Congenital portopulmonary shunt in a cat.

A 9-year-old spayed female crossbreed cat with chief complaints of anorexia and hypersalivation had high serum concentrations of ammonia and fasting and postprandial total bile acid. Therefore, she was referred to our hospital. On the first evaluation, haematology, serum chemistry, radiography and ultrasonography findings suggested that she had a congenital portosystemic shunt. CT revealed a shunt vessel from the left gastric vein to the left pulmonary vein. During median celiotomy and sternotomy, gross findings and mesenteric portography revealed abnormal vessel shunting from the left gastric vein to the left pulmonary vein. Complete ligation of the shunt vessel was achieved. She recovered without any complications. Postoperative serum chemistry revealed that ammonia and total bile acid levels decreased to within the reference intervals. This report is the first to describe the clinical features and surgical outcome of a cat with a congenital portopulmonary shunt.

Transarterial chemoembolisation for palliative treatment of renal cell carcinoma in two dogs with pulmonary metastasis.

Two dogs with anorexia and rapid weight loss were referred to our hospital due to a right renal mass and several pulmonary nodules. Both dogs underwent needle core biopsy of the mass, followed by transarterial chemoembolisation of the renal mass. A catheter was inserted from the femoral artery and advanced into the right renal artery. A suspension of carboplatin (100 mg/m2 ) and equivalent lipiodol was administered via the inserted multipurpose catheter. Immediately after, under fluoroscopic guidance, pulse injections of small amounts of gelatin particles (diameter 1 mm) dissolved in iohexol were administered until complete embolisation of the renal artery. Histopathologic diagnosis was renal cell carcinoma in both dogs. Clinical signs improved for 134 and 358 days after transarterial chemoembolisation. In addition, postoperative radiographs demonstrated a decrease in the tumour size. The dogs died 215 and 525 days after the initial evaluation, respectively. As a palliative treatment, transarterial chemoembolisation might help reduce the tumour volume and improve the quality of life in dogs with renal cell carcinoma and distant metastases.

Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men.

Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = -0.68) and intensity (R = -0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia.

A novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus.

The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl-d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 degrees C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers (betaIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl-d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl-d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl-d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.

Quantitative analysis of amyloid plaques in a mouse model of Alzheimer's disease by phase-contrast X-ray computed tomography.

Densely aggregated beta-amyloid peptides are believed to play a key role in the pathogenesis of Alzheimer's disease. Amyloid plaques are a potential target for molecular imaging to determine the clinical status of Alzheimer's disease. Phase-contrast X-ray imaging combined with computed tomography is a promising technique that can be used to visualize the physical density of structures in biological tissues non-invasively, and without the use of imaging agents. Using brain tissue isolated from a mouse model of Alzheimer's disease, we show that beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques, but not beta-amyloid 40-negative/beta-amyloid 42-positive amyloid plaques, exist as high-density aggregates that can be specifically detected by phase-contrast X-ray computed tomography. The phase-contrast X-ray computed tomography detected beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques in three-dimensions with an extremely high sensitivity comparable to that of histological analysis, and also enabled the load of amyloid plaques to be quantified. Furthermore, the use of phase-contrast X-ray computed tomography reveals that the physical density of beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques increases with age, and that the large volume, high-density, amyloid plaques that are specifically observed in aged Alzheimer's disease mice are closely associated with neuritic dystrophy. These results demonstrate that phase-contrast X-ray computed tomography is a highly sensitive imaging technique for analyzing dense-cored amyloid plaques in postmortem samples, and is beneficial in elucidating amyloid pathophysiology in Alzheimer's disease.

Greater development of 1,2-dimethylhydrazine-induced colon cancer in a rat model of type 2 diabetes mellitus.

Several clinical cohort and case-control studies have suggested a link between diabetes and colon cancer. Otsuka Long-Evans Tokushima Fat (OLETF) rats spontaneously develop type 2 diabetes mellitus and Long-Evans Tokushima Otsuka (LETO) rats are non-diabetic. The relationship between type 2 diabetes mellitus and colon cancer was examined in these rats. The carcinogen 1,2-dimethylhydrazine was administered subcutaneously once weekly for 10 weeks, and the animals were killed and necropsied in week 29. All OLETF rats and 80% of the LETO rats developed cancer. The number of colon cancers per rat was significantly greater in the diabetic than in the non-diabetic rats. Although the tumours tended to be larger in diabetic rats, the difference was not statistically significant. No significant differences were observed in the depth of invasion or histological type of cancer in the two groups. Type 2 diabetes mellitus may enhance the generation and growth of colon cancer.

Transcriptional regulation of osteopontin gene in vivo by PEBP2alphaA/CBFA1 and ETS1 in the skeletal tissues.

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2alphaA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2alphaA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2alphaA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2alphaA. In the present study, we determined the direct regulation of PEBP2alphaA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2alphaA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2alphaA or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2alphaA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2alphaA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2alphaA to MC3T3/E1 cells expressing endogenous PEBP2alphaA, ETS1 and opn. These findings suggest that PEBP2alphaA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.

A novel oligodendrocyte cell line OLP6 shows the successive stages of oligodendrocyte development: late progenitor, immature and mature stages.

The successive stages of development from oligodendrocyte progenitor to mature oligodendrocyte have been investigated in detail by using stage-specific antibodies. However, no cell lines are available that show stepwise differentiation from oligodendrocyte progenitors to mature oligodendrocytes. Here we show the establishment of an immortalized oligodendrocyte cell line, OLP6, from adult transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. The OLP6 cells had a fibroblastic morphology and continuously proliferated at 33 degrees C. They displayed growth arrest and multipolar morphology when they were cultured at 39 degrees C. They express the oligodendrocytic markers O4, 2'-3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebroside and second endothelial differentiation gene receptor-2 at 39 degrees C. The OLP6 cells underwent apoptosis upon serum withdrawal at 39 degrees C. Lysophosphatidic acid inhibited this apoptosis and promoted the expression of myelin basic protein. These results demonstrate that the activation of endothelial differentiation gene receptor-2 exerts anti-apoptosis and myelinogenesis effects on the OLP6 cells. Taken together, the OLP6 cells in the late oligodendrocyte progenitor stage can progress to the immature oligodendrocyte stage by shifting culture temperature. Furthermore, lysophosphatidic acid promoted the maturation of OLP6 cells in the immature oligodendrocyte stage. Such OLP6 cells should provide a potent model system for studying the precise mechanism involved in stepwise differentiation of oligodendrocytes.

Role of osteopontin in bone remodeling caused by mechanical stress.

Changes in the number and proportion of osteopontin mRNA (Opn) expressing osteocytes and osteoclasts caused by the mechanical stress applied during experimental tooth movement were examined in the present study. Opn expression was detected in the osteocytes on the pressure side at the early stage, and gradually spread to those on the tension side and also to the osteoblasts and bone-lining cells in the alveolar bone. Only 3.3% of the osteocytes located on the pressure side expressed Opn in the interradicular septum of control rats; in contrast, the value was increased to 87.5% at 48 h after the initiation of tooth movement. These results indicate that these cells responded to mechanical stress loaded on the bone with expression of the osteopontin gene. Following the increased expression of Opn in these cells, a 17-fold greater number of osteoclasts compared with the control and numerous resorption pits were observed on the pressure side of the alveolar bone. Injection of arginine-glycine-aspartic acid-serine peptide but not that of arginine-glycine-glutamic acid-serine peptide strongly inhibited the increase in the number of osteoclasts. Furthermore, an in vitro migration assay demonstrated the chemotactic activity of osteopontin (OPN) on the precursor of osteoclasts. Our study strongly suggests that OPN is an important factor triggering bone remodeling caused by mechanical stress.

Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease.

The membrane attack complex, C5b-9, is of considerable importance in many inflammatory reactions. It is the terminal, cytolytic component of both classical and alternative pathway activation, and its presence presupposes other potentially destructive complement constituents, including anaphylotoxins and opsonins. We have characterized C5b-9 and its C9 constituent in the Alzheimer's disease (AD) and nondemented elderly (ND) brain using immunohistochemistry at the light and electron microscopic levels, Western blot analysis, and the reverse transcriptase polymerase chain reaction. We have also conducted in vitro ELISA assays of amyloid beta-peptide-stimulated SC5b-9 production. C5b-9 is abundantly present in Alzheimer's disease cortex, associated with neurofibrillary tangle containing neurons, dystrophic neurites within neuritic plaques, and neuropil threads, but is weakly detected, if at all, in nondemented elderly cortex under the same conditions. Staining of Alzheimer's disease sections is abolished both by deletion of primary antibody or preabsorption with purified SC5b-9.

Expression of CD43 in human microglia and its downregulation in Alzheimer's disease.

CD43 (leukosialin, sialophorin) expression in brain tissue of neurologically normal and Alzheimer disease (AD) cases was studied immunohistochemically. Abundant CD43-like immunoreactivity was detected in ramified microglia of normal brain. It was also seen in residual leukocytes in capillaries and was faintly detectable on the surface of some normal appearing neurons. In AD brains, the overall expression of CD43 by microglia was markedly lower than in control brains. This was in contrast to HLA-DR which was sharply upregulated due to the activated state of the microglia. This is the first report of a microglial marker which is more highly expressed in the resting or ramified state. Such expression is consistent with theories that CD43 plays an anti-adhesional role, and that cleavage occurs during cellular activation.

Immunohistochemical localization of GABAA receptors in comparison with GABA-immunoreactive structures in the nucleus tractus solitarii of the rat.

The localization of GABAA receptors was studied by immunohistochemistry in the nucleus tractus solitarii of the rat using a monoclonal antibody (bd17) against the beta-subunit. The pattern of distribution was compared with that of GABA-immunoreactive axons and nerve terminals. Positive staining for GABAA receptors was confined to regions near the surface of neuronal somata and their processes. The highest density of positive staining for GABAA receptors was seen in the central part of the rostral nucleus tractus solitarii where GABA-positive terminals were also rather dense. At both intermediate and caudal levels of the nucleus tractus solitarii, a moderate density of positive staining for GABAA receptors was located in the ventrolateral part, including the ventrolateral subnucleus. In these regions, the density of GABA-positive terminals was low. In the medial nucleus tractus solitarii, including the medial subnucleus, very little or no positive staining for GABAA receptors was detected, although many GABA-positive terminals were observed. The results suggest that the central part of the rostral nucleus tractus solitarii is controlled by the GABAergic system via GABAA receptors, but in the medial subnucleus of the nucleus tractus solitarii the GABA neurons appear to act via receptors that are not detectable by the antibody used.

beta-amyloid deposits in transgenic mice expressing human beta-amyloid precursor protein have the same characteristics as those in Alzheimer's disease.

A transgenic mouse expressing the human beta-amyloid precursor protein with the "Swedish" mutation, Tg2576, was used to investigate the mechanism of amyloid-beta peptide (Abeta) deposition. We characterized Abeta deposits in the cerebral cortex biochemically and pathologically. A surface-enhanced laser desorption/ionization affinity mass spectrometric study using the 6E10 monoclonal antibody demonstrated that the major species of Abeta in a formic acid-extracted fraction of the cortex were Abeta(1-38), Abeta(1-40) and Abeta(1-42). Immunohistochemistry using antibodies to the carboxy-terminal epitopes of Abeta(1-40) and Abeta(1-42), as well as 6E10, showed that plaques containing Abeta(1-42) were more numerous than those containing Abeta(1-40) throughout the cortex. Laser confocal analysis of the immunoreactivities in the plaques demonstrated that Abeta(1-40) was preferentially located in the central part of the Abeta(1-42) positive plaques. Enzyme-linked immunosorbent assay measurements of Abeta(1-40) and Abeta(1-42) showed that Abeta(1-40) was several-fold more abundant than Abeta(1-42). From these data we suggest that Abeta(1-42) deposition may precede Abeta(1-40) deposition, while Abeta(1-40) begins to deposit in the central part of the plaques and accumulates there. Furthermore, localization of Abeta(1-40) corresponded almost exactly to congophilic structures, which were associated with aberrant swollen synapses detected with antibodies to synaptophysin and alpha-synuclein. Thus, Abeta deposits in Tg2576 mice have similar characteristics to those in Alzheimer's disease.

Edg-8 receptors are preferentially expressed in oligodendrocyte lineage cells of the rat CNS.

The messenger RNA for endothelial differentiation gene 8 receptors is known to be expressed almost exclusively in the rat CNS, but the nature of the expressing cells has not been defined. Using an antibody specific for endothelial differentiation gene 8, we investigated the immunohistochemical localization of endothelial differentiation gene 8 receptors in the rat CNS. Immunopositive staining was detected in a subset of glial cells distributed throughout the brain and spinal cord, including both gray and white matter, but not in the dorsal root ganglion. The distribution and morphological similarity in comparative immunostaining for endothelial differentiation gene 8 and various glial markers suggested that endothelial differentiation gene 8 is preferentially expressed in NG2-positive oligodendrocyte progenitor cells in adult rat brains. Counts of endothelial differentiation gene 8-positive cells and NG2-positive cells in the forebrain revealed that a subset of NG2-positive cells was endothelial differentiation gene 8-positive, and that the ratio of endothelial differentiation gene 8-positive cells to NG2-positive cells varied from region to region. In 17-day-old embryonic brains, the endothelial differentiation gene 8 distribution was similar to that of an oligodendrocytic marker, 2',3'-cyclic nucleotide 3'-phosphodiesterase. These data suggest that endothelial differentiation gene 8 receptors are preferentially expressed in oligodendrocyte lineage cells including oligodendrocyte progenitor cells and immature/maturating oligodendrocytes in rat CNS, and that they might have important functions in oligodendrocytic maturation and myelination.

Neuroprotective effects of NBQX on hypoxia-induced neuronal damage in rat hippocampus.

Models of cerebral ischaemia were used for analysis of mechanism of neuronal cell death and/or damage. Ischaemia is caused dominantly by severe hypoxia and hypoglycaemia: in the present study, we examined the influence of severe in vivo hypoxia (5% O2/95% N2 for 30 min at 22 degrees C). After hypoxia, neuronal damage was observed in the CA3 and dentate gyrus (DG) after 3 and 21 days of survival, but not in the CA1.2,3-Dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), an antagonist for AMPA/kainate receptors, showed neuroprotective effects in the CA3 and DG. These results suggest that hypoxia may induce neuronal damage in the CA3 and DG through activation of AMPA/kainate receptors.

Encephalitogenic peptide (EP) in human cerebrovascular white matter lesions.

The expression of encephalitogenic peptide (EP), a 68-86 amino acid sequence of guinea pig myelin basic protein (MBP), was investigated in autopsied brains with focal cerebral damage or with diffuse white matter (WM) lesions. EP immunoreactive fibers were distributed in parallel with fibers immunoreactive for amyloid protein precursor (APP), an indicator of WM damages. EP was expressed in the periphery of cerebral infarctions and hematoma in the acute and subacute stages, but was also distributed in diffuse WM lesions due to heterogeneous causes. These data indicate that EP epitopes are exposed specifically in ongoing WM damages, and that the destruction of myelin occurs sporadically in diffuse WM lesions of varying intensity.

Enhancement of immunoreactivity for NF-kappa B in the hippocampal formation and cerebral cortex of Alzheimer's disease.

The distribution of nuclear factor-kappa B (NF-kappa B) was investigated immunohistochemically in the hippocampal formation, entorhinal cortex, middle temporal gyrus and visual cortex of Alzheimer's disease (AD) and control postmortem cases using a polyclonal antibody against the NF-kappa B p65 subunit. In AD cases, prominent staining for NF-kappa B was seen in neurons and their processes, neurofibrillary tangles and dystrophic neurites. In control cases, only weak staining of some neurons was obtained. The neuronal staining observed in AD was strongest in the hippocampal formation and entorhinal cortex, less in the middle temporal gyrus and least in the visual cortex. There was no difference between AD and control cases in the staining of glial cells and vascular walls. These results suggest that enhanced expression of neuronal NF-kappa B occurs in areas affected by AD pathology.

Enhancement of immunoreactivity for NF-kappa B in human cerebral infarctions.

The distribution of the nuclear factor-kappa B (NF-kappa B) was investigated immunohistochemically in recently infarcted areas of postmortem human brain. Previously we reported that immunoreactivity for NF-kappa B was enhanced in neurons of Alzheimer disease brain in comparison with control cases. In the present study, a similar enhancement of immunoreactivity was observed in glial cells of infarcted areas, but not in the unaffected surround. Prominent staining for NF-kappa B was seen in some astrocytes, particularly in the penumbra or border zone between ischemic and non-ischemic areas. In some cases, positively stained macrophages were also observed in affected areas. Capillary staining for NF-kappa B was weak and did not differ significantly between affected and unaffected areas. These results suggest that enhanced expression of astrocytic NF-kappa B occurs in cerebral infarcted areas.

Neurons express proteins of the classical complement pathway in Alzheimer disease.

Occurrence of the classical pathway complement proteins C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9 was studied in human hippocampus and temporal cortex by immunohistochemistry and Western blotting. In Alzheimer disease (AD) cases, positive staining for all of these proteins was observed in pyramidal neurons and senile plaques. In control cases, weaker pyramidal neuron staining was observed except for C1q and C1s which were not detected. On Western blots of AD hippocampal extracts, bands corresponding to those detected in normal serum were found for each of the complement proteins. Comparable bands were also detected in normal hippocampal extracts with the exception of C1s which was not observed. The intensity of the bands was generally stronger in AD than in normal extracts, but, in the latter, there was considerable variability between cases and between bands in a single case. These data suggest that pyramidal neurons may be a source of the complement components known to be associated with Alzheimer lesions.

Delayed expression of c-fos protein in rat hippocampus and cerebral cortex following transient in vivo exposure to hypoxia.

The time course of c-fos protein expression after hypoxia was examined in rat hippocampus and cerebral cortex using an immunohistochemical method. The rats were exposed to in vivo hypoxia for 30 min in a chamber containing 5% O2 and 95% N2. Immediately after the treatment, c-fos protein-like immunoreactivity was observed in the granule cell layer of the dentate gyrus. The change was transient, and the density of immunoreactive cells returned quickly to a control level 3 h after the exposure. However, the density of positive cells was again increased 1 day after hypoxia and reached the maximum 7 days after. In the cerebral cortex, on the other hand, no change was detected in the pattern of staining at any time, with an exception on 21 days after hypoxia. At this period, positively stained neurons were significantly increased in both density and intensity throughout the entire extent of the cerebral cortex including the cingulate gyrus. These results clearly indicate that hypoxia induces different patterns of c-fos protein expression among various regions of the brain. The biphasic pattern seen in the dentate gyrus as well as the delayed expression in the cerebral cortex may be related to delayed neuronal damages induced by hypoxia.