Immunohistochemical localization of HCA1 receptor in placenta in presence of fetal growth restriction.

INTRODUCTION: Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Fetuses with intrauterine growth restriction are considered to become progressively lactacidemic as well as hypoxic. Roles of lactate in the placenta in the presence of fetal growth restriction (FGR) remain to be clarified. METHODS: Immunohistochemical localization of lactate-related substances, such as a receptor for lactate (hydroxy-carboxylic acid 1 receptor (HCA1 receptor/GPR81)), monocarboxylate transporters (MCTs) for lactate, lactate dehydrogenases (LDHs), and proteins expressed in syncytiotrophoblasts or cytotrophoblasts was examined in placentas of appropriate weight for gestational age (AGA) fetus and those showing FGR. RESULTS: Immunoreactivity for the HCA1 receptor was present in the cytoplasm of some trophoblasts, predominantly localized to their basal (fetus-facing) side, and was frequently colocalized with that for E-cadherin or serine peptidase inhibitor, Kunitz type 1 (SPINT1), a marker protein of cytotrophoblasts. Immunoreactivity for MCT1 and MCT4 was present on the basal and the microvillous (maternal-facing) membranes of trophoblasts in both groups, respectively. Clear immunoreactivity for LDHA and LDHB was also observed in the cytoplasm of trophoblasts, mainly localized to their basal side. However, there were no significant differences in immunohistochemically stained areas of lactate-related substances between AGA and late-onset FGR groups. On the other hand, there were correlations between coefficients of the presence of chorioamnionitis and the values of LDHB and E-cadherin. DISCUSSION: Immunohistochemical localization of the HCA1 receptor was predominantly observed in the cytoplasm located on the basal side of trophoblasts, suggesting a role of lactate in human placental development, including syncytialization.

Transporters, Ion Channels, and Junctional Proteins in Choroid Plexus Epithelial Cells.

The choroid plexus (CP) plays significant roles in secreting cerebrospinal fluid (CSF) and forming circadian rhythms. A monolayer of epithelial cells with tight and adherens junctions of CP forms the blood-CSF barrier to control the movement of substances between the blood and ventricles, as microvessels in the stroma of CP have fenestrations in endothelial cells. CP epithelial cells are equipped with several kinds of transporters and ion channels to transport nutrient substances and secrete CSF. In addition, junctional components also contribute to CSF production as well as blood-CSF barrier formation. However, it remains unclear how junctional components as well as transporters and ion channels contribute to the pathogenesis of neurodegenerative disorders. In this manuscript, recent findings regarding the distribution and significance of transporters, ion channels, and junctional proteins in CP epithelial cells are introduced, and how changes in expression of their epithelial proteins contribute to the pathophysiology of brain disorders are reviewed.

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKß/2 splice variants that co-localize with CaMKIV in spermatids.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca2+-dependent physiological and pathophysiological pathways. Further, CaMKKß/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKß/2 splice variants (CaMKKß-3 and ß-3x). RT-PCR analyses revealed that mouse CaMKKß-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKß-3 and ß-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKß-3 or ß-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKß-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKß-1. We also observed the co-localization of CaMKKß-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKß-3 exhibited Ca2+/CaM-induced kinase activity similar to CaMKKß-1. Conversely, we noted that CaMKKß-3x impaired Ca2+/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKß-1 or ß-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKß-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKß-1 and ß-3. Collectively, CaMKKß-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

E-Cadherin Is Expressed in Epithelial Cells of the Choroid Plexus in Human and Mouse Brains.

Evidence showing the functional significance of the choroid plexus is accumulating. Epithelial cells with tight and adherens junctions of the choroid plexus play important roles in cerebrospinal fluid production and circadian rhythm formation. Although specific types of cadherin expressed in adherens junctions of choroid plexus epithelium (CPE) have been examined, they remained uncertain. Recent mass spectrometry and immunolocalization analysis revealed that non-epithelial cadherins, P- and N-cadherins, are expressed in the lateral membrane of CPE, whereas E-cadherin expression has not been confirmed in CPE of humans or mice. In this study, we examined E-cadherin expression in CPE of mice and humans by RT-PCR, immunohistochemical-, and Western blotting analyses. We confirmed, by using RT-PCR analysis, the mRNA expression of E-cadherin in the choroid plexus of mice. The immunohistochemical expression of E-cadherin was noted in the lateral membrane of CPE of mice and humans. We further confirmed, in Western blotting, the specific immunoreactivity for E-cadherin. Immunohistochemically, the expression of E- and N-cadherins or vimentin was unevenly distributed in some CPE, whereas that of E- and P-cadherins or ß-catenin frequently co-existed in other CPE. These findings indicate that E-cadherin is expressed in the lateral membrane of CPE, possibly correlated with the expression of other cadherins and cytoplasmic proteins.

Distribution of Monocarboxylate Transporters in Brain and Choroid Plexus Epithelium.

The choroid plexus (CP) plays central roles in regulating the microenvironment of the central nervous system by secreting the majority of cerebrospinal fluid (CSF) and controlling its composition. A monolayer of epithelial cells of CP plays a significant role in forming the blood-CSF barrier to restrict the movement of substances between the blood and ventricles. CP epithelial cells are equipped with transporters for glucose and lactate that are used as energy sources. There are many review papers on glucose transporters in CP epithelial cells. On the other hand, distribution of monocarboxylate transporters (MCTs) in CP epithelial cells has received less attention compared with glucose transporters. Some MCTs are known to transport lactate, pyruvate, and ketone bodies, whereas others transport thyroid hormones. Since CP epithelial cells have significant carrier functions as well as the barrier function, a decline in the expression and function of these transporters leads to a poor supply of thyroid hormones as well as lactate and can contribute to the process of age-associated brain impairment and pathophysiology of neurodegenerative diseases. In this review paper, recent findings regarding the distribution and significance of MCTs in the brain, especially in CP epithelial cells, are summarized.

Partially hydrolyzed guar gum alleviates hepatic steatosis and alters specific gut microbiota in a murine liver injury model.

PURPOSE: The gut microbiota, via the gut-liver axis, plays an important role in the development of intestinal failure-associated liver disease. Here, we investigated whether partially hydrolyzed guar gum (PHGG), a dietary fiber could alleviate liver damage and modulate the gut microbiota in a murine liver injury (LI) model. METHODS: Liver injury was induced in 6-week-old male C57BL/6 mice using an enteral liquid diet composed of parenteral nutrition (LI group) and treated with 5% PHGG (LI/PHGG group). Liver histopathology was examined using oil red O and a tumor necrosis factor-α (TNF-α) labeling. The gut microbiota was examined using 16S rRNA gene sequencing. RESULTS: Lipid accumulation was significantly decreased in the LI /PHGG group when compared with that of the LI group. The area of TNF-α-positive cells was significantly higher in the LI group when compared with that of the control. The principal coordinate analysis (PCoA) revealed pronounced changes in the gut microbiota after PHGG treatment. Linear discriminant analysis of effect size showed that PHGG treatment significantly increased cecal abundance of Parabacteroides. CONCLUSIONS: PHGG alleviated hepatic steatosis following liver injury in mice. The protective effect of PHGG treatment could be associated with increased abundance of Parabacteroides in the cecum.

Constrictive pericarditis caused by pericardial metastasis from esophageal squamous cell carcinoma: a case report.

Constrictive pericarditis is a rare condition characterized by clinical signs of right heart failure subsequent to the loss of pericardial compliance. We report a case of constrictive pericarditis due to pericardial metastasis in a patient with a history of esophageal squamous cell carcinoma that had a pathological complete response (pCR) to preoperative chemoradiotherapy. A 66-year-old woman was referred to our division for the treatment of advanced esophageal cancer. Video-assisted thoracoscopic surgery esophagectomy (VATSE) with 3-field lymphadenectomy was performed after neoadjuvant chemoradiotherapy (NAC-CRT). Pathological examination revealed no residual tumor, lymph node metastasis, lymphatic invasion, or vessel invasion. The histological treatment effect of the chemoradiotherapy was pathological complete response (pCR). Five months after surgery, the patient was admitted to a nearby hospital for the treatment of acute pericarditis. However, a month after admission, acute pericarditis progressed to constrictive pericarditis, and she was referred to our hospital for further management. Subsequently, urgent pericardiectomy was performed through a lower half sternotomy incision. After surgery, heart failure improved for a while but worsened again. The patient died 7 days after the surgery. Pathological examination of the resected pericardium revealed evidence of metastasis from squamous cell carcinoma of the esophagus. An autopsy revealed the spread of esophageal cancer to the bilateral pleura, right lung, pericardium, diaphragm, soft tissue surrounding the tracheal bifurcation, and bilateral hilar lymph nodes. Similarly, tumor cells were found in the lymphatic vessels of the pericardium and pleura. Even if pCR is achieved with NAC-CRT, as in our case, esophageal cancer may metastasize and present as constrictive pericarditis within a short period; therefore, careful patient follow-up is essential.

Metabolites and Biomarker Compounds of Neurodegenerative Diseases in Cerebrospinal Fluid.

Despite recent advances in diagnostic procedures for neurological disorders, it is still difficult to definitively diagnose some neurodegenerative diseases without neuropathological examination of autopsied brain tissue. As pathological processes in the brain are frequently reflected in the components of cerebrospinal fluid (CSF), CSF samples are sometimes useful for diagnosis. After CSF is secreted from the choroid plexus epithelial cells in the ventricles, some flows in the brain, some is mixed with intracerebral interstitial fluid, and some is excreted through two major drainage pathways, i.e., the intravascular periarterial drainage pathway and the glymphatic system. Accordingly, substances produced by metabolic and pathological processes in the brain may be detectable in CSF. Many papers have reported changes in the concentration of substances in the CSF of patients with metabolic and neurological disorders, some of which can be useful biomarkers of the disorders. In this paper, we show the significance of glucose- and neurotransmitter-related CSF metabolites, considering their transporters in the choroid plexus; summarize the reported candidates of CSF biomarkers for neurodegenerative diseases, including amyloid-ß, tau, α-synuclein, microRNAs, and mitochondrial DNA; and evaluate their potential as efficient diagnostic tools.

Immunohistochemical expression of osteopontin and collagens in choroid plexus of human brains.

Evidence showing the functional significance of the choroid plexus is accumulating. Although it is clinically well-known that calcification is frequently seen in the choroid plexus of aged human brains, it is unclear why calcification occurs in the aged choroid plexus and what exert effects on the calcification has. In this study, immunohistochemical localizations of collagens and other molecules related to fibrosis or calcification were investigated on the choroid plexus of autopsied human brains. Densely fibrous or calcified materials were located in the stroma just below the epithelial cells of the choroid plexus of all human brains examined. Immunoreactivity for collagen type I was identified in the stroma just below the epithelial cells, consistent with the densely fibrous or calcified area, whereas that for collagen type III was observed in almost all stroma other than the densely fibrous or calcified areas. Linear or membranous immunoreactivity for collagen type IV was intermittently localized on the epithelium-facing side of the materials, suggesting an injured basement membrane. In addition, clear immunoreactivity for osteopontin was localized on the epithelium-facing side of the fibrous or calcified materials as well as in the cytoplasm of epithelial cells. These findings indicate that collagen type I exists in contact with osteopontin in and around the densely fibrous or calcified materials in the choroid plexus. They suggest that the densely fibrous or calcified materials are deposited in the subepithelial stroma just below an injured basement membrane of epithelial cells via the collagen type I and osteopontin.

Polymorphic SERPINA3 prolongs oligomeric state of amyloid beta.

Molecular chaperon SERPINA3 colocalizes with accumulated amyloid peptide in Alzheimer's disease (AD) patient's brain. From the QTL analysis, we narrowed down Serpina3 with two SNPs in senescence-accelerated mouse prone (SAMP) 8 strain. Our study showed SAMP8 type Serpina3 prolonged retention of oligomeric Aß 42 for longer duration (72 hr) while observing under transmission electron microscope (TEM). From Western blot results, we confirmed presence of Aß 42 oligomeric forms (trimers, tetramers) were maintained for longer duration only in the presences of SAMP8 type Serpina3. Using SH-SY5Y neuroblastoma cell line, we observed until 36 hr preincubated Aß 42 with SAMP8 type Serpina3 caused neuronal cell death compared to 12 hr preincubated Aß 42 with SAMR1 or JF1 type Serpina3 proteins. Similar results were found by extending this study to analyze the effect of polymorphism of SERPINA3 gene of the Japanese SNP database for geriatric research (JG-SNP). We observed that polymorphic SERPINA3 I308T (rs142398813) prolonged toxic oligomeric Aß 42 forms till 48 hr in comparison to the presence wild type SERPINA3 protein, resulting neuronal cell death. From this study, we first clarified pathogenic regulatory role of polymorphic SERPINA3 in neurodegeneration.

Immunoreactivity of receptor and transporters for lactate located in astrocytes and epithelial cells of choroid plexus of human brain.

Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Cerebral ischemia or hypoxia is believed to become progressively lactacidemic. Monocarboxylate transporters (MCTs) in endothelial cells are essential for the transport of lactate from the blood into the brain. In addition, it is considered that MCTs located in astrocytic and neuronal cells play a key role in the shuttling of energy metabolites between neurons and astrocytes. However, roles of lactate in the brain remain to be clarified. In this study, the localization of lactate transporters and a receptor for cellular uptake of lactate was immunohistochemically examined in autopsied human brains. Immunoreactivity for MCT1 was observed in the apical cytoplasmic membrane of some epithelial cells in the choroid plexus as well as astrocytes and the capillary wall, whereas that for MCT4 was found in the basolateral cytoplasmic membrane of small number of epithelial cells as well as astrocytes and the capillary wall. In addition, immunoreactivity for the hydroxy-carboxylic acid 1 receptor (HCA1 receptor), a receptor for cellular uptake of lactate, was also found on the basolateral cytoplasmic membrane of epithelial cells as well as astrocytic and neuronal cells. Immunoreactivity for lactate dehydrogenase (LDH)-B was observed in the cytoplasm of epithelial cells in the choroid plexus as well as astrocytes and the capillary wall. These immunohistochemical findings indicate the localization of MCT1, MCT4, the HCA1 receptor, and LDH-B in epithelial cells of the choroid plexus as well as astrocytes, and suggest the transport of intravascular lactate into the brain through epithelial cells of the choroid plexus as well as cerebral vessels and the possibility of lactate being utilized in epithelial cells.

Proteomic profile differentiating between mesial temporal lobe epilepsy with and without hippocampal sclerosis.

Hippocampal sclerosis (HS) is the most common neuropathological condition in adults with drug-resistant epilepsy and represents a critical feature in mesial temporal lobe epilepsy (MTLE) syndrome. Although epileptogenic brain tissue is associated with glutamate excitotoxicity leading to oxidative stress, the proteins that are targets of oxidative damage remain to be determined. In the present study we designed comprehensive analyses of changes in protein expression level and protein oxidation status in the hippocampus or neocortex to highlight proteins associated with excitotoxicity by comparing MTLE patients with relatively mild excitotoxicity (MTLE patients without HS, MTLE-non-HS) and those with severe excitotoxicity (MTLE patients with HS, MTLE-HS). We performed 2-dimensional fluorescence difference gel electrophoresis, 2D-oxyblot analysis, and mass spectrometric amino acid sequencing. We identified 16 proteins at 18 spots in which the protein expression levels differed between sclerotic and non-sclerotic hippocampi. In the sclerotic hippocampus, the expression levels of several synaptic proteins were decreased, and those of some glia-associated proteins increased. We confirmed histologically that all MTLE-HS cases examined exhibited severe neuronal cell loss and remarkable astrocytic gliosis in the hippocampi. In all MTLE-non-HS cases examined, neurons were spared and gliosis was unremarkable. Therefore, we consider that decreased synaptic proteins are a manifestation of loss of neuronal cell bodies and dendrites, whereas increased glia-associated proteins are a manifestation of proliferation and hypertrophy of astrocytes. These are considered to be the result of hippocampal sclerosis. In contrast, the expression level of d-3-phosphoglycerate dehydrogenase (PHGDH), an l-serine synthetic enzyme expressed exclusively by astrocytes, was decreased, and that of stathmin 1, a neurite extension-related protein expressed by neurons, was increased in the sclerotic hippocampus. These findings cannot be explained solely as the result of hippocampal sclerosis. Rather, these changes can be involved in the continuation of seizure disorders in MTLE-HS. In addition, the protein carbonylation detection, an indicator of protein oxidation caused by excitotoxicity of multiple seizures and/or status epilepticus, revealed that the carbonyl level of collapsin response mediator protein 2 (CRMP2) increased significantly in the sclerotic hippocampus. In conclusion, protein identification following profiling of protein expression levels and detection of oxidative proteins indicated potential pathognomonic protein changes. The decreased expression of PHGDH, increased expression of stathmin 1, and carbonylation of CRMP2 differentiate between MTLE with and without HS. Therefore, further investigations of PHGDH, stathmin 1 and CRMP2 are promising to study more detailed effects of excitotoxicity on epileptogenic hippocampal tissue.

Glucose, Fructose, and Urate Transporters in the Choroid Plexus Epithelium.

The choroid plexus plays a central role in the regulation of the microenvironment of the central nervous system by secreting the majority of the cerebrospinal fluid and controlling its composition, despite that it only represents approximately 1% of the total brain weight. In addition to a variety of transporter and channel proteins for solutes and water, the choroid plexus epithelial cells are equipped with glucose, fructose, and urate transporters that are used as energy sources or antioxidative neuroprotective substrates. This review focuses on the recent advances in the understanding of the transporters of the SLC2A and SLC5A families (GLUT1, SGLT2, GLUT5, GLUT8, and GLUT9), as well as on the urate-transporting URAT1 and BCRP/ABCG2, which are expressed in choroid plexus epithelial cells. The glucose, fructose, and urate transporters repertoire in the choroid plexus epithelium share similar features with the renal proximal tubular epithelium, although some of these transporters exhibit inversely polarized submembrane localization. Since choroid plexus epithelial cells have high energy demands for proper functioning, a decline in the expression and function of these transporters can contribute to the process of age-associated brain impairment and pathophysiology of neurodegenerative diseases.

Sodium/glucose cotransporter 2 is expressed in choroid plexus epithelial cells and ependymal cells in human and mouse brains.

Diabetes mellitus (DM) is now recognized as one of the risk factors for Alzheimer's disease (AD), and the disease-modifying effects of anti-diabetic drugs on AD have recently been attracting great attention. Sodium/glucose cotransporter 2 (SGLT2) inhibitors are a new class of anti-diabetic drugs targeting the SGLT2/solute carrier family 5 member 2 (SLC5A2) protein, which is known to localize exclusively in the brush border membrane of early proximal tubules in the kidney. However, recent data suggest that it is also expressed in other tissues. In the present study, we investigated the expression of SGLT2/SLC5A2 in human and mouse brains. Immunohistochemical staining of paraffin sections from autopsied human brains and C3H/He mouse brains revealed granular cytoplasmic immunoreactivity in choroid plexus epithelial cells and ependymal cells. Immunoblot analysis of the membrane fraction of mouse choroid plexus showed distinct immunoreactive bands at 70 and 26 kDa. Band patterns around 70 kDa in the membrane fraction of the choroid plexus were different from those in the kidney. Reverse transcription-polymerase chain reaction analysis confirmed the expression of Slc5a2 mRNA in the mouse choroid plexus. Our results provide in vivo evidence that SGLT2/SLC5A2 is expressed in cells facing the cerebrospinal fluid, in addition to early proximal tubular epithelial cells. These findings suggest that SGLT2 inhibitors may have another site of action in the brain. The effects of SGLT2 inhibitors on brain function and AD progression merit further investigation to develop better treatment options for DM patients.

Retinal ganglion cell loss in kinesin-1 cargo Alcadein α deficient mice.

Maintenance of retinal ganglion cells (RGCs) activity is relied on axonal transport conveying materials required for their survival such as neurotrophic factors. Kinesin-1 undergoes anterograde transport in axons, and Alcadein α (Alcα; also called calsyntenin-1) is a major cargo adaptor protein that can drive kinesin-1 to transport vesicles containing Alcα. The long-term effects of Alcα-deficiency on retinal morphology and survival of RGCs during postnatal development were examined in Alcα knockout mice. At 1.5, 3, 6, and 15 months postnatal, the number of retrogradely labeled RGCs was determined in flat-mounted retinas of Alcα-deficient and wild-type mice. Retinal damage was assessed histologically by determining the retinal thickness. Intraocular pressure (IOP) was measured with a Tonolab tonometer. At 1.5 months postnatal, the number of retrogradely labeled RGCs was not different between wild-type and Alcα-deficient mice. However, at 3, 6, and 15 months postnatal, the number of RGCs was significantly lower in Alcα deficient mice than those of wild-type mice (143 ± 41.1 cells/mm2 vs. 208 ± 28.4 cells/mm2, respectively, at 3 months; P < 0.01). No differences were seen in retinal thickness or IOP between the two types of mice at any postnatal age. Alcα-deficient mice showed spontaneous loss of RGCs but no elevation in IOP. These mice mimic normal-tension glaucoma and will be useful for investigating the mechanism of neurodegeneration in this disorder and for developing treatments for RGC loss that does not involve changes in IOP.

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains.

Iron plays essential roles in the central nervous system. However, how the iron level is regulated in brain cells including glia and neurons remains to be fully clarified. In this study, the localizations of hepcidin, ferroportin, and hephaestin, which are known to be involved in iron efflux, were immunohistochemically examined in autopsied human brains. Immunoreactivities for hepcidin and ferroportin were observed in granular structures within the cytoplasm of reactive astrocytes and epithelial cells of the choroid plexus. Granular structures showing immunoreactivities for hepcidin and ferroportin were also stained with antibodies for early endosome antigen 1 (EEA1). In addition, immunoreactivity for hephaestin was observed in the cytoplasm of epithelial cells of the choroid plexus as well as reactive astrocytes. Immunoreactivity for hephaestin in the cytoplasm of reactive astrocytes was occasionally colocalized with immunoreactivity for EEA1, while that of hephaestin was frequently observed in the cytoplasm showing no immunoreactivity for EEA1. These findings suggest that immunoreactivities for hepcidin and ferroportin are localized in close proximity to granular structures showing immunoreactivity for EEA1 in the cytoplasm of human brain astrocytes. They also suggest that immunoreactivity of hephaestin is localized in the cytoplasm of the choroid plexus epithelium as well as reactive astrocytes of human brains.

Partially hydrolyzed guar gum alleviates small intestinal mucosal damage after massive small bowel resection along with changes in the intestinal microbiota.

PURPOSE: Short bowel syndrome is associated with intestinal mucosal inflammation and microbial dysbiosis, leading to intractable complications. Partially hydrolyzed guar gum (PHGG) has trophic and anti-inflammatory effects on the intestine. We investigated whether PHGG ameliorates small intestinal mucosal damage and alters the intestinal microbiota using a rat small bowel resection (SBR) model. METHODS: Sprague Dawley rats were divided into sham operation (Sham), Sham/PHGG, SBR, and SBR/PHGG groups. On day 21, all rats were euthanized. To assess small intestinal mucosal damage, the degeneration rate was morphometrically evaluated and immunohistochemically examined using anti-CD45 antibodies. Analyses of fecal microbiota using 16S rRNA and short-chain fatty acid production were also performed. RESULTS: The mucosal degeneration rate was significantly higher in the SBR group than in the Sham or SBR/PHGG groups. The number of CD45-positive cells was significantly higher in the SBR group than in the Sham, Sham/PHGG, or SBR/PHGG groups. The relative abundance of family Lachnospiraceae was significantly higher in the SBR/PHGG group than in the SBR group. CONCLUSIONS: PHGG administration alleviated small intestinal mucosal damage which could be associated with modulation of the intestinal microbiota.

Neuroprotective activation of astrocytes by methylmercury exposure in the inferior colliculus.

Methylmercury (MeHg) is well known to induce auditory disorders such as dysarthria. When we performed a global analysis on the brains of mice exposed to MeHg by magnetic resonance imaging, an increase in the T1 signal in the inferior colliculus (IC), which is localized in the auditory pathway, was observed. Therefore, the purpose of this study is to examine the pathophysiology and auditory dysfunction induced by MeHg, focusing on the IC. Measurement of the auditory brainstem response revealed increases in latency and decreases in threshold in the IC of mice exposed to MeHg for 4 weeks compared with vehicle mice. Incoordination in MeHg-exposed mice was noted after 6 weeks of exposure, indicating that IC dysfunction occurs earlier than incoordination. There was no change in the number of neurons or microglial activity, while the expression of glial fibrillary acidic protein, a marker for astrocytic activity, was elevated in the IC of MeHg-exposed mice after 4 weeks of exposure, indicating that astrogliosis occurs in the IC. Suppression of astrogliosis by treatment with fluorocitrate exacerbated the latency and threshold in the IC evaluated by the auditory brainstem response. Therefore, astrocytes in the IC are considered to play a protective role in the auditory pathway. Astrocytes exposed to MeHg increased the expression of brain-derived neurotrophic factor in the IC, suggesting that astrocytic brain-derived neurotrophic factor is a potent protectant in the IC. This study showed that astrogliosis in the IC could be an adaptive response to MeHg toxicity. The overall toxicity of MeHg might be determined on the basis of the balance between MeHg-mediated injury to neurons and protective responses from astrocytes.

Disturbance of Intracerebral Fluid Clearance and Blood-Brain Barrier in Vascular Cognitive Impairment.

The entry of blood-borne macromolecular substances into the brain parenchyma from cerebral vessels is blocked by the blood-brain barrier (BBB) function. Accordingly, increased permeability of the vessels induced by insult noted in patients suffering from vascular dementia likely contributes to the cognitive impairment. On the other hand, blood-borne substances can enter extracellular spaces of the brain via endothelial cells at specific sites without the BBB, and can move to brain parenchyma, such as the hippocampus and periventricular areas, adjacent to specific sites, indicating the contribution of increased permeability of vessels in the specific sites to brain function. It is necessary to consider influx and efflux of interstitial fluid (ISF) and cerebrospinal fluid (CSF) in considering effects of brain transfer of intravascular substances on brain function. Two pathways of ISF and CSF are recently being established. One is the intramural peri-arterial drainage (IPAD) pathway of ISF. The other is the glymphatic system of CSF. Dysfunction of the two pathways could also contribute to brain dysfunction. We review the effects of several kinds of insult on vascular permeability and the failure of fluid clearance on the brain function.

Histomorphometry of ectopic mineralization using undecalcified frozen bone sections.

To investigate the correlation between mineral formation and enhanced expressions of some proteins using undecalcified frozen bone sections. Histological studies have revealed that some proteins, such as BMP2, BMPR1A, and Connexin 43, are expressed in and around sites of ectopic ossification. However, the relationship between the expressed proteins considered to be associated with the ossification and mineral formation in vivo is not clear. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1)-mutant spinal hyperostotic TWY mice and ICR mice as controls were euthanized after calcein labeling, and undecalcified frozen sections were obtained from the middle thoracic spine. Intervertebral disc areas were examined histologically and by measuring calcein-labeled areas and areas showing immunoreactivity for BMP2, BMPR1A, and Connexin 43. Calcein-labeled areas, indicating mineralization in the ectopic mineralization sites, were significantly larger in the mutant mice than in controls. The expression of Connexin 43 was elevated in the annulus fibrosus. Increases in the calcein-labeled areas was not correlated with increases in the areas showing immunoreactivity for Connexin 43 in the annulus fibrosus. There was no statistical correlation between enhanced immunohistochemical expression and elevated calcein-labeled areas. By applying the morphometrical analysis method using undecalcified frozen sections to ENPP1-mutant mice, quantitative evaluation of the mineralization and proteins expressed in the surrounding area in the same animal became possible.