Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.     

酸性磷酸酶在大鼠脉络丛上皮细胞内的亚细胞定位

研究大鼠脑室脉络丛上皮细胞内酸性磷酸酶(Acid phosphatase,AcPase)的超微结构分布.取成年SD大鼠侧脑室、第三脑室和第四脑室的脉络丛组织,按硝酸铅法进行酸性磷酸酶电镜酶细胞化学染色显示.透射电镜下ACPase阳性反应物仅见于脉络丛上皮细胞内的初级和次级溶酶体上.通过延长孵育时间和改变固定条件(戊二醛浓度为0.5%)在脉络丛上皮细胞内的高尔基复合体扁囊和囊泡中也观察到了ACPase阳性反应物.各脑室脉络丛上皮细胞酸性磷酸酶的分布未见明显差异.文中讨论了脉络丛上皮细胞内ACPase的分布特征及其功能.     

Different Involvement of Vimentin during Invasion by Listeria monocytogenes at the Blood–Brain and the Blood–Cerebrospinal Fluid Barriers In Vitro

The human central nervous system (CNS) is separated from the blood by distinct cellular barriers, including the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CFS) barrier (BCSFB). Whereas at the center of the BBB are the endothelial cells of the brain capillaries, the BCSFB is formed by the epithelium of the choroid plexus. Invasion of cells of either the BBB or the BCSFB is a potential first step during CNS entry by the Gram-positive bacterium Listeria monocytogenes (Lm). Lm possesses several virulence factors mediating host cell entry, such as the internalin protein family—including internalin (InlA), which binds E-cadherin (Ecad) on the surface of target cells, and internalin B (InlB)—interacting with the host cell receptor tyrosine kinase Met. A further family member is internalin (InlF), which targets the intermediate filament protein vimentin. Whereas InlF has been shown to play a role during brain invasion at the BBB, its function during infection at the BCSFB is not known. We use human brain microvascular endothelial cells (HBMEC) and human choroid plexus epithelial papilloma (HIBCPP) cells to investigate the roles of InlF and vimentin during CNS invasion by Lm. Whereas HBMEC present intracellular and surface vimentin (besides Met), HIBCPP cells do not express vimentin (except Met and Ecad). Treatment with the surface vimentin modulator withaferin A (WitA) inhibited invasion of Lm into HBMEC, but not HIBCPP cells. Invasion of Lm into HBMEC and HIBCPP cells is, however, independent of InlF, since a deletion mutant of Lm lacking InlF did not display reduced invasion rates.     

西方理性规划的发展脉络及其对我国的影响

从科学理性和价值理性两个方面把握理性规划的内涵,并展开对其发展脉络的分析,最后阐述了理性规划对中国城市规划实践的影响,以供参考。     

Residual deformations in ocular tissues

Residual deformations strongly influence the local biomechanical environment in a number of connective tissues. The sclera is known to be biomechanically important in healthy and diseased eyes, such as in glaucoma. Here, we study the residual deformations of the sclera, as well as the adjacent choroid and retina. Using freshly harvested porcine eyes, we developed two approaches of quantifying residual deformations in the spherically shaped tissues of interest. The first consisted of punching discs from the posterior wall of the eye and quantifying the changes in the area and eccentricity of these samples. The second consisted of cutting a ring from the equatorial sclera and making stress-relieving cuts in it. Measurements of curvature were made before and after the stress-relieving cuts. Using the first approach, we observed a 42% areal contraction of the choroid, but only modest contractions of the sclera and retina. The observed contractions were asymmetric. In the second approach, we observed an opening of the scleral rings (approx. 10% decrease in curvature). We conclude that residual bending deformations are present in the sclera, which we speculate may be due to radially heterogeneous growth and remodelling of the tissue during normal development. Further, residual areal deformations present in the choroid may be due to the network of elastic fibres in this tissue and residual deformations in the constituent vascular bed. Future studies of ocular biomechanics should attempt to include effects of these residual deformations into mechanical models in order to gain a better understanding of the biomechanics of the ocular wall.     

Altered Expression of AQP1 and AQP4 in Brain Barriers and Cerebrospinal Fluid May Affect Cerebral Water Balance during Chronic Hypertension

Hypertension is the leading cause of cardiovascular affection and premature death worldwide. The spontaneously hypertensive rat (SHR) is the most common animal model of hypertension, which is characterized by secondary ventricular dilation and hydrocephalus. Aquaporin (AQP) 1 and 4 are the main water channels responsible for the brain’s water balance. The present study focuses on defining the expression of AQPs through the time course of the development of spontaneous chronic hypertension. We performed immunofluorescence and ELISA to examine brain AQPs from 10 SHR, and 10 Wistar–Kyoto (WKY) rats studied at 6 and 12 months old. There was a significant decrease in AQP1 in the choroid plexus of the SHR-12-months group compared with the age-matched control (p < 0.05). In the ependyma, AQP4 was significantly decreased only in the SHR-12-months group compared with the control or SHR-6-months groups (p < 0.05). Per contra, AQP4 increased in astrocytes end-feet of 6 months and 12 months SHR rats (p < 0.05). CSF AQP detection was higher in the SHR-12-months group than in the age-matched control group. CSF findings were confirmed by Western blot. In SHR, ependymal and choroidal AQPs decreased over time, while CSF AQPs levels increased. In turn, astrocytes AQP4 increased in SHR rats. These AQP alterations may underlie hypertensive-dependent ventriculomegaly.     

Central Nervous System Stimulants Limit Caffeine Transport at the Blood–Cerebrospinal Fluid Barrier

Caffeine, a common ingredient in energy drinks, crosses the blood–brain barrier easily, but the kinetics of caffeine across the blood–cerebrospinal fluid barrier (BCSFB) has not been investigated. Therefore, 127 autopsy cases (Group A, 30 patients, stimulant-detected group; and Group B, 97 patients, no stimulant detected group) were examined. In addition, a BCSFB model was constructed using human vascular endothelial cells and human choroid plexus epithelial cells separated by a filter, and the kinetics of caffeine in the BCSFB and the effects of 4-aminopyridine (4-AP), a neuroexcitatory agent, were studied. Caffeine concentrations in right heart blood (Rs) and cerebrospinal fluid (CSF) were compared in the autopsy cases: caffeine concentrations were higher in Rs than CSF in Group A compared to Group B. In the BCSFB model, caffeine and 4-AP were added to the upper layer, and the concentration in the lower layer of choroid plexus epithelial cells was measured. The CSF caffeine concentration was suppressed, depending on the 4-AP concentration. Histomorphological examination suggested that choroid plexus epithelial cells were involved in inhibiting the efflux of caffeine to the CSF. Thus, the simultaneous presence of stimulants and caffeine inhibits caffeine transfer across the BCSFB.     

The Expression of Connexin 37, 40, 43, 45 and Pannexin 1 in the Early Human Retina and Choroid Development and Tumorigenesis

The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas. Expressions of both Cx37 and Cx43 increased during development but diminished in the postnatal period, being higher in the retina than in the choroid. Cx37 was highly expressed in the choroid of retinoblastoma, and Cx43 in epitheloid melanoma, while they were both increasingly expressed in mixoid melanoma. In contrast, mild retinal Cx40 expression during development increased to strong in postnatal period, while it was significantly higher in the choroid of mixoid melanoma. Cx45 showed significantly higher expression in the developing retina compared to other samples, while it became low postnatally and in all types of melanoma. Pnx1 was increasingly expressed in developing choroid but became lower in the postnatal eye. It was strongly expressed in epithelial and spindle melanoma, and particularly in retinoblastoma. Our results indicate importance of Cx37 and Cx40 expression in normal and pathological vascularization, and Cx43 expression in inflammatory response. Whereas Cx45 is involved in early stages of eye development, Pnx1might influence cell metabolism. Additionally, Cx43 might be a potential biomarker of tumor prognosis.     

Teriflunomide Inhibits JCPyV Infection and Spread in Glial Cells and Choroid Plexus Epithelial Cells

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.     

Microvasculature of the buffalo (Bubalus bubalis) choroid plexuses: structural, histochemical, and immunocytochemical study

The choroid plexuses (CPs) in mammals produce the cerebrospinal fluid (CSF). In the literature, the morphology of CPs and the process that regulates the production of CSF are virtually nonexistent for domestic ruminants. Thus this study has two aims: 1. to investigate the morpho-structure of the buffalo CP microvasculature utilizing light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques, and 2. to investigate the relationship between the blood vessels and both the elongated cells and the cells with multiple protrusions located in the CPs. SEM and TEM analyses of the CPs from buffalo brain showed morphological and structural features similar those reported in other mammalian species. Moreover the blood microvasculature is the major component responsible for the formation of the CSF, secreted by the encephalic CPs. In addition the chemical composition of this fluid depends on several morpho-functional characteristics of the vascularization of the CPs. These characteristics are as follows: two shapes of the vascular organization: lamina-like and ovoid-like elongated cells of the CPs, which connect the ventricular cavities to the blood capillaries; and the CP capillaries have diverse forms. In the present study the employment of NADPHd and NOS I was taken as indirect evidence for the presence of NO for investigation their specific role in CPs. Then NOS I immunoreactivity is found in the walls of CP blood vessels demonstrating indirectly the presence of NO with a vaso-dilatatory and autoregulation function of vascular tone by cholinergic nerve stimulation of blood vessel smooth muscle.