Age‐related ultrastructural changes of the basement membrane in the mouse blood‐brain barrier

The blood‐brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24‐month‐old mice was double as compared with that of 6‐month‐old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid‐rich BM regions are located in small pockets formed by the end‐feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.     

Structural and Cytochemical Peculiarities of Basement Membranes in the Zone of Formation of the Blood–Brain Barrier in Human Prenatal Ontogenesis

Structural and cytochemical organization of the basement membrane (BM) of the developing human brain at the embryonic period of prenatal ontogenesis was studied using light and electron microscopy and immunocytochemistry. Ultrastructural study has shown that the basement membranes of capillaries growing into the brain anlage are at the stage of formation (from the complete absence to the stage of broken membrane, without characteristic differentiation into layers). At the same time, BM of the brain surface looks sufficiently differentiated and prevents contacts of cells of the developing brain with meningocytes. These data are in contrast with results of immunocytochemical study, which show that the basement membranes (according to reactions for fibronectin, laminin, and type IV collagen) are clearly contoured around growing capillaries and, hence, exist from the very beginning of the intracerebral angiogenesis. The explanation of the established discrepancy between electron microscopic and cytochemical data seems to be searched for in spatial distribution and organization of BM components.     

Deoxynivalenol affects the composition of the basement membrane proteins and influences en route the migration of CD16+ cells into the intestinal epithelium

The numerous pores in the basement membrane (BM) of the intestinal villi are essential for the communication of enterocytes with cells in the lamina propria, an important mechanism for the induction of intestinal immune responses. The intestinal epithelial barrier is affected by the mycotoxin deoxynivalenol (DON) from both the apical (luminal) and basolateral (serosal) side. The pig is the most susceptible species to the anorectic and immune-modulating effects of DON, which is most prevalent in crops. We analysed in pigs the effect of DON-contaminated feed on the composition and perforation of the BM and the presence of CD16⁺ cells or their dendrites in the epithelium. In addition to in vivo experiments, in vitro studies were carried out. Using microarray analyses, the effects of DON on IPEC-J2 cells were studied with the focus on the BM. Our in vivo results showed in the control pigs: (1) a significant increased pore number (p?≤?0.001) in the jejunum in comparison to ileum, (2) no difference in the pore size, and (3) comparable frequency of intraepithelial CD16⁺ cells/dendrites in the jejunum and ileum. There was a marked trend that DON feeding increases: (1) the pore number in jejunum, and (2) the number of CD16⁺ cells/dendrites in the epithelium (Tukey–Kramer; p?=?0.055 and p?=?0.067, respectively). The in vivo results were extended with microarray analyses of epithelial cell (IPEC-J2 cells). The down-regulation of genes like syndecan, fibulin 6 and BM-40 was observed. These proteins are important factors in the BM composition and in formation of pores. Our results provide evidence that already low basolateral concentrations of DON (50 ng/mL) influence the production of the BM protein laminin by epithelial cells. Thus, DON affects the composition of the BM.     

Electron microscope observations of the melanocyte of the human epidermis

Proteins and colloidal materials, administered orally to suckling rats and mice, were ingested by columnar absorptive cells of the jejunum and ileum, but not of the duodenum. Bovine gamma globulin and ovalbumin were identified in the apical cytoplasm by staining with fluorescent antibody; trypan blue, Evans blue, saccharated iron oxide, and colloidal gold were detected intracellularly by their color, specific staining, and appearance in the electron microscope. Each substance was segregated in membrane-enclosed vacuoles, apparently part of a system of potentially interconnecting vacuoles and tubules in the apical cytoplasm which is continuous in places with the apical cell membrane. We postulate that ingestion of foreign materials was accomplished by pinocytosis, that is, by invagination of the apical cell membrane to form vacuoles containing material from the intestinal lumen. Approximately 18 days after birth columnar absorptive cells lost the ability to ingest proteins and colloids, and no longer contained large vacuoles and numerous tubules. At this age rats and mice lose the ability to absorb antibodies from the intestine in an immunologically intact form, and we conclude that cellular ingestion is part of the mechanism of absorption of intact proteins in suckling animals. Particulate fat apparently is absorbed in both newborn and adult animals by micropinocytosis. Thus adult animals may not have lost the capacity for pinocytosis, but rather have become selective as to what substances provoke it. Cortisone acetate, administered subcutaneously to rats 8 to 10 days old alters the columnar absorptive cells within 72 hours so that they resemble the cells in adult animals and no longer ingest proteins.     

The ultrastructure of the capillaries in the gingiva of alloxan-induced diabetic rats

The diabetic effects of alloxan (type I diabetes mellitus) were investigated in 40 Wistar albino rats (18 controls and 22 diabetics). Alloxan in sterile physiological saline was injected into animals intravenously. After the induction of diabetes with alloxan, the ultrastructure of the capillaries in the gingiva was examined by transmission electron microscopy. The thickness of the basement membranes was observed closely adherent to the endothelial cells of the capillary alloxan-diabetic rats. It was greatly thickened owing to the increase in its amorphous, granular and filamentous material with occasional scattered collagen fibres. In some sections, the capillary lumens of the diabetics were closed by epithelial cells. Loss of cytoplasmic material and hyalinization were seen in some smooth muscle cells. In addition, the mitochondrial cristae of smooth muscle cell and epithelial cells disappeared. There was endothelial integrity throughout the smooth muscle cells.     

The Ingestion of Proteins and Colloidal Materials by Columnar Absorptive Cells of the Small Intestine in Suckling Rats and Mice

Proteins and colloidal materials, administered orally to suckling rats and mice, were ingested by columnar absorptive cells of the jejunum and ileum, but not of the duodenum. Bovine gamma globulin and ovalbumin were identified in the apical cytoplasm by staining with fluorescent antibody; trypan blue, Evans blue, saccharated iron oxide, and colloidal gold were detected intracellularly by their color, specific staining, and appearance in the electron microscope. Each substance was segregated in membrane-enclosed vacuoles, apparently part of a system of potentially interconnecting vacuoles and tubules in the apical cytoplasm which is continuous in places with the apical cell membrane. We postulate that ingestion of foreign materials was accomplished by pinocytosis, that is, by invagination of the apical cell membrane to form vacuoles containing material from the intestinal lumen. Approximately 18 days after birth columnar absorptive cells lost the ability to ingest proteins and colloids, and no longer contained large vacuoles and numerous tubules. At this age rats and mice lose the ability to absorb antibodies from the intestine in an immunologically intact form, and we conclude that cellular ingestion is part of the mechanism of absorption of intact proteins in suckling animals. Particulate fat apparently is absorbed in both newborn and adult animals by micropinocytosis. Thus adult animals may not have lost the capacity for pinocytosis, but rather have become selective as to what substances provoke it. Cortisone acetate, administered subcutaneously to rats 8 to 10 days old alters the columnar absorptive cells within 72 hours so that they resemble the cells in adult animals and no longer ingest proteins.     

Ultrastructural changes in the brain capillaries after hypoxia

The authors describe ultrastructural changes in and around rat brain capillaries after hypoxia. The experimental animals breathed a mixture of 5% oxygen and 95% nitrogen for two or for three hours; a third group, which spent three hours in this atmosphere, was treated 24 h later. Cytoplasm processes and vesicles were observed on the luminal side in the endothelial cells, while the cytoplasm contained vacuoles and altered mitochondria. The basement membrane of the brain capillaries was uneven and longitudinal clear zones were formed in it. Altered mitochondria were present in the pericytes and astrocytes. The most pronounced changes were found in the astrocyte processes, which were light and hydrated and contained destroyed mitochondria and lamellar bodies resembling myelin. After 24 h, morphological changes still persisted, especially in the astrocyte processes.     

IFN-gamma production by intrinsic renal cells and bone marrow-derived cells is required for full expression of crescentic glomerulonephritis in mice

The contribution of IFN-gamma from bone marrow (BM) and non-BM-derived cells to glomerular and cutaneous delayed-type hypersensitivity (DTH) was studied in mice. Chimeric IFN-gamma mice (IFN-gamma(+/+) BM chimera), in which IFN-gamma production was restricted to BM-derived cells, were created by transplanting normal C57BL/6 (wild-type (WT)) BM into irradiated IFN-gamma-deficient mice. BM IFN-gamma-deficient chimeric mice (IFN-gamma(-/-) BM chimera) were created by transplanting WT mice with IFN-gamma-deficient BM. WT and sham chimeric mice (WT mice transplanted with WT BM) developed crescentic glomerulonephritis (GN) with features of DTH (including glomerular T cell and macrophage infiltration) in response to an Ag planted in their glomeruli and skin DTH following subdermal Ag challenge. IFN-gamma-deficient mice showed significant protection from crescentic GN and reduced cutaneous DTH. IFN-gamma(+/+) BM chimeric and IFN-gamma(-/-) BM chimeric mice showed similar attenuation of crescentic GN as IFN-gamma-deficient mice, whereas cutaneous DTH was reduced only in IFN-gamma(-/-) BM chimeras. In crescentic GN, IFN-gamma was expressed by tubular cells and occasional glomerular cells and was colocalized with infiltrating CD8(+) T cells, but not with CD4(+) T cells or macrophages. Renal MHC class II expression was reduced in IFN-gamma(+/+) BM chimeric mice and was more severely reduced in IFN-gamma-deficient mice and IFN-gamma(-/-) BM chimeric mice. These studies show that IFN-gamma expression by both BM-derived cells and intrinsic renal cells is required for the development of crescentic GN, but IFN-gamma production by resident cells is not essential for the development of cutaneous DTH.     

产毒性大肠杆菌毒素在豚鼠肠道定位的免疫组织化学研究

研究利用免疫组织化学方法对产毒性大肠杆菌（ETEC）感染豚鼠小肠组织中ETEC肠毒素的定位进行了研究,本研究结果表明,发病豚鼠从空肠到回肠明显充血,肿胀,但盲肠,结肠和直肠外观与对照组差别不明显,光镜下见到发病动物肠组织病变主要出现在空肠和回肠,以回肠最为严重。主要病理改变为水肿,炎症细胞浸润和充血,病变部位可以出现在粘膜层,粘膜下层,肌层和浆膜层,取回肠组织切片作免疫组织化学显示ETEC不耐热肠毒素（LT）和耐热肠毒素（ST）、可见回肠粘膜表层,粘膜肌层,肌层及浆膜层均呈LT和ST阳性反应,分布弥漫,空白对照和正常豚鼠回肠组织均呈阴性结果。本研究表明,ETEC主要作用于空肠和回肠,尤其是回肠;回肠组织各层都有病变,且与肠毒素的分布一致,证明毒素的作用并不仅限于肠粘膜细胞。     

Basement membrane attachment is dispensable for radial glial cell fate and for proliferation, but affects positioning of neuronal subtypes

Radial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogen-binding site of the laminin gamma1 chain is mutated, in the absence of alpha6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin gamma1III4-/- mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex.     

Mentha piperita (Linn) leaf extract provides protection against radiation induced alterations in intestinal mucosa of Swiss albino mice

Intestinal protection in mice against radiation injury by M. piperita (1 g/kg body weight/day) was studied from day 1 to day 20 after whole body gamma irradiation (8 Gy). Villus height, goblet cells/villus section, total cells, mitotic cells and dead cells/crypt section in the jejunum are good parameters for the assessment of radiation damage. There was significant decrease in the villus height, number of total cells and mitotic cells/crypt section, whereas goblet cells and dead cells showed significant increase after irradiation. Mentha pretreatment resulted in a significant increase in villus height, total cells and mitotic cells, whereas goblet cells and dead cells showed a significant decrease from respective irradiated controls at each autopsy day. The results suggest that Mentha pretreatment provides protection against radiation induced alterations in intestinal mucosa of Swiss albino mice.     

Differential expression of laminin chains in the human major salivary gland

Laminin represents a macromolecule family. The heterotrimeric isoforms of laminin can be determined by immunohistochemical demonstration of the single chains. The laminin chain heterogeneity of the basement membrane in adult human major salivary glands was evaluated in relation to cellular differentiation of the epithelia and the stromal compartment. Monoclonal antibodies to the laminin alpha1, alpha3 (BM165) chains and epiligrin reacted with the basement membranes of serous and mucous acini and of intercalated, striated and excretory ducts. As evidenced by a double-labelling technique, the alpha2 chain showed a spatial association with the myoepithelium of the acini, whereas the ductal basement membranes containing no myoepithelial cells were negative. Almost exclusively, beta1 chain was detected in acinar basement membrane, beta2 chain whereas in ductal basement membrane. gamma2 chain is a unique chain belonging to the laminin-5 isoform. It was restricted to the ductal basement membrane. alpha1, alpha2, beta1, beta2 and gamma1 chains were detected in nerves of salivary tissue and alpha1, alpha3, beta1, beta2 and gamma1 chains and epiligrin in blood vessels. Our results indicate that the acinar ductal unit contains basement membranes with different isoforms, which relate to cell differentiation and cell function. © 1998 Chapman & Hall     

Identification of lymph and blood capillaries by immunohistochemical staining for various basement membrane components.

In the present report we analyzed the presence and distribution of various basement membrane (BM) proteins in normal blood and lymph vessels with special emphasis on BM-associated heparan sulfate proteoglycan (HSPG) when compared to the BM-components collagen IV, laminin and fibronectin. We found that normal lymph capillaries have a BM that contains only collagen IV and small amounts of laminin, but almost no BM-associated HSPG and fibronectin, while blood capillaries showed a BM comprising of all components tested for. Larger lymphatics, however, were indistinguishable from blood vessels on the basis of BM staining. Lymphangiomas showed a BM pattern similar to that of lymph capillaries. Our findings provide evidence that the differential staining of BM-components may represent a reliable method for morphological distinction between blood and lymph capillaries. A comparison of these results with the BM-pattern in other functionally specialized blood vessels (glomerulus, sinusoids) provides evidence that the BM-composition may have some major impact on the functional properties. Thus, it is conceivable that the lack of HSPG in lymph capillaries may be essential for a free influx of fluid and proteins into these capillaries, which may have been extravasated into the interstitium.     

Identification of lymph and blood capillaries by immunohistochemical staining for various basement membrane components

Summary In the present report we analyzed the presence and distribution of various basement membrane (BM) proteins in normal blood and lymph vessels with special emphasis on BM-associated heparan sulfate proteoglycan (HSPG) when compared to the BM-components collagen IV, laminin and fibronectin. We found that normal lymph capillaries have a BM that contains only collagen IV and small amounts of laminin, but almost no BM-associated HSPG and fibronectin, while blood capillaries showed a BM comprising of all components tested for. Larger lymphatics, however, were indistinguishable from blood vessels on the basis of BM staining. Lymphangiomas showed a BM pattern similar to that of lymph capillaries. Our findings provide evidence that the differential staining of BM-components may represent a reliable method for morphological distinction between blood and lymph capillaries. A comparison of these results with the BM-pattern in other functionally specialized blood vessels (glomerulus, sinusoids) provides evidence that the BM-composition may have some major impact on the functional properties. Thus, it is conceivable that the lack of HSPG in lymph capillaries may be essential for a free influx of fluid and proteins into these capillaries, which may have been extravasated into the interstitium.     

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.     

Effects of reduced food intake on morphometry and cell production in the small intestine of the rat

The present study examined the effects of a 60% reduction in food intake on kinetic and morphometric parameters in the small intestine of adult male Lewis rats. We observed that, after 20 days, the wet weight of jejunum and ileum, thickness of muscularis externa of duodenum, crypt depth throughout the intestine, and DPM/mg and DPM/crypt in ileum were decreased in animals on reduced food intake when compared to their paired, normally fed controls. These results demonstrated that reduced food intake caused distinctive effects focused primarily in the ileum, thus opening to question the use of the technique of pair feeding as a control for studies of intestinal cell proliferation in which manipulations of the animals result in altered food intake or body weight.     

Cellular Proliferation of Intestinal Epithelia in the Rat Two Months after Partial Resection of the Ileum

Sprague-Dawley rats subjected 2 months previously to partial resection (10 per cent) of the small intestine and their controls were injected with tritiated thymidine and sacrificed at 2 and 23 hours. Segments of the duodenum, jejunum, and ileum were autoradiographed, and the migration of the labelled cells during the period between 2 and 23 hours was measured with an eyepiece micrometer. The cells had migrated 35, 42, and 34 per cent of the total distance from the crypts to the tips of the villi in the control segments of duodenum, ileum, and jejunum respectively, and 43, 90, and 82 per cent, respectively, in similar segments from resected animals. The rate of migration in the portion of the intestine remaining after resection was approximately three times the normal rate in the ileum, twice the normal rate in the jejunum, and showed an increase of one-third in the duodenum. These results demonstrate that the rate of cell renewal is considerably greater in the remaining portion of the intestine of resected animals than in normal intestine. The increased rate of migration after resection, together with the increase in the height of the villi, resulted in an increase in the rate of cell renewal amounting to 141 per cent in the ileum, 114 per cent in the jejunum, and 23 per cent in the duodenum when compared with control segments.     

Absorption of elastase through the jejunal mucosa of the rat. An immunocytochemical study

In order to reveal the absorption process of elastase from the intestine, hog pancreatic elastase was injected into the ligated jejunum lumen of the rat, and the tissues were cytochemically observed at various times after injection. The peroxidase anti-peroxidase (PAP) method using anti-hog-elastase rabbit antibody was used for light microscopy, and the anti-elastase Fab'-peroxidase conjugate was used for electron microscopy. The tissues stained by the PAP method exhibited a dense deposition of reaction products on the luminal surface of epithelial cells and a moderate deposition in the blood and lymph capillaries of the intestinal villi. Immunoelectron microscopy revealed that the reaction product was deposited on the surface of the microvilli and in their pocketing; some was found in the pinocytotic vesicles in the terminal-web area and on the inner surface of the enlarged smooth endoplasmic reticulum. Round droplets which gave a positive reaction were found in the widened intercellular cleft and the thick basement membrane lining the blood capillaries and lymphatics. The jejunum retained its normal ultrastructure. The results indicate that the elastase molecules, which were introduced into the rat jejunum lumen, were absorbed without being decomposed through healthy intestinal epithelial cells by pinocytosis and translocated into blood and lymph capillaries.     

Intestinal capillaries. II. Structural effects ofEDTA and histamine

Perfusion of the fenestrated capillaries of the intestinal mucosa of the rat with 0.05–0.1 M EDTA removes the diaphragms of the endothelial cells and detaches these cells from one another and from the basement membrane. The latter, even when completely denuded, retains effectively particles of 340 A (average) diameter. Perfusion with histamine (1 µg/ml) results in partial removal of fenestral diaphragms, occasional detachment of the endothelium from the basement membrane, and focal separation of endothelial intercellular junctions.     

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.