Characterization of the oral and faecal microbiota associated with atopic dermatitis in dogs selected from a purebred Shiba Inu colony.

Atopic dermatitis (AD) is a chronic and relapsing multifactorial inflammatory skin disease that also affects dogs. The oral and gut microbiota are associated with many disorders, including allergy. Few studies have addressed the oral and gut microbiota in dogs, although the skin microbiota has been studied relatively well in these animals. Here, we studied the AD-associated oral and gut microbiota in 16 healthy and 9 AD dogs from a purebred Shiba Inu colony. We found that the diversity of the oral microbiota was significantly different among the dogs, whereas no significant difference was observed in the gut microbiota. Moreover, a differential abundance analysis detected the Family_XIII_AD3011_group (Anaerovoracaceae) in the gut microbiota of AD dogs; however, no bacterial taxa were detected in the oral microbiota. Third, the comparison of the microbial co-occurrence patterns between AD and healthy dogs identified differential networks in which the bacteria in the oral microbiota that were most strongly associated with AD were related to human periodontitis, whereas those in the gut microbiota were related to dysbiosis and gut inflammation. These results suggest that AD can alter the oral and gut microbiota in dogs.

Prototyping the Experimental Setup to Quantify the Tissue Oxygen Consumption Rate and Its Preliminary Test.

In order to establish a reliable and practical method to make a diagnosis on the viability of an amputated extremity, we propose a method to evaluate the oxygen consumption rate. To validate this concept, we prototyped an experimental system with which the oxygen transfer rate into tissue can be assessed by the rate of change of the decrease in dissolved oxygen (DO) concentration within the buffer fluid surrounding the target tissue. The purpose of this study is to examine the feasibility of our prototyped experimental system by comparison between fresh and non-fresh rat skeletal muscles. The results show that the fresher tissue transferred more oxygen to the tissue, which suggests that tissue oxygen consumption is highly related to tissue freshness and can indirectly assess the tissue viability.

Expression of Autophagy-Related Proteins in the Spinal Cord of Pembroke Welsh Corgi Dogs With Canine Degenerative Myelopathy.

Canine degenerative myelopathy (DM) is a progressive neurodegenerative disease frequently found in Pembroke Welsh Corgi (PWC) dogs, and it has clinical and pathologic similarities to human amyotrophic lateral sclerosis. Autophagy is a major intracellular protein degradation system. Abnormalities of autophagy--resulting in cell death through mechanisms called type II programmed cell death--have recently been reported to occur in various neurodegenerative diseases, including amyotrophic lateral sclerosis. Thus, the distribution and expression levels of proteins involved in autophagy were examined in the spinal cords of 8 PWC dogs suffering from DM with superoxide dismutase mutation, 5 non-DM PWC dogs, and 6 Beagle dogs without neurologic signs. There was no significant difference in the ratio of neurons with microtubule-associated protein light chain 3 (LC3)-positive somata relative to those that were LC3 negative among the 3 groups, whereas the number of LC3-positive neurites was significantly increased in DM dogs. Punctate LC3 immunoreactivity did not colocalize with a lysosome marker, LAMP2 (lysosome-associated membrane protein 2). NBR1 (neighbor of BRCA gene 1) was localized mostly in reactive astrocytes, whereas there were p62 (p62/A170/SQSTM1)-positive foci in the neuropil of the spinal cord of DM dogs. Western blotting revealed in DM dogs the decreased expression of Beclin1 and Atg16 L, which are molecules involved in formation of the isolation membrane. These findings suggest that altered autophagosome degradation may result in LC3 and p62 accumulation in the DM spinal cord, whereas the early stage of membrane formation is likely to be downregulated.

Three-dimensional SAFT imaging for anisotropic materials using photoacoustic microscopy.

A pulsed laser illuminates a target zone that causes rapid thermoelastic expansion, generating broadband high-frequency ultrasonic wave (photoacoustic wave, PA wave). We developed a PA microscopy (PAM) with a confocal area of laser and ultrasonic wave for applications in nondestructive testing (NDT). The synthetic aperture focusing technique (SAFT) is applied in the PAM for the three-dimensional (3D) imaging of interior flaws. Here, we report proof-of-concept experiments for the NDT of a subsurface flaw in a thin laminar material. Graphical abstract (a) shows a specimen of carbon-fiber-reinforced plastic (CFRP) with an artificial delamination. Here, it should be noted that the group velocity varies directionally due to the strong anisotropy of the CFRP specimen (see Graphical abstract (b)). By considering the group velocity distribution in the SAFT, the shape and location of the subsurface delamination were accurately estimated as shown in Graphical abstract (c). Coating the surface of the CFRP specimen with a light-absorbent material improved the amplitude of the PA wave. This finding showed that the signal-to-noise ratio of the waves scattered from the flaws can be improved.

Patch sensor detection of glutamate release evoked by a single electrical shock.

We tried to detect minimal stimulation-induced glutamate overflow from the surface of a hippocampal slice using an outside-out patch electrode excised from pyramidal cell membranes. The amplitude of the stimulation-induced patch current was dependent on the distance between the slice surface and the tip of patch sensor. The current-voltage relations of the stimulation-induced patch current were similar to those of the current evoked puff by application of L-glutamate to the patch. This indicates that the stimulation-induced patch current was produced by glutamate released from presynaptic terminals, and thus this technique may be useful in the study of transmitter release evoked by minimal electrical stimulation in brain slices.

CLOCK gene T3111C polymorphism is associated with Japanese schizophrenics: a preliminary study.

The CLOCK gene has attracted attention due to its influence on the circadian rhythm, as well as its impacts on the dopaminergic system. We conducted a preliminary study to examine whether the T3111C single nucleotide polymorphism of the CLOCK gene is associated with the development of schizophrenia by examining samples from schizophrenics (n=145) and normal controls (n=128). Both genotype and allele frequencies were significantly different between schizophrenics and controls (p=0.022, p=0.015, respectively). Schizophrenics had a significantly higher frequency of the C allele compared to controls (odds ratio 1.76, 95% CI 1.12-2.75). In particular, disorganized and residual type schizophrenics had significantly higher C allele frequencies than controls (p=0.004 and p=0.037, respectively). Our results suggest that the T3111C polymorphism of the CLOCK gene is associated with schizophrenia. It is important to explore the association between CLOCK and dopamine function, and to examine the impact of CLOCK on phenotypes such as symptoms and drug response in patients with schizophrenia.

Survey of Two New (Kai 1 and Kai 2) and Other Blood Groups in Dogs of North America.

BACKGROUND: Based upon serology, >10 canine blood group systems have been reported. OBJECTIVE: We surveyed dogs for dog erythrocyte antigen (DEA) 1 and 2 new blood types (Kai 1 and Kai 2), and some samples also were screened for Dal and DEA 3, 4, and 7. METHODS: Blood samples provided by owners, breeders, animal blood banks, and clinical laboratories were typed for DEA 1 by an immunochromatographic strip technique with a monoclonal antibody and analysis of band intensity. Both new antigens, the Dal and other DEAs (except DEA 7 by tube method), were assessed by a gel column method with either monoclonal or polyclonal antibodies. The same gel column method was applied for alloantibody detection. RESULTS: Of 503 dogs typed, 59.6% were DEA 1+ with 4% weakly, 10% moderately, and 45.6% strongly DEA 1+. Regarding Kai 1 and Kai 2, 94% were Kai 1+/Kai 2-, 5% were Kai 1-/Kai 2- and 1% were Kai 1-/Kai 2+, but none were Kai 1+/Kai 2+. There was no relationship between Kai 1/Kai 2 and other blood types tested. Plasma from DEA 1-, Kai 1-, Kai 2- dogs, or some combination of these contained no detectable alloantibodies against DEA 1 and Kai 1 or Kai, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: The new blood types, called Kai 1 and Kai 2, are unrelated to DEA 1, 3, 4, and 7 and Dal. Kai 1+/Kai 2- dogs were most commonly found in North America. The clinical relevance of Kai 1 and Kai 2 in canine transfusion medicine still needs to be elucidated.

Molecular prevalence of multiple genetic disorders in Border collies in Japan and recommendations for genetic counselling.

Reproductive management is necessary to prevent deleterious genetic disorders in purebred dogs, but comprehensive studies aimed at prevention of multiple underlying genetic disorders in a single breed have not been performed. The aims of this study were to examine mutant allele frequencies associated with multiple genetic disorders, using Border collies as a representative breed, and to make recommendations for prevention of the disorders. Genotyping of known mutations associated with seven recessive genetic disorders was performed using PCR assays. More than half (56%) of the Border collies had no mutant alleles associated with any of the seven disorders, suggesting that these disorders can be removed from the population over several generations. Since frequencies of each mutant allele differed among disorders, reproductive management should be performed after the establishment of prevention schemes that are appropriate for each disorder, the type and specificity of genetic test available, and the effective population size in each breeding colony.

Feline cystinuria caused by a missense mutation in the SLC3A1 gene.

BACKGROUND: Cystinuria is an inherited metabolic disease that is relatively common in dogs, but rare in cats and is characterized by defective amino acid reabsorption, leading to cystine urolithiasis. OBJECTIVES: The aim of this study was to report on a mutation in a cystinuric cat. ANIMALS: A male domestic shorthair (DSH) cat with cystine calculi, 11 control cats from Wyoming, and 54 DSH and purebred control cats from elsewhere in the United States. METHODS: Exons of the SLC3A1 gene were sequenced from genomic DNA of the cystinuric cat and a healthy cat. Genetic screening for the discovered polymorphisms was conducted on all cats. RESULTS: A DSH cat showed stranguria beginning at 2 months of age, and cystine calculi were removed at 4 months of age. The cat was euthanized at 6 months of age because of neurological signs possibly related to arginine deficiency. Twenty-five SLC3A1 polymorphisms were observed in the sequenced cats when compared to the feline reference sequence. The cystinuric cat was homozygous for 5 exonic and 8 noncoding SLC3A1 polymorphisms, and 1 of them was a unique missense mutation (c.1342C>T). This mutation results in a deleterious amino acid substitution (p.Arg448Trp) of a highly conserved arginine residue in the rBAT protein encoded by the SLC3A1 gene. This mutation was found previously in cystinuric human patients, but was not seen in any other tested cats. CONCLUSIONS AND CLINICAL IMPORTANCE: This study is the first report of an SLC3A1 mutation causing cystinuria in a cat, and could be used to characterize other cystinuric cats at the molecular level.

The loss of GluR2(3) immunoreactivity precedes neurofibrillary tangle formation in the entorhinal cortex and hippocampus of Alzheimer brains.

Double-immunolabeling techniques were employed to examine the distribution of GluR2(3) subunits and markers of early cytoskeletal changes (mab MC1) within the entorhinal cortex (EC) and hippocampus of cases with varying degrees of Alzheimer disease (AD) pathology (stages I-VI by Braak and Braak). In addition near-adjacent tissue sections were double-immunolabeled using antibodies against GluR2(3) and a marker of normal neuronal cytoskeleton (MAP2). In those cases classified as stages I-II, most layer II neurons of the EC and pyramidal neurons in the CA1/subiculum were double-labeled with GluR2(3) and MAP2. An occasional MC1-labeled cell was observed, yet in no instance were these neurons double-labeled with GluR2(3). In cases with moderate AD pathology (stages III-IV), layer II of the EC and CA1/subiculum were characterized by a substantial loss of GluR2(3)-labeled neurons, while many were still immunoreactive to MAP2. Notably, the loss of GluR2(3) immunolabeling was accompanied by an increasing number of MC1-positive neurons. In no instance were GluR2(3) and MC1 co-localized within the same neuron. In cases with severe AD pathology (stages V-VI), the EC and CA1/subiculum were almost completely devoid of GluR2(3)-positive neurons. MAP2-labeled neurons also were reduced in number. In contrast, both regions contained an abundance of MC1-positive cells. That GluR2(3) and MC1 are not observed in the same neuron, together with the observation that the number of GluR2(3)-labeled neurons decreases as the number of MC1-positive cells increases, suggest that a loss of GluR2(3) immunolabeling precedes the appearance of MC1 immunolabeling.

GABAA receptor beta 2 and beta 3 subunits mRNA in the hippocampal formation of aged human brain with Alzheimer-related neuropathology.

Our work on the role of glutamate in Alzheimer's disease (AD)-related neuronal vulnerability and death provided significant insight into the potential contribution of the gamma-aminobutyric acid (GABA) neurotransmitter system as it participates in countering the neurotoxic effects of excessive glutamate receptor stimulation. Our previous studies demonstrate that beta2/3 GABAA receptor subunit immunoreactivity is relatively well preserved in hippocampi with AD pathology. To further elucidate the molecular basis for this observation, we employed in situ hybridization histochemistry to examine the levels of beta2 and beta3 receptor subunit mRNAs in the hippocampus of 19 elderly subjects presenting with a broad range of pathologic severity (i.e., Braak stage I-VI). Semi-quantitative analysis with film autoradiograms revealed that beta2 mRNA signal was highest in the granule cell layer, CA2 and CA1 subfields, while beta3 mRNA hybridization was highest in the granule cell layer, followed by CA2>/=CA3>/=CA1 regions. No significant difference in beta2 mRNA expression was detected among the pathologically mild, moderate or severe groups. In contrast, levels of beta3 mRNA in the pathologically severe group was significantly decreased compared to the mild group within all subregions examined except CA4. Our data suggest that alterations in the expression of GABAA receptor subunits in the AD hippocampus differ between specific receptor subunits with the amount of beta2 mRNA being relatively well-preserved, while beta3 mRNA levels were decreased.

Immunohistochemical study of the hnRNP A2 and B1 in the rat forebrain.

Immunohistochemical techniques were employed to examine the distribution of RNA-binding proteins A2 and B1 in the rat forebrain. Intense A2 and B1 immunolabeling were observed in the nucleoplasm of the neurons in the cerebral cortices, hippocampal formation, olfactory regions, caudate-putamen as well as the supraoptic nucleus of hypothalamus. In contrast, within the bed nucleus of the stria terminalis, as well as the medial and lateral habenular nucleus of thalamus, immunoreactivity for both proteins was weak. Within the globus pallidus and thalamic nucleus immunoreactivity for A2 was hardly detectable despite of intense B1 immunolabeling, while within the endopiriform nucleus and lateral and basolateral nucleus of amygdala intensity of B1 immunolabeling was relatively weak compared to A2. Our study suggests that the distribution of A2 and B1 are not constant throughout the forebrain and this diversity may reflect the post-transcriptional regulation of cell-specific gene expression of neuronal cells.

Alterations of GABA(A)beta2/3 immunoreactivity in the dentate gyrus after perforant pathway lesion.

Immunocytochemical techniques were employed to examine the changes in the GABA receptor subunits beta2/3 within the dentate gyrus of the rat brain 1, 3, 7, 14, 30 and 90 days after a unilateral perforant pathway lesion. Three days post-lesion we observed a decrease in beta2/3 immunolabeling in the inner molecular layer of the dentate gyrus followed by a comparable decrease in the outer molecular layer 7 days post-lesion. These decreases were transient; 30 and 90 days post-lesion, beta2/3 immunolabeling appeared similar to controls in the inner portion of the molecular layer, while in the outer region the labeling was increased. In this latter region we also observed a dense band of AChE fibers. Following survival times of 3 days we observed a diffuse staining of the neuropil in the hilar region, and a dense amorphous accumulation of peroxidase reaction product in the polymorphic region. These responses were transient and by 14 days the hilar/polymorphic region appeared indistinguishable from controls. These data suggest a unique pattern of immunoabeling in the molecular and polymorphic region in response to perforant pathway lesion. A putative explanation for this response is discussed.

Alterations of AMPA-selected glutamate subtype immunoreactivity in the dentate gyrus after perforant pathway lesion.

Immunocytochemical techniques were employed to examine the changes in immunolabeling of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the dentate gyrus 1, 3, 7, 14, 30, and 90 days after a unilateral perforant pathway lesion in the rat brain. Completeness of the lesion was confirmed following examination of Nissl-stained tissue sections at all times post-lesion and acetylcholinesterase (AChE)-stained sections 14, 30 and 90 days post-lesion, the latter providing evidence of compensatory sprouting of cholinergic fibers in the outer molecular layer of the dentate gyrus. Compared to the non-lesioned hippocampus there was no difference in the staining pattern of AMPA receptor subunits in the dentate gyrus of the deafferented hippocampus 1, 3, 7 and 14 days following lesioning of the perforant pathway. In contrast, 30 and 90 days post-lesion, GluR1 immunolabeling was increased in the outer molecular layer of the dentate gyrus (i.e., deafferented zone) ipsilateral to lesion. Likewise, GluR2/3 immunolabeling was increased within the same region although the intensity of the response was less than that which was observed for GluR1. These data suggest that the loss of the perforant pathway fibers results in a compensatory increase in GluR1 and to a lesser extent GluR2/3 immunolabeling of the outer molecular layer at 30 and 90 days post-lesion and further suggest that AMPA receptor subunits play a role in perforant pathway signal transduction.

Distribution of orexin neurons in the adult rat brain.

Orexin (ORX)-A and -B are recently identified neuropeptides, which are specifically localized in neurons within and around the lateral hypothalamic area (LHA) and dorsomedial hypothalamic nucleus (DMH), the regions classically implicated in feeding behavior. Here, we report a further study of the distribution of ORX-containing neurons in the adult rat brain to provide a general overview of the ORX neuronal system. Immunohistochemical study using anti-ORX antiserum showed ORX-immunoreactive (ir) neurons specifically localized within the hypothalamus, including the perifornical nucleus, LHA, DMH, and posterior hypothalamic area. ORX-ir axons and their varicose terminals showed a widespread distribution throughout the adult rat brain. ORX-ir nerve terminals were observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. We also observed strong staining of ORX-ir varicose terminals in areas outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, circumventricular organs (subfornical organ and area postrema), limbic system (hippocampus, amygdala, and indusium griseum), and brain stem (locus coeruleus and raphe nuclei). These results indicate that the ORX system provides a link between the hypothalamus and other brain regions, and that ORX-containing LHA and DMH neurons play important roles in integrating the complex physiology underlying feeding behavior.

Immunohistochemical study of GABAA receptor alpha1 subunit in the hippocampal formation of aged brains with Alzheimer-related neuropathologic changes.

Immunocytochemical techniques were employed to examine the distribution of the gamma-aminobutyric acid (GABA)A receptor alpha1 subunit within the hippocampus of 19 elderly subjects with Alzheimer-related neuropathologic changes. In mild cases (i.e., Braak stages I and II), the most intense neuropil immunolabeling was observed in the molecular layer of the dentate gyrus, the stratum pyramidale of the CA1 subregion and subiculum, while the weakest labeling was observed in the CA3 subfield. In CA4 region, the proximal dendrites and cell bodies of mossy cells were intensely alpha1 positive. Throughout the hippocampus, we observed a number of alpha1 labeled interneurons. These cells consisted of both large and small multipolar cells as well as small bipolar neurons. In moderate cases (i.e., Braak stages III and IV), the pattern and intensity of alpha1 immunolabeling appeared indistinguishable from mild cases. In severe cases (i.e., Braak stages V and VI), we observed a marked decrease in neuropil immunolabeling within the CA2, CA1 subregions and prosubiculum, while the labeling of the molecular layer of the dentate gyrus, subiculum proper and presubiculum was indistinguishable from mild and moderate cases. These data together with our previous immunocytochemical study in which we demonstrated a marked preservation of the GABAA receptor subunit beta2/3 suggest that responses of selected GABAA receptor subunits to AD pathology are variable with the alpha1 subunit displaying a high degree of vulnerability.

Some physiological and pharmacological properties of slow depolarization of substantia gelatinosa neurons by repetitive stimulation of C-fibers of dorsal root in adult rat spinal cord slices with dorsal root attached.

Transverse slices of the spinal cord with dorsal root attached were prepared from adult rats and used to record the response of the spinal substantia gelatinosa neurons evoked by repetitive stimulation of the dorsal root with a whole cell patch clamp method. The repetitive stimulation of 10 trains (20 pulses at 100 Hz/train) with an intensity necessary to activate C-fibers, but not A-fibers alone, evoked slow depolarization (SD) during it and the SD disappeared within 1 min after the termination of it. The SD was completely inhibited by 500 nM of tetrodotoxin (TTX), but not significantly changed by 50 nM of TTX, nor 20 microM CNQX + 50 microM AP5, nor 1 microM CP-99994. DAMGO inhibited the SD in a concentration dependent manner (10 nM-1 microM), but 1 microM DPDPE and 1 microM U-50488H did not. The inhibitory effect of DAMGO (1 microM) was reversed by naloxone (1 microM). These results suggest that the SD of substantia gelatinosa neurons evoked by repetitive stimulation of C-fibers of the dorsal root is an event relevant to nociception in the spinal dorsal horn.