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Fluorescence microscopic localization of F-actin, a -tubulin, and vimentin in the central canal. The ependymocytes of the central canal contain peripherally distributed F-actin ( A,D ). In the apical region, F-actin is arranged into a ring-like structure surrounding the lumen of the central canal. The lateral cell margins are also less intensely labeled. Positive signals for a -tubulin are seen predominantly in the apical region of the central canal, where they are present in the cytoplasmic protrusions of the ependymocytes (arrow in B ). C is merged from A and B . No F-actin labeling is found in the a -tubulin – positive structures protruding into the lumen (arrow in C ). Nuclei were stained blue with 4 ′ -6-diamidino-2-phenylindole (DAPI). Vimentin-positive components are organized into a polarized network in the central canal ( E ). In the apical region, vimentin filaments run circumferentially beneath the apical F-actin network. Basally, they extend along the peripheral processes of ependymocytes into the subependymal zone with a root-like appearance. F is merged from D and E . The inset shows the colocalization (yellow color) of vimentin with F-actin in the outer boundary of the F-actin ring. Nuclei were stained blue with DAPI. Bar in inset 5 5 m m.
Source publication
The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeli...
Contexts in source publication
Context 1
... Microscopic Studies on F-actin, Microtubules, and Intermediate Filaments in the Ependymocytes In the central canal, the apical region of the ependymo- cytes was intensely stained with FITC-conjugated phal- loidin ( Figures 1A and 1D). In transversely cut sections, apically localized F-actin formed a ring-like structure, surrounding the lumen of the central canal. ...
Context 2
... in the central canal was located mainly in the cytoplasmic protrusions of ependymocytes ( Figure 1B). Double labeling showed that the positive structures protruding into the lumen did not contain F-actin ( Figure 1C). ...
Context 3
... in the central canal was located mainly in the cytoplasmic protrusions of ependymocytes ( Figure 1B). Double labeling showed that the positive structures protruding into the lumen did not contain F-actin ( Figure 1C). Cytokeratins were not demon- strated immunocytochemically in the central canal with either anti-cytokeratin 8/18 or anti-pan cytokeratin antibodies (not shown). ...
Context 4
... were not demon- strated immunocytochemically in the central canal with either anti-cytokeratin 8/18 or anti-pan cytokeratin antibodies (not shown). Instead, vimentin was found as the major intermediate filamentous component (Figure 1E). Apically localized vimentin filaments ran circumferentially beneath the apical F-actin network, and partially overlapped in the outer boundary of the F-actin ring ( Figure 1F). ...
Context 5
... vimentin was found as the major intermediate filamentous component (Figure 1E). Apically localized vimentin filaments ran circumferentially beneath the apical F-actin network, and partially overlapped in the outer boundary of the F-actin ring ( Figure 1F). From this circumferential net- work, vimentin filament bundles radiated into the sub- ependymal zone and formed a root-like structure. ...
Citations
... In addition, the actin protein skeleton is relatively well preserved in the septum likely conducive to cilia regeneration. 48 iScience Article regulating the niche of EPC in the promotion of cytoskeleton remodeling and motile cilia recovery by certain adjuvant interventions may have far-reaching clinical significance in promoting CSF circulation, syringomyelia resolution, and the overall parenchyma microenvironment. ...
Syringomyelia is a common clinical lesion associated with cerebrospinal fluid flow abnormalities. By a reversible model with chronic extradural compression to mimic human canalicular syringomyelia, we explored the spatiotemporal pathological alterations during syrinx development. The most dynamic alterations were observed in ependymal cells (EPCs), oligodendrocyte lineage, and microglia, as a response to neuroinflammation. Among different cell types, EPC subtypes experienced obvious dynamic alterations, which were accompanied by ultrastructural changes involving the ependymal cytoskeleton, cilia, and dynamic injury in parenchyma primarily around the central canal, corresponding to the single-cell transcripts. After effective decompression, the syrinx resolved with the recovery of pathological damage and overall neurological function, implying that for syringomyelia in the early stage, there was still endogenous repair potential coexisting with immune microenvironment imbalance. Ependymal remodeling and cilia restoration might be important for better resolution of syringomyelia and parenchymal injury recovery.
... Apical microvilli found in LFCs and LPCs resembled those of ependymal cells. The microvilli of ependymocytes, which are considered to be the source of multipotent adult NSCs (Gritti et al., 2002;Lobo et al., 2003), provide the capability for cells to adapt to different external conditions (Li et al., 2009). It is possible that the microvilli of outer sheet cover cells of these spheroids have a similar function, which allows cells to become organized and positioned against external forces. ...
Neurospheres (NS) derived from adult stem cells of non‐neural tissues represent a promising source of neural stem cells (NSCs) and neural progenitor cells (NPCs) for autologous cell therapy. Knowing the fine structure of NS cells is essential for characterizing them during differentiation or oncogenic transformation. NS are generated by culturing ovarian cortical cells (OCCs) under specific conditions. To establish whether these OCCs exhibited a similar morphophenotype as those from the central nervous system (CNS) reported in the literature, sheep OCCs were cultured for 21 days to generate NS. Expression levels of pluripotency (Nanog, octamer‐binding transcription factor 4 [Oct4], and SRY‐box transcription factor 2 [Sox2]) and NSCs/NPCs (nestin, paired box 6 [Pax6], and p75 neurotrophin receptor [P75NTR]) transcripts were analyzed by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), the NSC/NPC antigens were immunolocalized, and structural and ultrastructural analyses were performed in OCC‐NS on Days 10, 15, and 21 in culture. Spheroids expressed transcripts and antigens of pluripotency as well as NSCs/NPCs. Cells were arranged into an inner core, with frequent apoptotic and degenerative events, and a peripheral epithelial‐like cover with abundant cytoplasmic organelles, apical microvilli, and filament bundles of cytoskeleton elements. Adherens junctions and apical tight and lateral loose interdigitations were found in peripheral cells that eventually lost apical–basal polarization, which might indicate their disengaging/aggregating from/to the NS. We can conclude that OCC‐NS shares the most structural and ultrastructural characteristics with CNS‐NS.
... Contiguous bands of tracer were detected between the subependymal microvasculature and the central canal ependymal cells. To confirm and further examine this putative pathway, the next step is to fluorescently label ependymal cells (such as with F-actin [24]) in future intravital studies of influx and efflux. Tanycytes and complex ependymal basement membranes (labyrinths) are thought to subserve this putative connection between CSF, ISF and the central canal [25]. ...
Background:
Disruption of cerebrospinal fluid (CSF)/interstitial fluid (ISF) exchange in the spinal cord is likely to contribute to central nervous system (CNS) diseases that involve abnormal fluid accumulation, including spinal cord oedema and syringomyelia. However, the physiological factors that govern fluid transport in the spinal cord are poorly understood. The aims of this study were to determine the effects of cardiac pulsations and respiration on tracer signal increase, indicative of molecular movement following infusion into the spinal cord grey or white matter.
Methods:
In Sprague Dawley rats, physiological parameters were manipulated such that the effects of spontaneous breathing (generating alternating positive and negative intrathoracic pressures), mechanical ventilation (positive intrathoracic pressure only), tachycardia (heart atrial pacing), as well as hypertension (pharmacologically induced) were separately studied. Since fluid outflow from the spinal cord cannot be directly measured, we assessed the molecular movement of fluorescent ovalbumin (AFO-647), visualised by an increase in tracer signal, following injection into the cervicothoracic spinal grey or white matter.
Results:
Tachycardia and hypertension increased AFO-647 tracer efflux, while the concomitant negative and positive intrathoracic pressures generated during spontaneous breathing did not when compared to the positive-pressure ventilated controls. Following AFO-647 tracer injection into the spinal grey matter, increasing blood pressure and heart rate resulted in increased tracer movement away from the injection site compared to the hypotensive, bradycardic animals (hypertension: p = 0.05, tachycardia: p < 0.0001). Similarly, hypertension and tachycardia produced greater movement of AFO-647 tracer longitudinally along the spinal cord following injection into the spinal white matter (p < 0.0001 and p = 0.002, respectively). Tracer efflux was strongly associated with all blood vessel types.
Conclusions:
Arterial pulsations have profound effects on spinal cord interstitial fluid homeostasis, generating greater tracer efflux than intrathoracic pressure changes that occur over the respiratory cycle, demonstrated by increased craniocaudal CSF tracer movement in the spinal cord parenchyma.
... We have previously developed an ultrastructual detection method of F-actin by use of a FITC-specific antibody and a highly probe specific for F-actin, phalloidin (Li et al., 2009(Li et al., , 2010Tan et al., 2017). In the present study, this technique was used to investigate the subcellular distribution of F-actin within the myelinated axons. ...
... Phalloidin staining and electron microscopy. The animals were perfused, and processed as described previously by us (Li et al., 2009). Spinal cords, DRGs, and spinal nerves were dissected out at the level of L4 ~ L5, and cut into sections about 50 mm thick by a vibratome. ...
... Spinal cords, DRGs, and spinal nerves were dissected out at the level of L4 ~ L5, and cut into sections about 50 mm thick by a vibratome. The sections were then stained with FITC-conjugated phalloidin, which were further converted to peroxidase/DAB products by a FITC-anti-FITC system (Li et al., 2009). ...
Despite the existence of a large amount of actin in the axons, the concentration F-actin was quite low in the myelinated axons and almost all the F-actin were located in the peripheries of the myelinated axons. Until now, the ultrastructural localization of F-actin has still not been reported in the myelinated axons, probably due to the lack of an appropriate detection method. In the present study, a phalloidin-based FITC-anti-FITC technique was adopted to investigate the subcellular localization of F-actin in the myelinated axons. By using this technique, F-actin is located in the outer and inner collars of myelinated cytoplasm surrounding the intermodal axon, the Schmidt-Lanterman incisures, the paranodal terminal loops and the nodal microvilli. In addition, the satellite cell envelope, which encapsulates the axonal initial segment of the peripheral sensory neuron, was also demonstrated as an F-actin-enriched structure. This study provided a hitherto unreported ultrastructural view of the F-actin in the myelinated axons, which may assist in understanding the unique organization of axonal actin cytoskeleton.
... In an uncomplicated early stage, findings like an altered sense of smell or hyposmia can be found. Once the virus caused respiratory manifestation, there will be neurological involvement with loss of involuntary control over breathing (90). ...
The newly discovered coronavirus disease 2019 (COVID-19) is an infectious disease introduced to humans for the first time. Following the pandemic of COVID-19, there is a major shift of practices among surgical departments in response to an unprecedented surge in reducing the transmission of disease. With pooling and outsourcing of more health care workers to emergency rooms, public health care services and medical services, further in-hospital resources are prioritised to those in need. It is imperative to balance the requirements of caring for COVID-19 patients with imminent risk of delay to others who need care. As Malaysia now approaches the recovery phase following the pandemic, the crisis impacted significantly on neurosurgical services throughout the country. Various emergency measures taken at the height of the crisis may remain as the new normal in the provision of neurosurgical services and practices in Malaysia. The crisis 142 has certainly put a strain on the effective delivery of services and as we approach the recovery era, what may have been a strain may prove to be a silver lining in neurosurgical services in Malaysia. The following details are various measures put in place as the new operational protocols for neurosurgical services in Malaysia.
... As a mushroom-derived toxin, phalloidin is a highly specific probe for filamentous actin (F-actin) that is the most abundant cytoskeletal component in the smooth muscular and endothelial cells (Wulf et al. 1979;Cooper 1987;Alarcon-Martinez et al. 2018). By conjugating with different fluorophores available, phalloidin labeling has been widely used in double-labeling experiments (Pankov et al. 2005;Li et al. 2007Li et al. , 2009Chazotte 2010;Hagan 2016). During antibody-based immunohistochemical staining, phalloidin-labeled fluorophores can be added in either the primary or secondary antibody incubation step. ...
The neurovascular structures in the cranial dura mater have been studied with various histological techniques in the past years. In order to obtain a proper approach to reveal the detailed structures, different labeling methods for the cranial vessels and nerve fibers were tested in this study. Firstly, the labeling characteristics of phalloidin, alpha smooth muscle actin (α-SMA), and CD31 were compared in rat whole-mount cranial dura mater by using fluorescent immunohistochemistry or histochemistry. Secondly, according to their properties, phalloidin and α-SMA were selected to combine with calcitonin gene-related peptide (CGRP) to further demonstrate the cranial neurovascular structure. By these approaches, a three-dimensional map of blood vessels and nerve fibers within the whole-mount rat cranial dura mater was obtained. The results showed that phalloidin, α-SMA, and CD31 were preferably expressed in the wall of cranial vessels, corresponding to the arteriors, venules, and capillaries, respectively. Additionally, CGRP + nerve fibers were clearly demonstrated together with phalloidin + or α-SMA + vessels, forming a delicate neurovascular network in the cranial dura mater. The thick nerve bundles ran closely to the phalloidin + or α-SMA + vessels in parallel pattern, while the thin nerve fibers branched off from the bundles tending to surround the phalloidin + arterioles rather than α-SMA + venules. These findings suggest that phalloidin could be an appropriate biochemical maker to be effectively used together with CGRP for experiments examining the detailed spatial correlation of cranial blood vessels and nerve fibers in a three-dimensional view, which may provide clues for understanding the underlying mechanisms of cranial neurovascular disorders.
... However, we observed a significant increase in the proportion of Wnt5a −/− nuclei oriented at an angle of <60 • to the basal surface, suggesting the loss of apicobasal polarity (Fig. 3J). The polarized CP epithelium is characterized by a prominent basal lamina and an actin-rich apical brush border (Li, et al. 2009a). Laminin immunolabeling revealed a well-defined continuous basement membrane underlying the E14.5 Wnt5a +/+ epithelia (tCP, Fig. 4A; dCP, hCP, see Supplementary Fig. S2A,C). ...
The choroid plexus (CP) is the predominant supplier of cerebral spinal fluid (CSF) and the site of the blood-CSF barrier and is thus essential for brain development and central nervous system homeostasis. Despite these crucial roles, our understanding of the molecular and cellular processes giving rise to the CPs within the ventricles of the mammalian brain is very rudimentary. Here, we identify WNT5a as an important regulator of CP development, where it acts as a pivotal factor driving CP epithelial morphogenesis in all ventricles. We show that WNT5a is essential for the establishment of a cohesive epithelium in the developing CP. We find that in its absence all CPs are substantially reduced in size and complexity and fail to expand into the ventricles. Severe defects were observed in the epithelial cytoarchitecture of all Wnt5a-/- CPs, exemplified by loss of apicobasally polarized morphology and detachment from the ventricular surface and/or basement membrane. We also present evidence that the WNT5a receptor, RYK, and the RHOA kinase, ROCK, are required for normal CP epithelial morphogenesis. Our study, therefore, reveals important insights into the molecular and cellular mechanisms governing CP development.
... Apical microvilli found in light cells of the outer sheet cover from the current study, resembles those of ependymal cells. Microvilli of ependymocytes, considered as the source of multipotent adult NSCs [10,63], provide the capability of adaptation of cells to different external conditions [57]. It is possible that microvilli of outer sheet cover cells of the spheroid have a similar function, that is to become organized and positioned against external forces. ...
Background: Neurospheres derived from adult stem cells of non-neural tissues represent a promising source of neural stem cells (NSCs) and neural progenitor cells (NPCs) for autologous cell therapy in neurological diseases. Analyzing the fine structure of neurospheres can provide pivotal information regarding the phenotype of spheroid-integrating cells, being useful for cell characterization during differentiation, and oncogenic transformation. Neurospheres can be generated by culturing ovarian cortical cells under particular conditions. In order to assess the reliability of use of these spheroids in regenerative medicine, developmental biology, and to state whether they share morphological features with central nervous system (CNS) derived neurospheres, they were analyzed by light and transmission electron microscopy. Sheep ovarian cortical cells were cultured in serum-free medium for 21 days to generate neurospheres. On days 10, 15 and 21 of culture, expression of pluripotency (Nanog, Oct4, Sox2), and NSC/NPC (nestin, Pax6, P75NTR) transcripts by qRT-PCR, immunolocalization of NSC/NPC antigens (nestin, Pax6, P75NTR), structure, and ultrastructure, were analyzed in spheroids.
Results: Spheroids expressed transcripts and antigens of pluripotency, and NSC/NPC. Structural analyses revealed that cells were arranged in a peripheral sheet cover comprised by a few layers of spindle-shaped cells exhibiting frequent mitoses, and large round cells; and an inner core, with intermediate-sized cells showing frequent events of apoptosis and necrosis. Ultrastructural analyses showed that in the inner core, most cells were electron dense (dark), showing frequent apoptotic and degenerative events; the outer epithelium-like sheet, was formed by low electron dense cells (light flat cells; LFCs) showing signs of intense metabolic activity, abundant cytoplasmic organelles, phagosomes, lysosome-like structures, apical microvilli, and filament bundles of cytoskeleton elements, indicating that LFCs play a role in extracellular exchanges. Apical-basal polarization of LFCs evidenced by frequent intercellular contacts, like adherens junctions and cytoplasmic interdigitations, was lost in some cells whose contacts with neighboring cells were loose intedigitations and expanded cytoplasmic processes. This might anticipate disengaging/aggregating of light protruding cells (LPCs) from/to the neurosphere, as consistently observed in cultures by inverted microscopy.
Conclusions: Neurospheres spontaneously generated from ovarian cortical cells in culture, share most structural and ultrastructural characteristics of neurospheres derived from the CNS.
... The specific staining reagent of F-actin is phalloidin which is also the marker adopted in this study. 10 It was shown from experimental results that F-actin marked by phalloidin in local tissues played a very important role in differentiating muscle tissue, dermis and epidermis, and it was an important tool to determine the dermal microvascular structure and distribution characteristics. Different from traditional HE staining, the composition labeled by phalloidin is relatively single; the vascular structure labeled by phalloidin is more distinct from other tissues in the specimen. ...
OBJECTIVE
To reveal the effects of Guasha (scraping therapy) on the histomorphology of scraped skins and on the expressions of calcitonin gene-related peptide (CGRP) and substance P (SP).
METHODS
50 rats, as experimental subjects, were randomly divided into 5 groups according to different observation time points. Dorsal setae were shaved for the exposure of skin on both sides of the spine. Even reinforcing and reducing method was applied to the rats in Guasha groups on the site equivalent to bladder meridian of human body on one side of the spine, and the skin was scraped from top to bottom until rash of measles occurred. The skin tissues with rash of measles were taken down after perfusion. The corresponding sites of rats in group A were also taken down as control. The tissues were made to sections and used for immunofluorescence histochemical staining and HE staining of antibodies such as SP and CGRP.
RESULTS
After Guasha, there were significant differences in appearance, hair follicle and blood vessel on local skin scraped on the back of rats when compared with control; In different time points, the differences reduced. There was no significant difference in expression of CGRP and SP when compared local skin scraped on the back of rats in different time points.
CONCLUSION
Guasha didn't significantly change the morphology of nerve fibers inside local skin, and the histomorphology of hair follicle, blood vessel and etc. However, after Guasha they basically returned to normal level within five days.
... To identify the apical membrane, we used two markers: phalloidin coupled with a fluorochrome (Figure 4c) and an antibody against aquaporin 1 (Figure 4g). Phalloidin binds to F-actin, which is predominantly present in the apical border of ependymocytes, 9,11 and aquaporin 1 has also been found predominantly on the apical membrane. 12,13 As shown earlier, the green fluorescence signal of GFPdockerin was distributed throughout the cytosol of the ependymocytes (Figure 4a,e). ...
Crossing the blood-brain and the blood-cerebrospinal fluid barriers (BCSFB) is one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because these barriers prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Based on this mechanism, we engineered a receptor/ligand system to overcome the brain barriers by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested the hybrid receptor in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the BCSFB, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.
