Hydrogen gas can ameliorate seizure burden during therapeutic hypothermia in asphyxiated newborn piglets.

BACKGROUND: We previously reported that hydrogen (H2) gas combined with therapeutic hypothermia (TH) improved short-term neurological outcomes in asphyxiated piglets. However, the effect on seizure burden was unclear. Using amplitude-integrated electroencephalography (aEEG), we compared TH + H2 with TH alone in piglets 24 h after hypoxic-ischemic (HI) insult. METHODS: After a 40-min insult and resuscitation, 36 piglets ≤24 h old were divided into three groups: normothermia (NT, n = 14), TH alone (33.5 ± 0.5 °C, 24 h, n = 13), and TH + H2 (2.1-2.7% H2 gas, 24 h, n = 9). aEEG was recorded for 24 h post-insult and its background pattern, status epilepticus (SE; recurrent seizures lasting >5 min), and seizure occurrence (Sz; occurring at least once but not fitting the definition of SE) were evaluated. Background findings with a continuous low voltage and burst suppression were considered abnormal. RESULTS: The percentage of piglets with an abnormal aEEG background (aEEG-BG), abnormal aEEG-BG+Sz and SE was lower with TH + H2 than with TH at 24 h after HI insult. The duration of SE was shorter with TH + H2 and significantly shorter than with NT. CONCLUSIONS: H2 gas combined with TH ameliorated seizure burden 24 h after HI insult. IMPACT: In this asphyxiated piglet model, there was a high percentage of animals with an abnormal amplitude-integrated electroencephalography background (aEEG-BG) after hypoxic-ischemic (HI) insult, which may correspond to moderate and severe hypoxic-ischemic encephalopathy (HIE). Therapeutic hypothermia (TH) was associated with a low percentage of piglets with EEG abnormalities up to 6 h after HI insult but this percentage increased greatly after 12 h, and TH was not effective in attenuating seizure development. H2 gas combined with TH was associated with a low percentage of piglets with an abnormal aEEG-BG and with a shorter duration of status epilepticus at 24 h after HI insult.

Genetic and histopathological analysis of spermatogenesis after short-term testicular torsion in rats.

BACKGROUND: Patients with testicular torsion (TT) may exhibit impaired spermatogenesis from reperfusion injury after detorsion surgery. Alteration in the expressions of spermatogenesis-related genes induced by TT have not been fully elucidated. METHODS: Eight-week-old Sprague-Dawley rats were grouped as follows: group 1 (sham-operated), group 2 (TT without reperfusion) and group 3 (TT with reperfusion). TT was induced by rotating the left testis 720° for 1 h. Testicular reperfusion proceeded for 24 h. Histopathological examination, oxidative stress biomarker measurements, RNA sequencing and RT-PCR were performed. RESULTS: Testicular ischemia/reperfusion injury induced marked histopathological changes. Germ cell apoptosis was significantly increased in group 3 compared with group 1 and 2 (mean apoptotic index: 26.22 vs. 0.64 and 0.56; p = 0.024, and p = 0.024, respectively). Johnsen score in group 3 was smaller than that in group 1 and 2 (mean: 8.81 vs 9.45 and 9.47 points/tubule; p = 0.001, p < 0.001, respectively). Testicular ischemia/reperfusion injury significantly upregulated the expression of genes associated with apoptosis and antioxidant enzymes and significantly downregulated the expression of genes associated with spermatogenesis. CONCLUSION: One hour of TT followed by reperfusion injury caused histopathological testicular damage. The relatively high Johnsen score indicated spermatogenesis was maintained. Genes associated with spermatogenesis were downregulated in the TT rat model. IMPACT: How ischemia/reperfusion injury in testicular torsion (TT) affects the expressions of genes associated with spermatogenesis has not been fully elucidated. This is the first study to report comprehensive gene expression profiles using next generation sequencing for an animal model of TT. Our results revealed that ischemia/reperfusion injury downregulated the expression of genes associated with spermatogenesis and sperm function in addition to histopathological damage, even though the duration of ischemia was short.

Impact of hydrogen gas inhalation during therapeutic hypothermia on cerebral hemodynamics and oxygenation in the asphyxiated piglet.

We previously reported the neuroprotective potential of combined hydrogen (H2) gas ventilation therapy and therapeutic hypothermia (TH) by assessing the short-term neurological outcomes and histological findings of 5-day neonatal hypoxic-ischemic (HI) encephalopathy piglets. However, the effects of H2 gas on cerebral circulation and oxygen metabolism and on prognosis were unknown. Here, we used near-infrared time-resolved spectroscopy to compare combined H2 gas ventilation and TH with TH alone. Piglets were divided into three groups: HI insult with normothermia (NT, n = 10), HI insult with hypothermia (TH, 33.5 ± 0.5 °C, n = 8), and HI insult with hypothermia plus H2 ventilation (TH + H2, 2.1-2.7%, n = 8). H2 ventilation and TH were administered and the cerebral blood volume (CBV) and cerebral hemoglobin oxygen saturation (ScO2) were recorded for 24 h after the insult. CBV was significantly higher at 24 h after the insult in the TH + H2 group than in the other groups. ScO2 was significantly lower throughout the 24 h after the insult in the TH + H2 group than in the NT group. In conclusion, combined H2 gas ventilation and TH increased CBV and decreased ScO2, which may reflect elevated cerebral blood flow to meet greater oxygen demand for the surviving neurons, compared with TH alone.

Concomitant attenuation of HMGCR expression and activity enhances the growth inhibitory effect of atorvastatin on TGF-ß-treated epithelial cancer cells.

Epithelial-mesenchymal transition (EMT) in primary tumor cells is a key prerequisite for metastasis initiation. Statins, cholesterol-lowering drugs, can delay metastasis formation in vivo and attenuate the growth and proliferation of tumor cells in vitro. The latter effect is stronger in tumor cells with a mesenchymal-like phenotype than in those with an epithelial one. However, the effect of statins on epithelial cancer cells treated with EMT-inducing growth factors such as transforming growth factor-ß (TGF-ß) remains unclear. Here, we examined the effect of atorvastatin on two epithelial cancer cell lines following TGF-ß treatment. Atorvastatin-induced growth inhibition was stronger in TGF-ß-treated cells than in cells not thusly treated. Moreover, treatment of cells with atorvastatin prior to TGF-ß treatment enhanced this effect, which was further potentiated by the simultaneous reduction in the expression of the statin target enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Dual pharmacological targeting of HMGCR can thus strongly inhibit the growth and proliferation of epithelial cancer cells treated with TGF-ß and may also improve statin therapy-mediated attenuation of metastasis formation in vivo.

Meganuclease-Based Artificial Transcription Factors.

Embedding middle-scale artificial gene networks in live mammalian cells is one of the most important future goals for cell engineering. However, the applications of the highly orthogonal and conventional artificial transcription factors currently available are limited. In this study, we present a scalable pipeline to produce artificial transcription factors based on homing endonucleases, also known as meganucleases. The introduction of mutations at critical sites for nuclease activity renders these homing endonucleases a simple but highly specific DNA binding domain for their specific DNA target. The introduction of inactivated meganucleases linked to transcriptional activator domains strongly induced reporter gene expression, while their fusion to transcriptional repressor domains suppressed them. In addition, we show that inactivated meganuclease-based transcription factors could be embedded in the synthetic membrane receptor synNotch and used to construct synthetic circuits. These results suggest that inactivated meganucleases are useful DNA-binding domains for the construction of synthetic transcription factors in mammalian cells.

In vitro evaluation of gene expression changes for gonadotropin-releasing hormone 1, brain-derived neurotrophic factor and neurotrophic tyrosine kinase, receptor, type 2, in response to bisphenol A treatment.

We evaluated the effects of bisphenol A (BPA) on embryonic mouse hypothalamic cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) indicated that gonadotropin-releasing hormone 1 (Gnrh1) expression in 0.02-20 µM BPA-treated cells did not differ from that in control cells but decreased significantly in 200 µMBPAtreated cells. The mRNA level for brain-derived neurotrophic factor (Bdnf), which participates in GNRH1 secretory system development, decreased significantly in 200 µM BPA-treated cells, but that for neurotrophic tyrosine kinase, receptor, type 2 (Ntrk2), did not change. This indicates that Gnrh1 gene expression in mice fetuses is not affected by exposure to <20 µM BPA and that the adverse effects of BPA on the BDNF-NTRK2 neurotrophin system are induced by decrease in the mRNA level of the ligand, not of its receptor.

Microarray and gene ontology analyses reveal downregulation of DNA repair and apoptotic pathways in diethylstilbestrol-exposed testicular Leydig cells.

This study investigated the deleterious effects of the synthetic non-steroidal estrogen diethylstilbestrol (DES) on testicular Leydig cells and compared these effects with those of the natural estrogen 17ß-estradiol (E2). For that purpose, we performed microarray analysis of a mouse Leydig cell line (TTE1) treated with these estrogens, followed by Gene Ontology (GO) analysis and parametric analysis of gene set enrichment (PAGE). Most notably, GO analysis revealed a significant decrease in the biological processes of the GO categories "DNA repair" and "apoptotic program" in DES-exposed cells. PAGE showed that "cell death," which is a superior GO category including apoptosis in the GO tree structure, significantly decreased in DES-exposed cells but significantly increased in E2-exposed cells. Interestingly, only 2 genes (Tia1 and Gas1) with altered expression patterns in the "cell death" category were common between DES- and E2-treated cells. The downregulation of apoptotic cell death pathways and DNA repair capability of DES-exposed cells implies that DES promotes carcinogenic processes more strongly than E2 does. These findings suggest that molecular events that occur following DES and E2 treatments differ substantially in Leydig cells, and that the effects of synthetic estrogen and natural estrogen differ more substantially than previously suspected.

A high cholesterol diet given to apolipoprotein E-knockout mice has a differential effect on the various neurotrophin systems in the hippocampus.

Apolipoprotein E (apoE) is one of the major transporters of cholesterol in the body and is essential for maintaining various neural functions in the brain. Given that hypercholesterolemia is a risk factor in Alzheimer's disease (AD), it has been suggested that altered cholesterol metabolism may be involved in the development of the pathogenesis, including neural degeneration, commonly observed in AD patients. Neurotrophic factors and their receptors, which are known to regulate various neural functions, are also known to be altered in various neurodegenerative diseases. We therefore hypothesized that cholesterol metabolism may itself influence the neurotrophin system within the brain. We decided to investigate this possibility by modulating the amount of dietary cholesterol given to apoE-knockout (apoE-KO) and wild-type (WT) mice, and examining the mRNA expression of various neurotrophin ligands and receptors in their hippocampal formations. Groups of eight-week-old apoE-KO and WT mice were fed a diet containing either "high" (HCD) or "normal" (ND) levels of cholesterol for a period of 12 weeks. We found that high dietary cholesterol intake elevated BDNF mRNA expression in both apoE-KO and WT mice and TrkB mRNA expression in apoE-KO animals. On the other hand, NGF and TrkA mRNA levels remained unchanged irrespective of both diet and mouse type. These findings indicate that altered cholesterol metabolism induced by HCD ingestion combined with apoE deficiency can elicit a differential response in the various neurotrophin ligand/receptor systems in the mouse hippocampus. Whether such changes can lead to neural degeneration, and the mechanisms that may be involved in this, awaits further research.

Gender-dependent differences in the incidence of ochratoxin A-induced neural tube defects in the Pdn/Pdn mouse.

Genetic polydactyly/arhinencephaly mouse embryo, Pdn/Pdn, exhibits suppression of Gli3 gene expression. Ochratoxin A (OTA) is a teratogen that causes neural tube defects (NTD) in mice. We investigated gender-dependent differences in the incidence of NTD induced by OTA in the Pdn/Pdn mouse. After administering 2 mg/kg OTA to Pdn/+ female mice, mated with Pdn/+ males, on day 7.5 of gestation, we examined the genotypes, sex and NTD of fetuses on day 18. Non-treated Pdn/Pdn had a 15.8% risk of NTD, and all NTD fetuses were female. When Pdn/Pdn embryos were exposed to OTA, the incidence of NTD increased to 16 (51.6%) of 31 Pdn/Pdn fetuses, and 10 (71.4%) of 14 male Pdn/Pdn fetuses exhibited NTD. From these results, it was speculated that NTD in OTA-treated male Pdn/Pdn were due to the synergistic effect between depressed Gli3 and altered sex-correlated gene expression from OTA treatment. After treatment with OTA, the embryos were recovered on day 9 and gene expressions, which were correlated with Gli3, telencephalic morphogenesis, formation of gonadal anlage, and gender-dependent differentiation were investigated. From real-time polymerase chain reaction analysis results, it was suggested that the manifestation of NTD in the male OTA-treated Pdn/Pdn might be due to the complicated altered gene expressions among Gli3, Wnt7b, Wnt8b, Fez1, Barx1, Lim1, Dmrt1, Igf1, Fog2, Dax1 and Sox9, and in particular, upregulation and gender-dependent difference in Barx1 and gender-dependent difference in Sox9 gene expressions might be noteworthy findings.

Genetic susceptibility in the neural tube defects induced by ochratoxin A in the genetic arhinencephaly mouse, Pdn/Pdn.

It is well known that ochratoxin A (OTA) induces neural tube defects (NTDs) in mice. In the present study, OTA was administered to the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) to investigate the synergistic effect between gene and environmental toxin. OTA treatment on day 7.5 of gestation increased NTDs in the Pdn/Pdn mouse. The responsible gene for Pdn/Pdn is Gli3. So, it was speculated that specific susceptibility for OTA in the Pdn/Pdn mouse embryo may be due to the severe depression of Gli3 gene expression. As correlated genes, Gli3, Shh and Fgf8 gene expressions were examined in the Pdn mouse embryo on day 9 of gestation after administration of OTA on day 7.5. No alteration of Shh expression was observed in the non-treated Pdn/Pdn, and OTA-treated +/+ and Pdn/Pdn. Fgf8 signal was observed at the anterior neural ridge (ANR) in the non-treated +/+, and that was elongated in the non-treated Pdn/Pdn, and further elongated and more intensive in the OTA-treated Pdn/Pdn. It was suggested that Fgf8 gene expression was affected by the depression of Gli3, and alteration of Fgf8 gene expression was accelerated by the toxicity of OTA in the Pdn/Pdn.

Exencephaly induction by valproic acid in the genetic polydactyly/arhinencephaly mouse, Pdn/Pdn.

Non-treated homozygous polydactyly/arhinencephaly (Pdn/Pdn) mouse fetuses exhibited exencephaly in 16.7% of cases. Treatment of Pdn/Pdn mice with 350 mg/kg of valproic acid (VPA) on days 8.5 and 9.5 of gestation increased the rate of exencephaly to 66.7%. The responsible gene for the Pdn mouse phenotype has been determined to be Gli3, and the suppression of Gli3 gene expression has been documented in Pdn/Pdn embryos. We investigated how the sonic hedgehog (Shh) and Fgf8 genes, the correlated genes of Gli3, are expressed in the VPA-treated exencephalic Pdn/Pdn embryos on day 10 of gestation, using whole mount in situ hybridization (WISH) and real-time PCR methods. We could not detect any alterations in Shh expression by real-time PCR, or WISH in the non-treated Pdn/Pdn and VPA-treated exencephalic Pdn/Pdn embryos. Altered Fgf8 expression patterns were observed in the commissural plate and dorsal isthmal neuroepithelium in the non-treated Pdn/Pdn embryos. We speculated that the altered expression of Fgf8 might be the result of down-regulation of Gli3 in Pdn/Pdn embryos. Fgf8 gene expression in the commissural plate and dorsal isthmal neuroepithelium exhibits wide or altered signal patterns in the VPA-treated exencephalic Pdn/Pdn embryo. From these findings, it was suggested that down-regulation of Gli3 gene expression induced the altered expression of Fgf8 in the Pdn/Pdn embryos, and that VPA treatment accelerated the alterations of Fgf8 gene expression in the Pdn/Pdn embryos. It was further speculated that altered expression of Fgf8 in the commissural plate may be the fundamental cause of exencephaly, and that the synergistic effect between gene and drug shown in this experiment may explain the differences of sensitivity in the side-effects of the drug.

Application of the AMeX method to the evaluation of HER-2 status in breast carcinomas: comparison with the results of HercepTest.

The recent development of the recombinant humanized monoclonal antibody against HER-2 oncoprotein requires a simple and accurate method for the evaluation of HER-2 status in patients with breast cancers. We here report that the evaluation of the HER-2 status is improved by the use of the acetone-methanol-xylene (AMeX) method. Compared with an ordinary test of HercepTest, 25 out of 63 cases (39.7%) were scored upwards by the AMeX method. In addition, the HER-2 gene amplification was easily estimated by fluorescence in situ hybridization (FISH) using the AMeX method. Thus, the AMeX method is likely to provide more improved data about the HER-2 status in breast carcinoma specimens.