity and nonneuronal plasticity, while repeated ECS induced long-lasting changes in the efficacy of synaptic plasticity and nonneuronal plasticity at least up to 3 months after ECS.

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease.

Mouse models of age-related macular degeneration (AMD) such as Ccl2-/- and Ccl2-/-/Cx3cr1-/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch's membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD's pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch's membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

Consequences of being phenotypically mismatched with the environment: rapid muscle ultrastructural changes in cold-shocked black-capped chickadees (Poecile atricapillus).

Phenotypic flexibility has received considerable attention in the last decade; however, whereas many studies have reported amplitude of variation in phenotypic traits, much less attention has focused on the rate at which traits can adjust in response to sudden changes in the environment. We investigated whole animal and muscle phenotypic changes occurring in black-capped chickadees (Poecile atricapillus) acclimated to cold (-5°C) and warm (20°C) temperatures in the first 3 h following a 15°C temperature drop (over 3 h). Before the temperature change, cold-acclimated birds were consuming 95% more food, were carrying twice as much body fat, and had 23% larger pectoralis muscle fiber diameters than individuals kept at 20°C. In the 3 h following the temperature drop, these same birds altered their pectoralis muscle ultrastructure by increasing the number of capillaries per fiber area and the number of nuclei per millimeter of fiber by 22%, consequently leading to a 22% decrease in myonuclear domain (amount of cytoplasm serviced per nucleus), whereas no such changes were observed in the warm-acclimated birds. To our knowledge, this is the first demonstration of such a rapid adjustment in muscle fiber ultrastructure in vertebrates. These results support the hypothesis that chickadees maintaining a cold phenotype are better prepared than warm-phenotype individuals to respond to a sudden decline in temperature, such as what may be experienced in their natural wintering environment.

Capillary facilitation of skeletal muscle function in health and disease.

Skeletal muscle is highly vascularized, with perfusion being tightly regulated to meet wide-ranging metabolic demands. For decades, the capillary supply has been explored mainly in terms of evaluating the capillary numbers and their function in the supply of oxygen and substrates and the removal of metabolic byproducts. This review will focus on recent discoveries concerning the role played by capillaries in facilitating other aspects of cell regulation and maintenance, in health and disease, as well as alterations during the aging process. Novelty Capillaries play a central role in the coordination of the vascular response that controls blood flow during contraction and the cellular responses to which they feed into. Nitric oxide is an important regulatory compound within the cardiovascular system, and a significant contributor to skeletal muscle capillary angiogenesis and vasodilatory response to agonists. The microvascular network between muscle fibres may play a critical role in the distribution of signalling factors necessary for optimal muscle satellite cell function.

Fenestral diaphragms and PLVAP associations in liver sinusoidal endothelial cells are developmentally regulated.

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.

Clinical and ultrastructural findings in an ataxic variant of Kufor-Rakeb syndrome.

INTRODUCTION: Kufor-Rakeb syndrome (KRS) is a rare autosomal recessive neurodegenerative disorder manifesting as juvenile-onset atypical parkinsonism with pyramidal signs, supranuclear gaze palsy, dementia and characteristic minimyoclonus, with a notable phenotype variability. The responsible gene ATP13A2 was also associated with hereditary spastic paraplegia, uncomplicated early - or late-onset parkinsonism and a form of neuronal ceroid lipofuscinosis. We present clinical and ultrastructural findings in a 28-year-old woman with novel biallelic ATP13A2 mutations. MATERIAL AND METHODS: An ultrastructural study of the skin and muscle sample was carried out. Sequence analysis of all protein coding exons and exon-intron boundaries of genes was performed on patient's genomic DNA. A proprietary oligonucleotide-selective sequencing method was used for capturing genomic targets and sequencing was performed using Illumina sequencing system. RESULTS: The patient presented with juvenile-onset progressive parkinsonian syndrome and cognitive deterioration, accompanied by mild spastic paraplegia, supranuclear gaze palsy, cerebellar syndrome, peripheral neuropathy and fine myoclonus. Plentiful and varied osmiophilic deposits were found in skin and muscle biopsy. Sequence analysis identified two novel heterozygous variants in ATP13A2: a nonsense variant c.2209C>T, p.(Gln737*) and a 2-bp deletion c.2366_2367delTC, p.(Leu789Argfs*15) causing a frameshift leading to a premature stop codon. Oral levodopa treatment was initiated resulting in marked improvement of bradykinesia, rigidity, speech and swallowing. CONCLUSIONS: We report two novel ATP13A2 pathogenic mutations, further expanding the phenotype of Kufor-Rakeb syndrome with the unusual features of ataxia and polyneuropathy. We thoroughly describe ultrastructural findings and document a meaningful response to levodopa.