Pore alterations of the endothelial lining of rat fenestrated intestinal capillaries exposed to acute stress.

Stress-induced inflammatory responses in the portal system are characterized by elevations in serum concentrations of interleukin-6 (IL-6) and endotoxins such as lipopolysaccharides (LPS). LPS translocation from the intestinal to the capillary lumen occurs via LPS endocytosis by the capillary endothelium. Because the capillary endothelium of the small intestinal submucosa is fenestrated, we determined the role of pore modifications within the fenestrated endothelium in relaying inflammatory stress responses in the portal vein. We evaluated changes in the diameter and density of endothelial pores of the lamina propria of intestinal villi induced by continuous light (CL) exposure for 48 h and the correlation between these changes and serum IL-6 concentration in the portal vein in a rat model. We found significant increases in both the pore diameter and density, accompanied by a significant increase in portal IL-6 concentration; these changes were significantly attenuated by pretreatment with propranolol, a beta adrenergic receptor antagonist. In contrast, intravenous noradrenaline administration mimicked CL-induced modifications of the diameter and density of pores and the elevation of portal vein IL-6 concentration. These findings suggested that stress-induced inflammatory responses in the portal system may be a part of the modifications of the endothelial pores triggered by sympathetic activation.

Distribution of histaminergic neuronal cluster in the rat and mouse hypothalamus.

Histidine decarboxylase (HDC) catalyzes the biosynthesis of histamine from L-histidine and is expressed throughout the mammalian nervous system by histaminergic neurons. Histaminergic neurons arise in the posterior mesencephalon during the early embryonic period and gradually develop into two histaminergic substreams around the lateral area of the posterior hypothalamus and the more anterior peri-cerebral aqueduct area before finally forming an adult-like pattern comprising five neuronal clusters, E1, E2, E3, E4, and E5, at the postnatal stage. This distribution of histaminergic neuronal clusters in the rat hypothalamus appears to be a consequence of neuronal development and reflects the functional differentiation within each neuronal cluster. However, the close linkage between the locations of histaminergic neuronal clusters and their physiological functions has yet to be fully elucidated because of the sparse information regarding the location and orientation of each histaminergic neuronal clusters in the hypothalamus of rats and mice. To clarify the distribution of the five-histaminergic neuronal clusters more clearly, we performed an immunohistochemical study using the anti-HDC antibody on serial sections of the rat hypothalamus according to the brain maps of rat and mouse. Our results confirmed that the HDC-immunoreactive (HDCi) neuronal clusters in the hypothalamus of rats and mice are observed in the ventrolateral part of the most posterior hypothalamus (E1), ventrolateral part of the posterior hypothalamus (E2), ventromedial part from the medial to the posterior hypothalamus (E3), periventricular part from the anterior to the medial hypothalamus (E4), and diffusely extended part of the more dorsal and almost entire hypothalamus (E5). The stereological estimation of the total number of HDCi neurons of each clusters revealed the larger amount of the rat than the mouse. The characterization of histaminergic neuronal clusters in the hypothalamus of rats and mice may provide useful information for further investigations.

A clinical approach to brown adipose tissue in the para-aortic area of the human thorax.

BACKGROUND: Human thoracic brown adipose tissue (BAT), composed of several subdivisions, is a well-known target organ of many clinical studies; however, the functional contribution of each part of human thoracic BAT remains unknown. The present study analyzed the significance of each part of human thoracic BAT in the association between regional distribution, cellularity, and factors involved in the functional regulation of thoracic BAT. METHODS: We analyzed 1550 healthy adults who underwent medical check-ups by positron-emission tomography and computed tomography (PET-CT) imaging, 8 cadavers, and 78 autopsy cases in an observational study. We first characterized the difference between the mediastinum and the supraclavicular areas using counts of BAT detection and conditions based on PET-CT outcomes. The measurable important area was then subjected to systematic anatomical and immunohistochemical analyses using anti-uncoupling protein 1 (UCP1) antibody to characterize the cellularity in association with age and sex. RESULTS: In PET-CT scanning, the main site of thoracic BAT was the mediastinum rather than the supraclavicular area (P < 0.05). Systemic macroanatomy revealed that the thumb-sized BAT in the posterior mediastinal descending para-aortic area (paBAT) had feeding vessels from the posterior intercostal arteries and veins and sympathetic/parasympathetic innervation from trunks of the sympathetic and vagus nerves, respectively. Immunohistochemical analysis indicated that the paBAT exhibited immunoreactivity for tyrosine hydroxylase and vesicular acetylcholine transporter located in the pericellular nervous fibers and intracellular UCP1. The brown adipose cells of paBAT showed age-dependent decreases in UCP1 expression (P < 0.05), accompanied by a significant increase in vacuole formation, indicating fat accumulation (P < 0.05), from 10 to 37 years of age (P < 0.01). CONCLUSIONS: paBAT may be one of the essential sites for clinical application in BAT study because of its visible anatomy with feeding vessels and sympathetic/parasympathetic innervation functionally affected by outer condition and senescence.

Reduced Tyk2 gene expression in ß-cells due to natural mutation determines susceptibility to virus-induced diabetes.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic ß-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by ß-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic ß-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Significance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis.

Myofibroblast transdifferentiation plays a crucial role in the development and progression of renal tubulointerstitial fibrosis. However, the significance of α-smooth muscle actin (α-SMA) expression, which is the major morphological characteristic of myofibroblasts, remains to be determined in detail. The effect of α-SMA expression on fibrosis tissue was examined by using a fibrosis model (collagen gel) in vitro. The transdifferentiation of fibroblasts into myofibroblasts was triggered in the culture medium with 0.5% fetal bovine serum (FBS)+transforming growth factor (TGF)-ß1, but not with 10% FBS+TGF-ß1. The TGF-ß1-induced gel contraction caused by myofibroblasts was greater than that by fibroblasts. Gel contraction by myofibroblasts involved the Ca²+-dependent myosin light chain kinase pathway, as well as the activation of Rho kinase and p38 mitogen-activated protein kinase (MAPK). Taken together, these findings suggest that α-SMA expression in renal interstitial fibroblasts, i.e., myofibroblast transdifferentiation, accelerates the contraction of the tubulointerstitial fibrosis tissue via the Ca²+-dependent pathway, in addition to the pathways involved in fibroblast contraction; this event may lead to renal atrophy and renal failure.

Human skeletal muscles replaced to a high degree by white adipose tissue.

Extreme replacement of skeletal muscles by adipose tissue was found in an 86-year old Japanese male cadaver during dissection practice for medical students at Oita University School of Medicine. Especially, the bilateral sartorius muscles looked overall like adipose tissue. The man had suffered from diabetes mellitus, renal failure, hypertension and hypothyroidism before his death. He was also an alcohol drinker. He had been bedridden late in life. The cause of death was renal failure. In microscopy, the adipose tissue-like sartorius muscle was shown to consist of leptin-positive adipocytes with a small number of degenerated muscle fibers. Fatty replacement, or fatty degeneration, appears to result from endocrine and metabolic disorders, and being bedridden leads to muscle atrophy and damage, although the origin of the adipocytes which emerged in the degenerated muscles is unknown.

Role of the CXCL12/CXCR4 axis in milky spots of rats bearing ascitic-type hepatoma.

Rat ascitic-type hepatoma AH7974 cells express CXCR4 mRNA and protein at high levels and also show vigorous migratory responses to its ligand CXCL12. We have shown that AMD3100 (a specific CXCR4 antagonist) effectively reduced tumor invasion into the milky spot in Sprague-Dawley rats inoculated with AH7974 cells. A histological analysis revealed that the milky spots from AMD3100-treated rats were both smaller and consisted of fewer constituent cells and blood vessels than those from the AH7974 inoculated rats. Alkaline phosphatase staining also showed a statistically significant reduction in the area of the milky spots in the AMD3100-treated rats in comparison to the AH7974 inoculated rats (P < 0.0001). Green fluorescence protein (GFP)-tagged AH7974 cells were constructed to detect the localization of the tumor cells in the milky spots. There were fewer GFP-tagged AH7974 cells in the AMD3100-treated rats than in the AH7974 inoculated rats. The number of eosinophils and mast cells increased in the milky spots of AH7974-inoculated rats, and angiogenesis was also seen. In comparison, both cell proliferation and angiogenesis were inhibited in the milky spots of the AMD3100-treated rats. Collectively, our results strongly suggest that the CXCR4/CXCL12 axis plays an important role in the development of peritoneal carcinomatosis. As such, CXCR4 may be a potential therapeutic target for peritoneal carcinomatosis.

Morphological and functional changes of the rat parotid glandular cells by clipping and reopening the parotid duct, using HAM8 antibody.

The purpose of this experiment is to examine the proliferative process of rat acinar cells after parotid duct ligation and reopening. Two experimental groups were observed. The first group was killed from 0 to 14 days after the duct ligation. In the second group, the duct was clipped for 14 days, and it was reopened. Following a period of from 2 to 28 days after removal of the clip, the glands were removed to perform a histological analysis, including hematoxylin-eosin (HE), immunofluorescent staining using HAM8 antibody, which recognizes connexin 32, and transmission electron microscopy (TEM). In the experimental gland from the 1st group at 6 days after ligation (I-6D), the acinar cells disappeared. In the tissue from the 2nd group 8 days after reopening (II-8D), newly formed acinar cells were found again. Lobular structure of the parotid glands recovered in the II-21D. HAM8 signals were observed between normal acinar cells, while they declined in the tissue from I-1D, and they were not observed in the I-2D. HAM8 signals were first observed in the II-25D and then subsequently returned to normal levels in the II-28D. These results suggest that the intercellular communication and functional recovery was not complete 25 days after reopening of the duct.In conclusion, the recovery of the acinar structure was recognized during an extended period of duct ligation, however, a time lag between the morphological and functional recovery was found to exist.

Contraction of tubulointerstitial fibrosis tissue in diabetic nephropathy, as demonstrated in an in vitro fibrosis model.

Tubulointerstitial fibrosis in diabetic nephropathy (DN) was investigated using an in vitro tissue model of remodeling, to determine the pathogenic mechanism of fibrosis that leads to renal atrophy, i.e., renal failure. The remodeling model consisted of a renal fibroblast-populated collagen lattice (FPCL). The overexpression of transforming growth factor (TGF)-beta1 in the diabetic kidney gave rise to FPCL contraction. FPCL relaxation was induced by the subsequent addition of cytochalasin D. The FPCL failed to contract when exposed to TGF-beta1 plus Y27632, a Rho kinase inhibitor. TGF-beta1 induced the phosphorylation of myosin light chains, and Y27632 blocked this activity. TGF-beta1-induced FPCL contraction was suppressed by the addition of 2,3-butanedione monoxime, a myosin ATPase inhibitor. As shown in the video, the contraction rate of the projections of the cells in the FPCL was significantly greater in the TGF-beta1 group than in the control group. Collectively, these results indicate that TGF-beta1-induced FPCL contraction is attributable to actin-myosin interactions in the fibroblasts through the activation of Rho kinase, the phosphorylation of myosin light chains, and the subsequent activation of myosin ATPase. We propose that via these mechanisms, tubulointerstitial fibrosis generates tissue contraction that leads to renal atrophy and renal failure in DN.

Intracellular formation of collagen microfibrils in granulation tissue.

It is important to determine the biosynthesis process of collagen fibers to elucidate the mechanism by which granulation tissue is induced after injury. The purpose of this study is to investigate whether collagen microfibrils can be formed not only outside but also inside a cell. Fibroblast-like cells in granulation tissue resulting from incision and ligation were examined. The cells possessed vesicles containing collagen microfibrils. The vesicles were present in connection with Golgi apparatus or the rough endoplasmic reticulum. Furthermore, the vesicles were exhibited to be secretory granules with the secretory granule marker Rab3A. The fibroblast-like cells were also indicated to be myofibroblasts, using conventional transmission electron microscopy and immunoelectron microscopy for the myofibroblast marker alpha smooth muscle actin. In conclusion, it was demonstrated that collagen microfibrils could be formed in the cell in the case of collagen fiber overproduction.

Glomerular podocyte endocytosis of the diabetic rat.

We used immunoelectron microscopy to examine whether glomerular podocytes have the endocytotic function of macromolecular proteins in the early stage of diabetic nephropathy. Diabetes was induced by injecting streptozocin 60 mg kg wt(-1) into rats. Creatinine clearance but not urinary protein excretion was increased after four weeks of diabetes. The kidneys were morphologically studied 1 h after goat serum injection. In conventional electron microscopy, lysosomes were conspicuous in the podocytes of diabetic rats. Immunoelectron microscopy revealed that endogenous rat IgG and exogenous goat IgG were present in the lysosomes of podocytes from diabetic rats. The results indicated that the podocytes had an increased capacity for endocytosis in the early stage of diabetic nephropathy without increased urinary protein excretion.

Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy.

The developmental mechanism of tubulointerstitial fibrosis in diabetic nephropathy (DN) has not been elucidated. Tubulointerstitial fibrosis, as well as glomerulosclerosis, occurs in DN. Myofibroblasts which overproduce extracellular matrix are present in the renal interstitium in diabetics, although they are almost never seen in normal kidneys. The myofibroblasts appear to originate from interstitial fibroblasts. In addition, transforming growth factor-beta1 (TGF-beta 1), which can evoke myofibroblast transformation, is detected in interstitial cells in the diabetic kidney, but not in the normal kidney. Taken together, these findings led us to speculate that TGF-beta 1 induces the transformation of interstitial fibroblasts into myofibroblasts, followed by tubulointerstitial fibrosis. Based on this speculation, we discuss the developmental mechanism of tubulointerstitial fibrosis in this review.

Characteristic distribution of gap junctions in rat lacrimal gland in vivo and reconstruction of a gap junction in an in vitro model.

We investigated localization of the gap junction in rat lacrimal gland in vivo and in vitro using electron microscopy and immunostaining with anti-connexin32 (Cx32) monoclonal antibody (HAM8). In immunofluorescence study of lacrimal gland tissues, Cx32 protein appeared to exist not only at the intercellular borders of acinar cells, but also in the basal regions, where there apparently was no contact with adjacent acinar cells. Thin sectioning and immunoelectron microscopy revealed that lacrimal acinar cells formed autocellular gap junctions (reflexive gap junctions) in the basal regions and intercellular gap junctions with adjacent acinar cell membranes. In immunofluorescence study of primary culture, Cx32 protein was found on the free surfaces of isolated acinar cells at the early stage of culture. With culturing time, cell aggregates were formed. We observed Cx32 immunoreactivity between acinar cells in these aggregates, but not on their free surface. Electron microscopic study confirmed that these aggregates possessed intercellular gap junctions and morphologically differentiated acinar-like structures. However, reflexive gap junctions were not observed in these aggregates. In conclusion, lacrimal acinar cells form intercellular and reflexive gap junctions in vivo. On the other hand, the existence of an acinar-like structure and intercellular gap junctions indicates that acinar cells differentiated in vitro morphologically. The cells may communicate with each other through these junctions, organizing themselves into an acinar cell network in an in vitro situation.