Amylin-like immunoreactivity in the extra-islet peptide YY-producing and glucagon-immunoreactive cells in Japanese quail pancreas.

In this study, we investigated amylin-like substance distribution in the pancreas of Japanese quail (Coturnix japonica) using a specific anti-rat amylin serum. We detected amylin-immunoreactive cells dispersed in the pancreatic extra-islet region but not in the islet region. The synthetic rat amylin-containing serum pre-absorption abolished the staining profile. Almost all amylin-immunoreactive cells were immuno-positive for peptide YY (PYY). In addition, certain amylin-immunoreactive cells stained immuno-positive for glucagon. Amylin and PYY co-secreted from the extra-islet cells might participate in the insulin and glucagon release regulation in the pancreas and food intake modulation through the central nervous system.

Acute stress transiently activates macrophages and chemokines in cervical lymph nodes.

Acute restraint stress (RS) is routinely used to study the effects of psychological and/or physiological stress. We evaluated the impact of RS on cervical lymph nodes in rats at molecular and cellular levels. Male Sprague-Dawley rats were subjected to stress by immobilization for 30, 60, and 120 min (RS30, RS60, and RS120, respectively) and compared with rats of a no-stress control (C) group. The expression of genes encoding chemokines CXCL1/CXCL2 (Cxcl1 and Cxcl2) and their receptor CXCR2 (Cxcr2) was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and microarray analyses. Immunohistochemistry and in situ hybridization were performed to determine the expression of these proteins and the macrophage biomarker CD68. Microarray analysis revealed that the expression of 514 and 496 genes was upregulated and downregulated, respectively, in the RS30 group. Compared with the C group, the RS30 group exhibited a 23.0-, 13.0-, and 1.6-fold increase in Cxcl1, Cxcl2, and Cxcr2 expression. Gene Ontology analysis revealed the involvement of these three upregulated genes in the cytokine network, inflammation, and leukocyte chemotaxis and migration. RT-qPCR analysis indicated that the mRNA levels of Cxcl1 and Cxcl2 were significantly increased in the RS30 group but were reverted to normal levels in the RS60 and RS120 groups. Cxcr2 mRNA level was significantly increased in the RS30 and RS120 groups compared with that in the C group. RS-induced CXCL1-immunopositive cells corresponded to B/plasma cells, whereas CXCL2-immunopositive cells corresponded to endothelial cells of the high endothelial venules. Stress-induced CXCR2-immunopositive cells corresponded to macrophages. Psychological and/or physiological stress induces an acute stress response and formation of an immunoreactive microenvironment in cervical lymph nodes, with the CXCL1/CXCL2-CXCR2 axis being pivotal in the acute stress response.

Orexin-B-like immunoreactivity localizes in both luteinizing-hormone-containing cells and melanin-concentrating hormone-containing fibers in the red-bellied piranha (Pygocentrus nattereri) pituitary.

We examined orexin-like immunoreactivity in the pituitary of the red-bellied piranha (Pygocentrus nattereri). Orexin-B-immunoreactive (IR) cells corresponded to luteinizing hormone (LH)-containing cells in the pars distalis, and orexin-B-IR fibers corresponded to melanin-concentrating hormone (MCH)-containing fibers in the pars nervosa. In the pars distalis, orexin-B-IR puncta that were also immunoreactive for MCH were observed around the orexin-B-IR cells. In the ventral hypothalamus, orexin-B-IR and MCH-IR neurons were found in the nucleus lateralis tuberis. Immunoelectron-microscopic analysis revealed that the orexin-B-like substance co-localized with LH in secretory granules and with MCH in MCH-containing neurons. Some of the MCH secreted in the pituitary might participate in the modulation of LH secretion from the gonadotrophs, together with orexin-B, leading to food intake by the stimulation of growth hormone secretion from the somatotrophs.

Occlusal Disharmony Transiently Impairs Learning and Memory in the Mouse by Increasing Dynorphin A Levels in the Amygdala.

Occlusal disharmony sometimes causes not only stiffness of neck but also psychiatric depression, suggesting that the condition of oral cavity may affect the central nervous system. Dynorphin A is an endogenous opioid peptide that specifically binds the κ-opioid receptor and has a protective role against stress. Dynorphinergic nervous system is intensely distributed in the amygdala and hippocampus that are coping areas with stress. As a model of malocclusion, we placed dental resin on the molars to increase the occlusal vertical dimension (bite-raise). After various survival times, we analyzed the amygdala and hippocampus by immunohistochemistry and immunosorbent assay (ELISA). Furthermore, the effects on learning and memory were assessed by Morris water maze test. In the amygdala, the levels of dynorphin A were increased on the 1st day after increasing the vertical dimension as indicated by immunohistochemical and ELISA assessments. The levels of dynorphin A returned to control levels on the 5th day. In the hippocampus, there were no noticeable changes in dynorphin A levels. The water maze test indicated that increasing the vertical dimension caused longer escape latency times on the 3rd day compared to those of sham-operated group. However, the bite-raised mice treated with a dynorphin antagonist, nor-binaltorphimine, showed similar escape latency times to the times of sham-operated group, even on the 3rd day. These results suggest that occlusal disharmony causes stress resulting in a transient increase of dynorphin A levels at least in the amygdala and that the increased dynorphin A levels transiently impair learning and memory.

CXCL14-like immunoreactivity in somatostatin-producing cells of the Japanese quail (Coturnix japonica) pancreas.

This study examines chemokine CXCL14-like peptide distribution in the Japanese quail (Coturnix japonica) pancreas using a specific anti-human CXCL14 antibody. CXCL14-immunoreactive cells were observed in the pancreatic islet peripheral region. The staining was abolished after pre-absorbing the antibody with recombinant human CXCL14. CXCL14-immunoreactive cells were immuno-positive for somatostatin, but not glucagon and insulin. CXCL14 secreted from somatostatin-producing cells might participate in insulin secretion modulation together with somatostatin. In addition, CXCL14 might participate in glucose homeostasis in co-operation with somatostatin and growth hormone.

Detection of SARS-CoV-2 and Its Related Factors on the Mucosal Epithelium of the Tongue.

SARS-CoV-2 infects a variety of tissues, including the oral cavity. However, there are few reports examining the association of SARS-CoV-2 with tongue mucosal tissues with sticky tongue debris. This study investigated the presence of SARS-CoV-2 and its associated molecules by dissecting tongue tissue from autopsy specimens of 23 patients who died of COVID-19-related illness (pneumonia). Immunohistochemical staining, electron microscopy, and PCR analysis were performed on the tongue tissue specimens. The mucosal epithelium of the tongue formed a very thick keratinized with well-developed filiform papillae in all cases. ACE2 and TMPRSS2 were consistently co-expressed in all samples in the epithelium. The S-protein was strongly expressed in basal cells and the epithelial surface. S-protein-positive viral particles were detected in the tongue's stratified squamous epithelium via an immunoelectron microscope. Based on PCR amplification of the N1 and N2 regions, the SARS-CoV-2 gene was detected on the tongue epithelium, tongue submucosa, and in tongue debris. This suggests that tongue debris, including the squamous epithelial tissue, could be a source of SARS-CoV-2 in saliva. Furthermore, removing tongue debris may decrease the amount of SARS-CoV-2 in the oral cavity.

Age estimation in forensic sciences: application of combined aspartic acid racemization and radiocarbon analysis.

Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster because the age at death, birth date, and year of death as well as gender can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization, has shown reproducible and more precise results. In this study, we analyzed teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that aboveground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ((14)C), which has been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel, and 10 of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R(2) = 0.66, p < 0.05). Radiocarbon analysis showed an excellent precision with an overall absolute error of 1.0 +/- 0.6 years. Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 +/- 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

The Galectin-1 level in serum as a novel marker for stress.

Galectin-1(Gal-1), a carbohydrate-binding protein with an affinity for beta-galactoside, is widely expressed in various normal and pathological tissues and it also plays an important role in regulating immune cell homeostasis and tumorigenesis. This study investigated the effects of restraint stress on serum Gal-1 by Western blot analyses and enzyme-linked immunosorbent assays. The Gal-1 levels of the restraint-stress group were significantly higher than those of the control group. However, this increase by stress was not obvious in adolescent rats. The pattern of these changes was similar to that of corticosterone. Furthermore, this Gal-1 increase in the serum was prevented by pre-treatment with a neurotoxin 6-hydroxydopamine (6-OHDA), which destroys the noradrenergic nerve terminals. However, a bilateral adrenalectomy (ADX) had no effect on the Gal-1 increase. These results suggest that Gal-1 is a candidate stress marker protein and that the stress-induced increase of Gal-1 in serum is regulated by the sympathetic nervous system under stress conditions.

Accuracy of pulse checks in terms of basic life support by lifesavers, as lay persons.

BACKGROUND: The lifesavers responsible for lifesaving at the waterside routinely undergo cardiopulmonary resuscitation (CPR) training, but in Japan, they are considered as lay persons. Lifesavers are likely to have better basic life support skills than lay persons. The objective of this study is to demonstrate that the accuracy of carotid pulse checks by lifesavers is not inferior to that of paramedics and is superior to that of lay persons by using CPR training mannequins. METHODS AND RESULTS: This was an observational study in which the subjects included 48 lifesavers certified by the Japan Lifesaving Association, as well as 16 paramedics and 15 lay persons. The accuracy of the examinees' answers and the time taken to answer in the 3 groups were compared. The accuracy rate was 93% in lifesavers, 94% in paramedics, and the difference was not significant (P=1). The accuracy rate of the lay persons' answers was 63%, with significant differences between this group and the lifesavers (P<0.001) and the paramedics (P<0.001). The average time taken to answer the questions was 6.6 s for the lifesavers and 7.0 s for the paramedics, and the difference was not significant (P=0.44). The average time taken to answer the questions from the lay persons group was 20.5 s, with significant differences between this group and the lifesavers (P<0.001) and the paramedics (P<0.001). CONCLUSIONS: The results of this evaluation, using CPR-training mannequins, to test the accuracy of carotid pulse checks by lifesavers were equivalent to those of paramedics and superior to those of lay persons.

Hypothermic and normothermic ischemia-reperfusion activate microglia differently in hippocampal formation.

Using immunohistochemical methods, we investigated microglial profiles under normothermic ischemia and hypothermic ischemia using an anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody. In the early stages of ischemia-reperfusion, Iba-1-immunoreactive microglial cells under normothermic ischemia were characterized by swollen somata with short and thick processes, while fine long-branched processes in greater numbers were seen emanating from microglial somata under hypothermic ischemia. In animals subjected to hypothermic ischemia, immunoreactive microglial areas in the hippocampal CA1 sector were significantly increased after 5 and 8 h of reperfusion when compared with those under normothermic ischemia. In the dentate gyrus, an increase in the microglial area under hypothermic ischemia was already evident at 2 h after reperfusion; this increased level was maintained up to 8 h. Considering the various neuroprotective roles of hypothermic ischemia, the characteristic features of microglia under hypothermic ischemia may be associated with the formation of a neuroprotective environment.

Occlusal disharmony in mice transiently activates microglia in hippocampal CA1 region but not in dentate gyrus.

Occlusal disharmony is induced by various conditions such as the loss of teeth and inappropriate vertical dimension of crowns, bridges, or dentures. Occlusal disharmony sometimes causes indefinite complaint syndromes, which may be associated with astrocytic hypertrophy and the reduction of numbers of neuronal somata and their dendritic spines in the hippocampus. Microglia monitors the condition of neurons and responds to their degeneration accompanying with astrocytes. However, the effect of occlusal disharmony on the microglia has not yet been investigated. We artificially increased the occlusal vertical dimension by placing dental resin on the upper molars in mice and immunohistochemically investigated the effects of the increase in the vertical dimension on microglia of the hippocampal formation using an antibody against ionized calcium-binding adaptor molecule 1 (Iba-1), a marker protein for microglia. We measured the area occupied by Iba-1-immunoreactive microglia in the hippocampal CA1 region and dentate gyrus 1, 3, and 5 days after increasing the vertical dimension, and compared it with that of control mice. The hippocampal CA1 region contains vulnerable neurons and the dentate gyrus durable neurons. We found that the areas occupied by microglia in the hippocampal CA1 region increased, with the peak on the third day after increasing the vertical dimension, and it gradually declined by the fifth post-operative day. However, such an increase of the area occupied by microglia was not seen in the dentate gyrus. In conclusion, abnormal mastication may activate microglia in the area harboring vulnerable neurons, but not in the area harboring durable neurons.

Chemokine CXCL14-like immunoreactivity in the αMSH-producing cells and PRL-producing cells of the flat-tailed house gecko pituitary.

The distribution pattern of chemokine CXCL14-immunoreactive cells was examined by immunohistochemistry in the pituitary of the gecko Hemidactylus platyurus. Immunoreactive cells were observed in the pars intermedia and pars distalis of the pituitary, but not in the pars nervosa. All α-melanocyte-stimulating hormone (αMSH)-producing cells were immunoreactive for CXCL14 in the pars intermedia. The CXCL14-immunoreactive cells corresponded to prolactin (PRL)-producing cells but not to other adenohypophyseal-hormone-producing cells in the pars distalis. CXCL14 secreted from αMSH-producing cells and PRL-producing cells may regulate insulin release from ß cells in the pancreatic islets as well as glucose uptake in the muscle cells together with αMSH and/or PRL. In addition, secreted CXCL14 with αMSH and/or PRL may act as a bioactive factor regulating hormone release in the adenohypophyseal cells of the reptilian pars distalis.

The Chemokine CXCL14-like Immunoreactivity Co-exists with Somatostatin, but not NPY in the Rat Dorsal Horn and Has Intimate Association with GABAergic Neurons in the Lateral Spinal Nucleus.

Recent studies have proposed that the chemokine CXCL14 not only has a chemotactic activity, but also functions as a neuromodulator and/or neurotransmitter. In this study, we investigated the distribution of CXCL14 immunoreactive structures in the rat spinal cord and clarified the association of these structures with somatostatin, glutamic acid decarboxylase (GAD; a marker for GABAergic neurons), and neuropeptide Y (NPY). CXCL14 immunoreactive fibers and puncta were observed in lamina II, which modulates somatosensation including nociception, and the lateral spinal nucleus of the spinal dorsal horn at cervical, thoracic, and lumber spinal cord levels. These CXCL14 immunoreactive structures were also immuno-positive for somatostatin, but were immuno-negative for GAD and NPY. In the cervical lateral spinal nucleus, CXCL14 immunoreactive puncta, which were also immuno-positive for somatostatin, existed along the proximal dendrites of some of GABAergic neurons. Together, these results suggest that CXCL14 contributes to the modulation of somatosensation in concert with somatostatin. Neurons targeted by the CXCL14 fiber system include GABAergic neurons located in the lateral spinal nucleus suggesting that CXCL14 with somatostatin can influence the GABAergic neuron function.

Chewing augments stress-induced increase of pERK-immunoreactive cells in the rat cingulate cortex.

The eff ;ects of chewing during restraint stress on the anterior, mid- and posterior cingulate cortices were investigated in rats using immunohistochemistry to detect the expression of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2), a marker of responding cells. The rats were divided into three groups: control (no immobilization), stress-only (immobilized), and stress-with-chewing (immobilized and allowed to chew a wooden stick). Significant increases in the number of pERK1/2-immunoreactive cells in the anterior, mid- and posterior cingulate cortices were noted in the stress-only group when compared with the control group (p < 0.05). Furthermore, the number of pERK1/2-immunoreactive cells in the anterior, mid- and posterior cingulate cortices in the stress-with-chewing group was also significantly higher than that in the stress-only group (p < 0.05). These findings indicate that the cingulate cortex plays a role in the negative-feedback effect and might be an essential part of the brain where the ameliorating effects of chewing against stress are produced.

Orexin-B-like immunoreactivity in pituitary αMSH-producing cells and median eminence GnRH-containing fibres of the flat-tailed house gecko.

We examined the distribution of the orexin-like peptides in the pituitary and median eminence of the flat-tailed house gecko (Hemidactylus platyurus) using immunohistochemistry. Orexin-B-like, but not orexin-A-like, immunoreactivity was detected in the pituitary, specifically in the pars intermedia, and these cells corresponded to alpha-melanocyte-stimulating hormone (αMSH)-producing cells. Orexin-B and αMSH secreted from pars intermedia may modulate secretion of adenohypophyseal cells in the pars distalis. In the median eminence, orexin-B-immunoreactive puncta and fibres were observed, and these structures corresponded to gonadotropin-releasing hormone (GnRH)-immunoreactive puncta and fibres. Orexin-B secreted from GnRH-containing neurons in the hypothalamus may affect thyrotropin-releasing hormone-containing neurons resulting in modulation of αMSH secretion of melanotrophs in the pars intermedia.

Distribution, nature, and origin of CXCL14-immunoreactive fibers in rat parotid gland.

The distribution and nature of CXCL14-immunoreactive nerve fibers in salivary glands, especially the parotid gland was immunohistochemically investigated. Furthermore, the origin of parotid CXCL14-immunoreactive nerve fibers was determined by retrograde tracing experiments. CXCL14-immunoreactive nerve fibers were localized in the parotid, submandibular, and sublingual glands, particularly in the parotid gland. Double staining using identical sections revealed that a subpopulation of cells neuropeptide Y (NPY)-containing fibers was immunopositive for CXCL14 in the parotid gland. In the peripheral regions of acinar cells, CXCL14-immunoreactive fibers tended to coexist with NPY; however, perivascular NPY-immunoreactive fibers tended to be immunonegative for CXCL14. Parotid CXCL14-immunoreactive fibers were immunopositive for tyrosine hydroxylase (TH) but immunonegative for choline acetyltransferase and vasoactive intestinal peptide (VIP). After injection of horseradish peroxidase-labeled wheat germ agglutinin (WGA-HRP) in the parotid gland, retrogradely labeled neurons were seen in the superior cervical ganglion (SCG) and otic ganglion. Some of the WGA-immunoreactive somata in the SCG were immunopositive for CXCL14; however, no doubly-labeled somata were noted in the otic ganglion. These results indicate that CXCL14-immunoreactive nerve fibers originate in the SCG, and are sympathetic in nature. The coexistence of CXCL14 with NPY/TH suggests that CXCL14 may be associated with NPY/TH functions as a neuromodulatory chemokine in the parotid gland. The localization of CXCL14 nerve fibers around the acinar cells of the parotid gland indicates its involvement in acinar cell function, but not vasoconstriction.

Effect of social isolation stress on saliva BDNF in rat.

Salivary glands produce various compounds, including brain-derived neurotrophic factor (BDNF), which serve as biomarkers of stress-related disorders. Social isolation-induced stress models a form of chronic mild stress that induces neurodegenerative changes in the brain and behavioral alterations. This study employed a rat model to determine whether social isolation stress affects BDNF levels in saliva. Male Sprague-Dawley rats were randomly allocated to social isolation stress (1 animal/cage) or control (3-4 animals/cage) groups and reared for 8 weeks. The concentration of BDNF was quantified in specific brain regions, blood, and saliva using enzyme-linked immunosorbent assay (ELISA). The levels of expression of Bdnf and tyrosine kinase B (TrkB) mRNA were quantified using reverse transcriptase-polymerase chain reaction. Behavioral alterations were analyzed using the open-field and elevated plus maze assays. The BDNF concentration was lower in the hippocampus, prefrontal cortex, blood, and saliva of the stress group than in those of the controls. Trkb expression in the hippocampus and prefrontal cortex was decreased by social isolation stress. Moreover, the social isolation stress group showed behavioral deficits in both tests. In conclusion, these findings indicate that social isolation stress may reduce the expression of BDNF protein in blood and saliva, thus providing a potentially valuable biomarker for diagnosis of stress-related disorders.

Orexin-A immunoreactive cells and fibers in the central nervous system of the axolotl brain and their association with tyrosine hydroxylase and serotonin immunoreactive somata.

Orexin-A-like immunoreactivity in the axolotl brain was investigated by immunohistochemistry. Immunoreactive somata formed a single group in the hypothalamus, but were distributed beyond several nuclei, namely, the ventral aspect of the nucleus preopticus posterior, dorsal aspect of the nucleus suprachiasmaticus and anterior aspect of the pars ventralis hypothalami. Immunoreactive fibers were distributed throughout the brain from the olfactory bulb to the spinal cord except the cerebellum. The densest immunoreactive fibers were seen in the medial forebrain bundle and caudal lateral forebrain bundle. The largest number of immunoreactive puncta were seen in the mesencephalic tectum in addition to the hypothalamus. Immunoelectron microscopic analysis revealed the presence of synaptoid connections of immunoreactive fibers on neuronal somata in the tectum. The function of the mesencephalic system in the urodele seems to be sensory integration, suggesting that the orexin-A nervous system is associated with the modulation of sensory inputs. Orexin-A immunoreactive puncta were also observed on catecholaminergic and serotonergic somata. In view of the restricted somatic distribution in the hypothalamus, wide distribution of fibers throughout the central nervous system (CNS), and intimate association with monoaminergic somata, the orexin nervous system in the axolotl CNS is similar to those of other vertebrates, suggesting that this system is essential for brain functions throughout vertebrates.

The bite-raised condition in aged SAMP8 mice induces dendritic spine changes in the hippocampal region.

The present study examined the effect of the bite-raised condition on the number of dendritic spines on hippocampal pyramidal cells in SAMP8 mice and related the results with learning ability in a water maze test. Aged mice in the bite-raised condition had reduced learning ability and a lower number of CA1 pyramidal cell dendritic spines. The results suggest that the bite-raised condition exacerbates the age-related spatial learning impairment, and that this may be due to the degeneration of hippocampal dendritic spines.

Overexpression of the mitotic spindle assembly checkpoint genes hBUB1, hBUBR1 and hMAD2 in thyroid carcinomas with aggressive nature.

BACKGROUND: The mitotic spindle assembly checkpoint (MSAC) genes are responsible for preventing chromosome missegregation. MSAC gene expressions have been reported to be associated with tumor cell proliferation or unfavorable cancer behavior. The present study was conducted to preliminary investigate the MSAC gene expressions in thyroid neoplasms. MATERIALS AND METHODS: Expression levels of MSAC genes (hBUB1, hBUBR1, hBUB3 and hMAD2) were evaluated in 9 follicular thyroid adenomas (FAs), 9 follicular thyroid carcinomas (FTCs), 21 papillary thyroid carcinomas (PTCs), 5 anaplastic (undifferentiated) thyroid carcinomas (ATCs) and 3 adjacent normal thyroid tissues (NTs) by real-time quantitative RT-PCR. These gene expressions were compared between undifferentiated thyroid carcinomas (ATCs) and differentiated thyroid carcinomas (DTCs) and between advanced DTCs and non-advanced DTCs. DTCs included PTCs and FTCs. Advanced DTCs were defined as carcinoma with aggressive nature such as extrathyroid extension, distant metastasis, recurrence or death from the disease. RESULTS: MSAC gene expressions varied in different thyroid tumors and fell in the order of ATC, DTC (PTC and FTC), FA and NT Carcinomas had higher expression compared to adenoma or normal tissue. hBUB1, hBUBR1 and hMAD2 expressions in ATCs were significantly higher than those in DTCs (p<0.005). hBUBR1 and hMAD2 expressions in advanced DTCs were significantly higher than those in non-advanced DTCs (p<0.05). CONCLUSION: The MSAC genes were overexpressed in thyroid carcinomas with aggressive nature. Further studies are required to clarify the relationship between the MSAC gene expressions and thyroid cancer behavior.