Development and validation of a novel overhead method for anteroposterior radiographs of fractured rat femurs.

We aimed to establish a new method of obtaining femur anteroposterior radiographs from live rats. We used five adult male Sprague-Dawley rats and created a femoral fracture model with an 8 mm segmental fragment. After the surgery, we obtained two femoral anteroposterior radiographs, a novel overhead method, and a traditional craniocaudal view. We obtained the overhead method three times, craniocaudal view once, and anteroposterior radiograph of the isolated femoral bone after euthanasia. We compared the overhead method and craniocaudal view with an isolated femoral anteroposterior view. We used a two-sample t-test and intraclass correlation coefficient (ICC) to estimate the intra-observer reliability. The overhead method had significantly smaller differences than the craniocaudal view for nail length (1.53 ± 1.26 vs. 11.4 ± 3.45, p < 0.001, ICC 0.96) and neck shaft angle (5.82 ± 3.8 vs. 37.8 ± 5.7, p < 0.001, ICC 0.96). No significant differences existed for intertrochanteric length/femoral head diameter (0.23 ± 0.13 vs. 0.23 ± 0.13, p = 0.96, ICC 0.98) or lateral condyle/medial condyle width (0.15 ± 0.16 vs. 0.13 ± 0.08, p = 0.82, ICC 0.99). A fragment displacement was within 0.11 mm (2.4%). The overhead method was closer to the isolated femoral anteroposterior view and had higher reliability.

Cardioprotective effect of St. Thomas' Hospital No. 2 solution against age-related changes in aquaporin-7-deficient mice.

OBJECTIVE: This study aimed to investigate whether St. Thomas' Hospital No. 2 solution (STH2) is equally effective in both young and aged aquaporin-7-knockout (AQP7-KO) mice and the mechanisms by which the intra-myocardial adenosine triphosphate (ATP) content is altered during ischemia without aquaporin-7. METHODS: In study 1, isolated hearts of male wild-type (WT) and AQP7-KO mice (< 12 weeks old) were Langendorff perfused with 5-min STH2 prior to a 20-min global ischemia (GI) or 25-min GI without STH2. Similarly, in Study 2, hearts from WT and AQP7-KO mice (≥ 24 weeks old) were subjected to 2-min STH2 infusion prior to GI. In study 3, intra-myocardial ATP content was compared before (sham) and after (control or STH2) ischemia in mature WT and AQP7-KO mice. RESULTS: In study 1, troponin T levels (ng/g wet weight) of WT and AQP7-KO hearts were significantly lower in the STH2 groups (75.6 ± 45.9 and 80.2 ± 52.2, respectively) than in the GI groups (934.0 ± 341.1 and 1089.3 ± 182.5, respectively). In Study 2, troponin T levels in aged WT and AQP7-KO mice were 566.5 ± 550.0 and 547.8 ± 594.3, respectively (p = 0.9561). In Study 3, ATP levels (µmol/g protein) in the sham, control, and STH2 AQP7-KO mice groups were 4.45, 2.57, and 3.37, respectively(p = 0.0005). CONCLUSIONS: The present study revealed the cardio-protective efficacy of STH2 in an experimental model of isolated AQP7-KO young and aged murine hearts. Further, STH2 preserved intra-myocardial ATP during ischemia with Krebs-Henseleit buffer perfusion in the Langendorff setting.

Class III Alcohol Dehydrogenase Plays a Key Role in the Onset of Alcohol-Related/-Associated Liver Disease as an S-Nitrosoglutathione Reductase in Mice.

Lipid accumulation in the liver due to chronic alcohol consumption (CAC) is crucial in the development of alcohol liver disease (ALD). It is promoted by the NADH/NAD ratio increase via alcohol dehydrogenase (ADH)-dependent alcohol metabolism and lipogenesis increase via peroxisome proliferator-activated receptor γ (PPARγ) in the liver. The transcriptional activity of PPARγ on lipogenic genes is inhibited by S-nitrosylation but activated by denitrosylation via S-nitrosoglutathione reductase (GSNOR), an enzyme identical to ADH3. Besides ADH1, ADH3 also participates in alcohol metabolism. Therefore, we investigated the specific contribution of ADH3 to ALD onset. ADH3-knockout (Adh3-/-) and wild-type (WT) mice were administered a 10% ethanol solution for 12 months. Adh3-/- exhibited no significant pathological changes in the liver, whereas WT exhibited marked hepatic lipid accumulation (p < 0.005) with increased serum transaminase levels. Adh3-/- exhibited no death during CAC, whereas WT exhibited a 40% death. Liver ADH3 mRNA levels were elevated by CAC in WT (p < 0.01). The alcohol elimination rate measured after injecting 4 g/kg ethanol was not significantly different between two strains, although the rate was increased in both strains by CAC. Thus, ADH3 plays a key role in the ALD onset, likely by acting as GSNOR.

Cesarean section delivery is a risk factor of autism-related behaviors in mice.

Cesarean section (C/S) is one way of delivering babies, and is chosen when mothers or babies are facing problems or life-threatening conditions during pregnancy. Many meta-analyses have suggested an etiological relationship between C/S delivery and autism spectrum disorders (ASDs). However, as a risk factor for ASDs, C/S delivery has not yet been well studied. Because C/S deliveries have been increasing, it is very important to investigate the causal association between C/S and ASDs. Here, using three approaches, we showed experimentally that C/S delivery induced ASD-like traits in offspring mice, and that some of these changes were ameliorated by one-time oxytocin (OXT) treatment. Treatment with OXT receptor antagonists before natural delivery also induced ASD-related behaviors. Moreover, wild-type mice born to OXT-KO dams showed similar changes. Thus, insufficient OXT exposure from dams to offspring during delivery may be a trigger for ASD-related behaviors.

Sodium Hypochlorite is Effective against Biofilms in Dialysis Equipment.

To test the efficacy of chemical disinfectants against bacterial biofilms in hemodialysis equipment, a Center for Disease Control and Prevention (CDC)-Biofilm Reactor was used to create biofilms. Methylobacterium radiotolerance was isolated from the hemodialysis fluid and used as the test organism. We examined the efficacy of sodium hypochlorite (NaOCl) in elimination of planktonic cells compared to that in the case of biofilms. Planktonic bacteria were completely eliminated at 50 parts per million (ppm) of NaOCl, which is the lowest concentration for clinical use. The viable cell count in the biofilm reached its minimum value around a logarithmic reduction value (LRV) of 6, when the concentration was raised to 1000 ppm and the reaction time was extended by 1 hour or more. Furthermore, at 200 ppm, the LRV was elevated depending on the time. And the LRV while maintaining static conditions for 6 hours at 200 ppm was similar to that of short time at 1000 ppm. These results suggest that NaOCl has sufficient bactericidal activity even for biofilms at a practical concentration and reaction time, and that the CDC-Biofilm Reactor is an effective tool for finding useful disinfection conditions.

Nrf2 Lowers the Risk of Lung Injury via Modulating the Airway Innate Immune Response Induced by Diesel Exhaust in Mice.

In the present study, we investigated the role of Nrf2 in airway immune responses induced by diesel exhaust (DE) inhalation in mice. C57BL/6J Nrf2+/+ and Nrf2-/- mice were exposed to DE or clean air for 8 h/day and 6 days/week for 4 weeks. After DE exposure, the number of neutrophils and macrophage inflammatory protein (MIP)-2 level in bronchoalveolar lavage fluid (BALF) and interleukin (IL)-17 level in the lung tissue increased in Nrf2-/- mice compared with Nrf2+/+ mice; however, the lack of an increase in the level of tumor necrosis factor (TNF)-α in the lung tissue in Nrf2+/+ mice and mild suppression of the level of TNF-α in Nrf2-/- mice were observed; the level of granulocyte macrophage colony-stimulating factor (GM-CSF) in the lung tissue decreased in Nrf2-/- mice than in Nrf2+/+ mice; the number of DE particle-laden alveolar macrophages in BALF were larger in Nrf2-/- mice than in Nrf2+/+ mice. The results of electron microscope observations showed alveolar type II cell injury and degeneration of the lamellar body after DE exposure in Nrf2-/- mice. Antioxidant enzyme NAD(P)H quinone dehydrogenase (NQO)1 mRNA expression level was higher in Nrf2+/+ mice than in Nrf2-/- mice after DE exposure. Our results suggested that Nrf2 reduces the risk of pulmonary disease via modulating the airway innate immune response caused by DE in mice.

Roles of Two Major Alcohol Dehydrogenases, ADH1 (Class I) and ADH3 (Class III), in the Adaptive Enhancement of Alcohol Metabolism Induced by Chronic Alcohol Consumption in Mice.

AIMS: It is still unclear which enzymes contribute to the adaptive enhancement of alcohol metabolism by chronic alcohol consumption (CAC). ADH1 (Class I) has the lowest Km for ethanol and the highest sensitivity for 4-methylpyrazole (4MP) among ADH isozymes, while ADH3 (Class III) has the highest Km and the lowest sensitivity. We investigated how these two major ADHs relate to the adaptive enhancement of alcohol metabolism. METHODS: Male mice with different ADH genotypes (WT, Adh1-/- and Adh3-/-) were subjected to CAC experiment using a 10% ethanol solution for 1 month. Alcohol elimination rate (AER) was measured after ethanol injection at a 4.0 g/kg dose. 4MP-sensitive and -insensitive AERs were measured by the simultaneous administration of 4MP at a dose of 0.5 mmol/kg in order to estimate ADH1 and non-ADH1 pathways. RESULTS: AER was enhanced by CAC in all ADH genotypes, especially more than twofold in Adh1-/- mice, with increasing ADH1 and/or ADH3 liver contents, but not CYP2E1 content. 4MP-sensitive AER was also increased by CAC in WT and Adh3-/- strains, which was greater in Adh3-/- than in WT mice. The sensitive AER was increased even in Adh1-/- mice probably due to the increase in ADH3, which is semi-sensitive for 4MP. 4MP-insensitive AER was also increased in WT and Adh1-/- by CAC, but not in Adh3-/- mice. CONCLUSION: ADH1 contributes to the enhancement of alcohol metabolism by CAC, particularly in the absence of ADH3. ADH3 also contributes to the enhancement as a non-ADH1 pathway, especially in the absence of ADH1.

Effect of H2 treatment in a mouse model of rheumatoid arthritis-associated interstitial lung disease.

Rheumatoid arthritis (RA)-associated interstitial lung disease (ILD), a primary cause of mortality in patients with RA, has limited treatment options. A previously established RA model in D1CC transgenic mice aberrantly expressed major histocompatibility complex class II genes in joints, developing collagen II-induced polyarthritis and anti-cyclic citrullinated peptide antibodies and interstitial pneumonitis, similar to those in humans. Molecular hydrogen (H2 ) is an efficient antioxidant that permeates cell membranes and alleviates the reactive oxygen species-induced injury implicated in RA pathogenesis. We used D1CC mice to analyse chronic lung fibrosis development and evaluate H2 treatment effects. We injected D1CC mice with type II collagen and supplied them with H2 -rich or control water until analysis. Increased serum surfactant protein D values and lung densities images were observed 10 months after injection. Inflammation was patchy within the perilymphatic stromal area, with increased 8-hydroxy-2'-deoxyguanosine-positive cell numbers and tumour necrosis factor-α, BAX, transforming growth factor-ß, interleukin-6 and soluble collagen levels in the lungs. Inflammatory and fibrotic changes developed diffusely within the perilymphatic stromal area, as observed in humans. H2 treatment decreased these effects in the lungs. Thus, this model is valuable for studying the effects of H2 treatment and chronic interstitial pneumonia pathophysiology in humans. H2 appears to protect against RA-ILD by alleviating oxidative stress.

Development of blastocyst complementation technology without contributions to gametes and the brain.

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.

The Contribution of Alcohol Dehydrogenase 3 to the Development of Alcoholic Osteoporosis in Mice.

BACKGROUND: Alcohol dehydrogenase 3 (ADH3) plays major roles not only in alcohol metabolism but also in nitric oxide metabolism as S-nitrosoglutathione reductase (GSNOR). ADH3/GSNOR regulates both adipogenesis and osteogenesis through the denitrosylation of peroxisome proliferator-activated receptor γ. The current study investigated the contribution of ADH3 to the development of alcoholic osteoporosis in chronic alcohol consumption (CAC). METHODS: Nine-week-old male mice of different ADH genotypes [wild-type (WT) and Adh3-/-] were administered a 10% ethanol solution for 12 months. The femurs were evaluated by histochemical staining and computed tomography-based bone densitometry. The mRNA levels of ADH3 were evaluated in the WT mice by reverse transcription-quantitative polymerase chain reaction. RESULTS: The Adh3-/- control mice exhibited increased activities of both osteoblasts and osteoclasts and lower bone masses than the WT control mice. CAC exhibited no remarkable change in osteoblastic and osteoclastic activities, but decreased bone masses were observed in WT mice despite an increase in the mRNA levels of ADH3. Conversely, bone masses in the Adh3-/- control mice were not reduced after CAC. CONCLUSIONS: The Adh3-/- control mice exhibited a high turnover of osteoporosis since osteoclastogenesis dominated osteoblastogenesis; however, bone resorption was not enhanced after CAC. In comparison, CAC lead to alcoholic osteoporosis in WT mice, accompanied by increased mRNA levels of ADH3. Hence, ADH3 can prevent osteoporosis development in normal ADH genotypes with no alcohol ingestion. However, ADH3 contributes to the development of alcoholic osteoporosis under CAC by participating in alcohol metabolism, increasing metabolic toxicity, and lowering GSNO reducing activity.

Metabolic pharmacokinetics of early chronic alcohol consumption mediated by liver alcohol dehydrogenases 1 and 3 in mice.

BACKGROUND AND AIM: Alcohol dehydrogenases (ADHs) 1 and 3 are responsible for systemic alcohol metabolism. The current study investigated the contribution of liver ADH1 and ADH3 to the metabolic pharmacokinetics of chronic alcohol consumption (CAC). METHODS: The 9-week-old male mice of different ADH genotypes (wild-type [WT], Adh1-/- , and Adh3-/- ) were administered with 10% ethanol solution for 1 month, followed by acute ethanol administration (4.0 g/kg). The alcohol elimination rate (AER), area under the blood alcohol concentration curve (AUC), and the maximum blood alcohol concentration (Cmax ) were calculated. The liver content, activity, and mRNA levels of ADH were evaluated. RESULTS: Chronic alcohol consumption increased the AER and reduced the AUC in all ADH genotypes. The increased ADH1 content was correlated with AER in WT mice but not in the Adh3-/- mice. Similarly, the increased ADH3 content was also correlated with AER in both WT and Adh1-/- mice. The Cmax was significantly higher in Adh3-/- control mice than in WT control mice. It decreased in the Adh1-/- mice by CAC along with an increase in the ADH3 content. CONCLUSIONS: Alcohol dehydrogenases 1 and 3 would accomplish the pharmacokinetic adaptation to CAC in the early period. ADH1 contributes to the metabolic pharmacokinetics of CAC with a decrease in AUC in conjunction with an increase of AER by increasing the enzyme content in the presence of ADH3. ADH3 also contributes to a decrease in AUC in conjunction with not only an increase in AER but also a decrease in Cmax by increasing the enzyme content.

Adiponectin deficiency-induced diabetes increases TNFα and FFA via downregulation of PPARα.

Expression of peroxisome proliferator-activated receptor (PPAR) α was investigated in adiponectin knockout mice to elucidate the relationship between PPARα and adiponectin deficiency-induced diabetes. Adiponectin knockout (Adp-/-) mice were generated by gene targeting. Glucose tolerance test (GTT), insulin tolerance test (ITT), and organ sampling were performed in Adp-/- mice at the age of 10 weeks. PPARα, insulin, triglyceride, free fatty acid (FFA), and tumor necrosis factor α (TNFα) were analyzed from the sampled organs. Adp-/- mice showed impaired glucose tolerance and insulin resistance. Additionally, PPARα levels were decreased and plasma concentration of triglyceride, FFA and TNFα were increased. These data may indicate that insulin resistance in Adp-/- mice is likely caused by an increase in concentrations of TNFα and FFA via downregulation of PPARα.

Vomeronasal signal deficiency enhances parental behavior in socially isolated male mice.

We previously reported that social isolation promotes parental care in sexually naïve male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an adjacent compartment. In the present study, we examined whether the chemosensory signals detected in the vomeronasal organ (VNO) are involved in parental behavior by using mice deficient for a VNO-specific ion channel (Trpc2-/-) and thus impaired in VNO-input signaling. We housed virgin homozygous Trpc2-/- and heterozygous Trpc2± males for 3weeks during puberty (5-8weeks old) alone or in groups of 3-5 males. At 8weeks of age, the mice were placed with three pups in an observation cage and tested for parental behavior. The Trpc2-/- males housed under isolated conditions spent significantly longer in the vicinity of pups than did the Trpc2-/- males than had been group housed, whereas no isolation effect was observed in heterozygous Trpc2± males. Both Trpc2 knockout and isolation housing significantly increased the time males spent licking pups and crouching (arched back posture over pups to enable nursing), whereas only isolation housing increased the incidence of retrieval behavior. These results demonstrated that social signals transmitted not only through the VNO but also from other modalities, independent of each other, suppress the expression of parental behavior during puberty in sexually naïve males.

A new in vivo analysis model to detect sexually dimorphic rat liver cytochrome P450 gene expression dependent on growth hormone secretory patterns.

Several drug-metabolizing cytochrome P450 (CYP) enzymes exhibit sexual dimorphism depending on the pituitary growth hormone (GH) secretory patterns. However, the mechanism underlying CYP sexual dimorphism remains unclear. We previously established a transgenic (Alb-DsRed2 Tg) rat that expressed red fluorescent DsRed2 protein, particularly in hepatocytes, to visualize cell differentiation and multiplication and found that hepatic DsRed2 expression exhibited sexual dimorphism that was limited to adult males. In this study, we compared the expression patterns between sexual dimorphic Cyps and DsRed2 in Tg rats after experimentally reversing the GH secretory patterns in males and females. Postnatal day 1 male and female Tg rats were gonadectomized and then testosterone propionate (0.25 mg/rat) was subcutaneously administered to ovariectomized females immediately after surgery. Cyp mRNA and DsRed2 expression levels were quantified using RT-PCR and an in vivo imaging system, respectively. GH-dependent Cyps and hepatic DsRed2 expression patterns were reversed in males and females at 9 weeks after birth and were significantly correlated (P<0.05). This suggested that DsRed2 expression in these Tg rats depended on GH secretory patterns. Based on DsRed2 fluorescence, this Tg rat model could become a tool to readily and effectively evaluate changes in GH-dependent Cyp expression.

Alcohol dehydrogenase 3 contributes to the protection of liver from nonalcoholic steatohepatitis.

Nutritional steatohepatitis is closely associated with dysregulation of lipid metabolism and oxidative stress control. ADH3 is a highly conserved bifunctional enzyme involved in formaldehyde detoxification and termination of nitric oxide signaling. Formaldehyde and nitric oxide are nonenzymatically conjugated with glutathione, which is regenerated after ADH3 metabolizes the conjugates. To clarify roles of ADH3 in nutritional liver diseases, we placed Adh3-null mice on a methionine- and choline-deficient (MCD) diet. The Adh3-null mice developed steatohepatitis more rapidly than wild-type mice, indicating that ADH3 protects liver from nutritional steatohepatitis. NRF2, which is a key regulator of cytoprotective genes against oxidative stress, was activated in the Adh3-null mice with liver damage. In the absence of NRF2, the Adh3 disruption caused severe steatohepatitis by the MCD diet feeding accompanied by significant decrease in glutathione, suggesting cooperative function between ADH3 and NRF2 in the maintenance of cellular glutathione level for cytoprotection. Conversely, with enhanced NRF2 activity, the Adh3 disruption did not cause steatohepatitis but induced steatosis, suggesting that perturbation of lipid metabolism in ADH3-deficiency is not compensated by NRF2. Thus, ADH3 protects liver from steatosis by supporting normal lipid metabolism and prevents progression of steatosis into steatohepatitis by maintaining the cellular glutathione level.

Expression of the glucose-sensing receptor T1R3 in pancreatic islet: changes in the expression levels in various nutritional and metabolic states.

We reported recently that the taste type 1 receptor 3 (T1R3), a subunit of the sweet taste receptor, functions as a cell-surface glucose-sensing receptor in pancreatic ß-cells. In the present study, we investigated the expression of T1R3 in pancreatic islets. mRNA for T1R2 and T1R3 was detected in mouse pancreatic islets. Quantitatively, the mRNA expression level of T1R2 was less than 1% of that of T1R3. Immunohistochemically, T1R3 was abundantly expressed in mouse islets whereas T1R2 was barely detected. Most immunoreactive T1R3 was colocalized with insulin and almost all ß-cells were positive for T1R3. In addition, T1R3 was expressed in some portion of α-cells. Immunoreactivity of T1R3 in ß-cells was markedly reduced in fed mice compared to those in fasting mice. In contrast, mRNA for T1R3 was not different in islets of fasting and fed mice. Glucose-induced insulin-secretion was higher in islets obtained from fasting mice compared to those from fed mice. The expression of T1R3 was markedly reduced in islets of ob/ob mice compared to those of control mice. Similarly, the expression of T1R3 was reduced in islet of db/db mice. In addition, the expression of T1R3 was markedly reduced in ß-cells of fatty diabetic rats and GK rats, models of obese and non-obese type 2 diabetes, respectively. These results indicate that T1R3 is expressed mainly in ß-cells and the expression levels are different depending upon the nutritional and metabolic conditions.

Time-dependent changes in cardiovascular function during copulatory behavior induced by the hand method in the male dog.

PURPOSE: Ejaculation in the male dog consists of three fractions. Observation of behavior and measurement of heart rate (HR), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were researched sequentially, and a fundamental examination of the features of sympathetic nerve activity during copulatory behavior induced by the hand method in the male dog was undertaken. METHODS: We investigated the breeding capability of male dogs. HR, plasma NA level and plasma Ad levels were measured during ejaculation induced by the hand method. RESULTS: HR was 125.8 ± 6.0 beats/min at rest, and peaked during mounting at 195.2 ± 8.2 beats/min. Moreover, HR at 3 min after the first fraction decreased to values similar to those at rest. Plasma NA and Ad concentrations during copulatory behavior induced by the hand method did not differ significantly from those at rest. However, although there was no significant difference, plasma NA concentration during ejaculation of the third fraction peaked at about 1.8 times the baseline value. CONCLUSIONS: In the male dog, excitation of sympathetic nerves of long duration during erection of the penis and ejaculation is questionable. However, inhibition of sympathetic nerves and activation of parasympathetic nerves is thought to occur during erection of the penis and ejaculation.

Antioxidant enzyme, 3-mercaptopyruvate sulfurtransferase-knockout mice exhibit increased anxiety-like behaviors: a model for human mercaptolactate-cysteine disulfiduria.

Human mercaptolactate-cysteine disulfiduria (MCDU) was first recognized and reported in 1968. Most cases of MCDU are associated with mental retardation, while the pathogenesis remains unknown. To investigate it, we generated homozygous 3-mercaptopyruvate sulfurtransferase (MST: EC 2.8.1.2) knockout (KO) mice using C57BL/6 embryonic stem cells as an animal model. The MST-KO mice showed significantly increased anxiety-like behaviors with an increase in serotonin level in the prefrontal cortex (PFC), but not with abnormal morphological changes in the brain. MCDU can be caused by loss in the functional diversity of MST; first, MST functions as an antioxidant protein. MST possessing 2 redox-sensing molecular switches maintains cellular redox homeostasis. Second, MST can produce H2S (or HS(-)). Third, MST can also produce SOx. It is concluded that behavioral abnormality in MST-KO mice is caused by MST function defects such as an antioxidant insufficiency or a new transducer, H2S (or HS(-)) and/or SOx deficiency.

Direct and specific effect of sevoflurane anesthesia on rat Per2 expression in the suprachiasmatic nucleus.

BACKGROUND: Our previous studies revealed that application of the inhalation anesthetic, sevoflurane, reversibly repressed the expression of Per2 in the mouse suprachiasmatic nucleus (SCN). We aimed to examine whether sevoflurane directly affects the SCN. METHODS: We performed in vivo and in vitro experiments to investigate rat Per2 expression under sevoflurane-treatment. The in vivo effects of sevoflurane on rPer2 expression were examined by quantitative in situ hybridization with a radioactively-labeled cRNA probe. Additionally, we examined the effect of sevoflurane anesthesia on rest/activity rhythms in the rat. In the in vitro experiments, we applied sevoflurane to SCN explant cultures from Per2-dLuc transgenic rats, and monitored luciferase bioluminescence, representing Per2 promoter activity. Bioluminescence from two peripheral organs, the kidney cortex and the anterior pituitary gland, were also analyzed. RESULTS: Application of sevoflurane in rats significantly suppressed Per2 expression in the SCN compared with untreated animals. We observed no sevoflurane-induced phase-shift in the rest/activity rhythms. In the in vitro experiments, the intermittent application of sevoflurane repressed the increase of Per2-dLuc luminescence and led to a phase delay in the Per2-dLuc luminescence rhythm. Sevoflurane treatment did not suppress bioluminescence in the kidney cortex or the anterior pituitary gland. CONCLUSION: The suppression of Per2-dLuc luminescence by sevoflurane in in vitro SCN cultures isolated from peripheral inputs and other nuclei suggest a direct action of sevoflurane on the SCN itself. That sevoflurane has no such effect on peripheral organs suggests that this action might be mediated through a neuron-specific cellular mechanism or a regulation of the signal transduction between neurons.

Involvement of matrix metalloproteinase-2 in the development of renal interstitial fibrosis in mouse obstructive nephropathy.

Renal fibrosis is a common finding in progressive renal diseases. Matrix metalloproteinases (MMPs) are involved in epithelial-to-mesenchymal transition (EMT). We investigated the role of MMP-2 and the effect of inhibition of MMPs on the development of renal fibrosis. Renal fibrosis was induced in MMP-2 wild-type (MMP-2⁺/⁺) mice by unilateral ureteral obstruction (UUO). Renal histopathology, EMT-associated molecules, and activity of MMP-2 and MMP-9 were examined during the development of interstitial fibrosis. UUO-renal fibrosis was also induced in MMP-2 deficient (MMP-2⁻/⁻) and MMP-2⁺/⁺ mice treated with minocycline (inhibitor of MMPs). In MMP-2⁺/⁺ mice, MMP-2 and MMP-9 were expressed in damaged tubules, and their activities increased in a time-dependent manner after UUO. Interstitial fibrosis was noted at day 14, with deposition of types III and I collagens and expression of markers of mesenchymal cells (S100A4, vimentin, α-smooth muscle actin, and heat shock protein-47) in damaged tubular epithelial cells, together with F4/80+ macrophage infiltration. Fibrotic kidneys expressed EMT-associated molecules (ILK, TGF-ß1, Smad, Wnt, ß-catenin, and Snail). In contrast, the kidneys of MMP-2⁻/⁻ mice and minocycline-treated MMP-2⁺/⁺ mice showed amelioration of renal fibrosis with reduced expression of markers of mesenchymal cells in tubular epithelial cells, inhibition of upregulated EMT-associated molecules, and suppression of macrophage infiltration. The results suggested that MMP-2 have a pathogenic role in renal interstitial fibrosis, possibly through the induction of EMT and macrophage infiltration. Inhibition of MMPs may be beneficial therapeutically in renal fibrosis.