Adult ICR Mice Research Articles

Effects of phenolic acid metabolites formed after chlorogenic acid consumption on retinal degeneration in vivo.

Although ingestion of coffee and its constituent chlorogenic acid (CGA) protects the retina from oxidative stress, the bioaccessibility and bioavailability of coffee metabolites are not well understood. The aim of this study was to determine which coffee metabolites reach the retina and protect against retinal degeneration. UPLC-MS/MS was used to detect CGA and coffee metabolites in the rat eye. The methyl thiazolyl tetrazolium assay and double staining with Hoechst and propidium iodide showed that CGA, caffeic acid (CA), and dihydrocaffeic acid (DHCA) protect retinal ganglion cells from hypoxia-induced damage. Western blots showed that treatment with coffee metabolites up-regulated anti-apoptotic proteins such as Bcl-2 and Bcl-XL and down-regulated pro-apoptotic proteins such as Bad, PARP, and cleaved caspase 3. Adult ICR mice were subjected to optic nerve crush-induced retinal ganglion cell death with intravitreal pre-treatment with coffee metabolites 1 day before and 1 h after the procedure. Retrograde Fluorogold(TM) labeling showed severe retinal ganglion cell loss after optic nerve crushing, and coffee metabolites significantly reduced damage to retinal ganglion cells. CGA and coffee metabolites, especially, CA, and DHCA, reach the eye, where they can significantly reduce apoptosis induced by hypoxia and optic nerve crush stress, and thus prevent retinal degeneration.

Effect of licorice flavonoids on kainic acid-induced seizure in mice

To investigate the effect of licorice flavonoid (LF) on kainic acid (KA)-induced seizure in mice and its mechanism. Male adult ICR mice were injected with 25 mg/kg KA to induce temporal lobe seizure. LF was administrated 7 d before seizure induction (pre-treatment) or 24 h after seizure induction (post-treatment) for 7 d. Acute seizure latency, seizure stage and duration were observed and compared between LF- and vehicle-treated mice. From d2 on, mice with status epilepticus were video-monitored for spontaneous seizures, 10 h/d for 6 w. Immunohistochemical analysis of BrdU and Timm staining was conducted to detect the neurogenesis and mossy fiber sprouting, respectively. No significant difference was observed in acute seizure latency, seizure stage and duration between LF-and vehicle-treated mice. KA-induced acute seizure resulted in spontaneous seizure in mice, and the seizure frequency was increased with time. Pre- and post-treatment with LF decreased seizure frequency from w3 after modeling [(0.58±0.15)/d, (0.38±0.38)/d vs (1.23±0.23)/d, P <0.05]. Furthermore, KA-induced seizure resulted in robust neurogenesis and mossy fiber sprouting, while treatment with LF both pre- and post- KA injection significantly inhibited neurogenesis (15.6±2.6, 17.1±3.1 vs 28.9±3.5, P <0.05) and mossy fiber sprouting (1.33±0.31, 1.56±0.42 vs 3.0±0.37, P <0.05). LF has no significant anti-seizure effect. However, it can decrease epileptogenesis through inhibition of neurogenesis and mossy fiber sprouting.

Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation.

Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2–4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.

Effects of pregabalin on D‐serine content and NMDA receptor‐mediated synaptic transmission in the mouse spinal cord (1129.9)

The anticonvulsant pregabalin (PGB) is used to relieve neuropathic pain. It has been shown that PGB binds to presynaptic calcium channels and subsequently inhibits transmitter release. Additionally, a preponderance of the evidence raises the possibility of other mechanisms independent of binding to calcium channels. In the present experiments, we have studied the effects of PGB on D‐serine content and NMDA receptor‐mediated synaptic transmission in the spinal cord dorsal horn.Experiments were performed on adult ICR mice. The sciatic nerve was partially ligated under halothane anesthesia. Mechanical allodynia was assessed by von Frey filaments. PGB (30‐50 mg/kg body weight) was intraperitonealy injected daily for 5‐7 days. The lumbar spinal cord was removed, homogenized, and ultrafiltrated. Supernatant samples were treated with Marfey's reagent and analyzed with liquid chromatography‐mass spectrometry for D‐serine concentration. Tight‐seal whole‐cell recordings were made from neurons in the superficial dorsal horn of spinal cord slices. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation.D‐serine content was 9.9±1.0 nM/g of wet tissue in sciatic nerve‐ligated mice (n=6), which was significantly larger than that in sham‐operated mice (7.9±0.8 nM/g, p &lt; 0.05). PGB considerably decreased D‐serine content to 6.0±0.9 nM/g (n=6, p &lt; 0.05, vs. sciatic nerve‐ligated mice). PGB decreased the NMDA/non‐NMDA ratio of the EPSC amplitude, and shortened the decay phase of the NMDA component of the EPSCs. These observations suggest that PGB decreases D‐serine concentration and thereby affects synaptic plasticity.Grant Funding Source: Supported by a grant from the Japan Society for the Promotion of Science (23500468 to Y.H.).

HISTOPATHOLOGICAL CHANGES IN MICE INFECTED WITH RIVER WATER CONTAMINATED BY &lt;i&gt;PASTEURELLA MULTOCIDA &lt;/i&gt;TYPE B:2

Haemorrhagic Septicaemia (HS) is a fatal systemic disease of bovines typified by an acute, highly fatal septicaemic disease with high morbidity and mortality. In this study, sixty five adult ICR mice were used for the study. In the first phase, 5 mice each were inoculated with 109 cfu of P. multocida type B:2 through the intraperitoneal route. After development of classical signs of HS, the mice were euthanised and dropped into a tank of river waterfor 24, 48 and 72 h. Contaminated water from the tank were collected at the three different times (24, 48 and 72 h) and was used for intraperitoneal, oral and aerosol inoculation of 15 mice each with a dose of 1 mL-1 109 cfu (intraperitoneal and aerosol) and 0.4 mL-1 109cfu (oral). The control group (n = 5) was given phosphate buffered saline. All mice in the three groups and control were culled after 72 h post inoculation and the vital organs (liver, spleen, lung, kidney, heart, brain) were collected in 10% buffered formalin, procesed, sectioned and stained with H&E. Degeneration and necrosis, hemorrhage and infiltration of inflammatory cells were the most observed lesions in all the organs, while thrombosis and oedema were least observed. The lesion severity was moderate to severe using contaminated water at 72 h post contamination and mild to moderate when inoculated with water 24 and 48 h post contamination. Mice inoculated through the oral and intraperitoneal routes recorded more severe lesion compared with those inoculated through the aerosol route. However, using contaminated water at 24 h, mice inoculated through the aerosol route had the highest distribution of inflammatory cells in the lungs, which declined following inoculation with contaminated water at 48 and 72 h, respectively.This study has shown that contamination of water bodiesby carcasses of HS infected animals is a likely source of infection to heathy animals in the wild and that development of lesions severity is time dependant and significantly higher using contaminated water at 72 h than water contaminated for 24 and 48 h. This further indicates the danger of leaving decomposing carcasses in water bodies for prolonged periods.

Immunomodulation Stimulates the Innervation of Engineered Tooth Organ

The sensory innervation of the dental mesenchyme is essential for tooth function and protection. Sensory innervation of the dental pulp is mediated by axons originating from the trigeminal ganglia and is strictly regulated in time. Teeth can develop from cultured re-associations between dissociated dental epithelial and mesenchymal cells from Embryonic Day 14 mouse molars, after implantation under the skin of adult ICR mice. In these conditions however, the innervation of the dental mesenchyme did not occur spontaneously. In order to go further with this question, complementary experimental approaches were designed. Cultured cell re-associations were implanted together with trigeminal ganglia for one or two weeks. Although axonal growth was regularly observed extending from the trigeminal ganglia to all around the forming teeth, the presence of axons in the dental mesenchyme was detected in less than 2.5% of samples after two weeks, demonstrating a specific impairment of their entering the dental mesenchyme. In clinical context, immunosuppressive therapy using cyclosporin A was found to accelerate the innervation of transplanted tissues. Indeed, when cultured cell re-associations and trigeminal ganglia were co-implanted in cyclosporin A-treated ICR mice, nerve fibers were detected in the dental pulp, even reaching odontoblasts after one week. However, cyclosporin A shows multiple effects, including direct ones on nerve growth. To test whether there may be a direct functional relationship between immunomodulation and innervation, cell re-associations and trigeminal ganglia were co-implanted in immunocompromised Nude mice. In these conditions as well, the innervation of the dental mesenchyme was observed already after one week of implantation, but axons reached the odontoblast layer after two weeks only. This study demonstrated that immunodepression per se does stimulate the innervation of the dental mesenchyme.

The Kidneys of Infant Mice are not Sensitive to the Food Mycotoxin Contaminant Nivalenol.

Nivalenol (NIV) is a trichothecene mycotoxin produced by Fusarium fungi that frequently contaminates agricultural commodities. Dietary administration of NIV to adult mice affects the renal glomeruli, but data about NIV toxicity in human infants are limited. To evaluate the effects of NIV on infant kidneys, 3-week-old male ICR-derived glomerulonephritis (ICGN) and ICR mice were administered 0, 4, 8 or 16 ppm NIV in diet for 4 weeks, and their renal status was compared with age-matched or adult ICR mice. In ICGN mice, the number of glomeruli showing mesangial expansion and α-smooth muscle actin (SMA)-positive mesangial cells was higher with 16 ppm NIV compared with controls. No other significant differences were observed in ICGN mice. In infant ICR mice, the IgA serum concentrations were significantly elevated without glomerular morphological changes in the 16 ppm NIV group. There was no difference in NIV sensitivity in the kidneys of infant ICGN and ICR mice. These data suggest that the kidneys in infant mice are not sensitive to nivalenol under the present conditions.

Delivery of Human EV71 Receptors by Adeno-Associated Virus Increases EV71 Infection-Induced Local Inflammation in Adult Mice

Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

Comparison of seizure induced by different drugs in ICR Mice

To compare seizure induced by different epileptic drugs in ICR mice. Male adult ICR mice were injected with pilocarpine (Pilo), kainic acid (KA) and pentylenetetrazole (PTZ) to induce status epilepticus (SE). After 2 h of SE, seizures were terminated by injection of diazepam. Mice were sacrificed and sectioned for assessment of neuronal cell death by Fluro-Jade B staining after 7 d and mossy fiber sprouting by Timm staining after 28 d, respectively. Spontaneous seizures were detected by video for 28 d. Pilo and KA induced typical SE in ICR mice, which was identical to those observed in rats and C57/BL6 mice. Timm staining showed evident mossy fiber sprouting in both Pilo and KA treated mice. The incidences of spontaneous seizure were 57.1% and 35.7% in Pilo and KA treated mice, respectively. Mice treated with PTZ represented kindling model. No mossy fiber sprouting and spontaneous seizures were observed. No cell death was detected in all three groups. Similar seizure pattern is observed in ICR mice as in rats and C57/BL6 mice. Both Pilo and KA model are the ideal models for chronic temporal lobe epilepsy. ICR mice can be widely used as a cheaper substitute in epilepsy research.

シュワン細胞株をもちいた末梢神経再生機構の解析

We have established spontaneously immortalized Schwann cell lines from adult ICR mice [IMS32] and Fischer344 rats [IFRS1]. IMS32 cells display distinct Schwann cell phenotypes such as a spindle-shaped morphology and the expression of glial cell markers (e.g. S100, glial fibrillary acidic protein (GFAP), p75 low-affinity neurotrophin receptor (p75(NTR))) and neurotrophic factors. In addition, conditioned medium obtained from IMS32 cells enhances neurite elongation of PC12 cells and mouse dorsal root ganglion (DRG) neurons. IMS32 cells have been utilized to investigate the action mechanisms of various molecules that accelerate peripheral nerve regeneration (e.g. ciliary neurotrophic factor, sonic hedgehog, galectin-1). Like IMS32 cells, IFRS1 cells retain the characteristic features of mature Schwann cells as described above. Furthermore, IFRS1 cells have been shown to myelinate neurites in coculture with adult rat DRG neurons and PC12 cells. Our current investigation with IFRS1 cells focuses on the molecular mechanisms of myelination-inducible factors, such as soluble neuregulin-1 type III and exendin-4. These Schwann cell lines can be valuable tools for exploring neuron-Schwann cell interactions, pathobiology of axonal degeneration and regeneration in the peripheral nervous system, and novel therapeutic approaches against neurological disorders in patients with relevant diseases.

Alternanthera sessilis and Alternanthera bettzickiana Improved Superoxide Dismutase and Catalase Activities in the Livers of Ovariectomized Mice

Background: Oxidative stress from reactive oxygen species can cause the damages to several cells or tissues. Ovariectomized (OVX) mice have been used as the oxidative stress model. Alternanthera sessilis (AS) and Alternanthera bettzickiana (AB), the Southeast Asia edible plants, have been traditionally used for promoting lactation, anti-inflammatory, wound healing, and antioxidation. Objective: Impacts of AS and AB crude extracts on the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) including the levels of malondialdehyde (MDA) formation and glutathione (GSH) content were investigated in the livers of ovariectomized induced oxidative stress in mice to clarify their anti-oxidative potentials, compared to 17b-estradiol (E2). Materials and Methods: Adult ICR mice were ovariectomized before treated with E2 (1 mg/kg/day, i.p.), or AS or AB (250, 500 mg/kg/day, p.o.), daily for eight weeks. The activity of CAT, SOD, and GPx and the levels of MDA and GSH in the mouse livers were determined. Results: Ovariectomy significantly reduced the activities of hepatic CAT, SOD, and GPx enzymes while the level of MDA was increased. Extensive decrease of total GSH content, reduced GSH, and the ratio of GSH/GSSG in the OVX mice demonstrated occurrence of hepatic oxidative stress. E2, AS, and AB restored the CAT and SOD activities while that of GPx activity was not modified. The MDA level was extensively suppressed by E2, AS, and AB. The significant increase of the GSH/GSSG ratio was achieved as a result from the marked abatement of GSSG content by E2, AS, and AB, though the level of total GSH content and the reduced GSH was not accrued. Conclusion: AS and AB possessed antioxidant benefit to improve the hepatic oxidant-antioxidant balance in the ovariectomized mice comparable to E2 did.

Acetazolamide impairs fear memory consolidation in rodents

Acetazolamide (AZ) is an carbonic anhydrase inhibitor, which has been used in the treatment of seizures, mountain sickness and glaucoma. Memory impairment by AZ has been reported in patient interviews; however, the related mechanism is unclear. We applied two fear conditioning paradigms, shuttle avoidance and passive avoidance, in both rats and mice to investigate this clinical anecdote. Adult Wistar rats receiving AZ 1 h before the shuttle avoidance test showed decreased avoidance rates, especially at high dosage. Adult ICR mice receiving AZ both before and after acquisition trials showed the decreased step-through latencies during the passive avoidance test. This impairment of fear memory was corroborated with decreased LTP by AZ in the amygdala. AZ only inhibited fear conditioning-induced ERK phosphorylation and had no effect on Akt phosphorylation. In conclusion, our study confirmed the adverse cognitive effect of AZ in animal and electrophysiological studies. In clinical practice, clinicians should be aware of this side effect in patients taking AZ. In addition, this inhibition of fear memory by AZ could potentially be applied to patients with posttraumatic stress disorder.

Di-(2-ethylhcxyl) phthalate reduces progesterone levels and induces apoptosis of ovarian granulosa cell in adult female ICR mice

Di-(2-ethylhexyl) phthalate (DEHP) as an environmental endocrine disruptor is a known reproductive toxicant and carcinogen. The purpose of this study was to evaluate the female reproductive toxicity of DEHP. Sixty ICR female mice were randomized into four groups dosed with 0, 125, 500, or 2 000mg/kg DEHP by gavage for 16 weeks, 6 days/week. DEHP treatment prolonged duration of the estrous cycle in mice at 500 and 2000mg/kg DEHP, but no significant effects on estrus phase of the cycle in each group were detected. Exposure to DEHP inhibited secretion of serum progesterone. DEHP arrested granulosa cells at G0/G1 phases and increased proportion of apoptosis cells at 500 and 2000mg/kg DEHP. There was no significant difference in P450arom mRNA expression among groups. Results demonstrate that DEHP can produce toxicity in female reproductive system.

HIGH CAPACITY COUNTER CURRENT CHROMATOGRAPHY FOR THE ISOLATION OF ACTIVE INGREDIENTS FROM MEDICINAL PLANTS

s. PHYTOPHARM 2012 M 60 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 Schisandrols A and b, schisantherin A, schisandrins A and b were detected in Schisandra oil extract by hPTlC (fig. 1A). These lignans are considered to be the main active substances of Schizandra chinensis. lignans antioxidant activity plays a significant role in pharmacological activity of Schizandra drugs (3). We established that these lignans don’t have fRSA by dPPh. They don’t bleach background of the plate after dPPh solution derivatization (fig. 1b). fRSA of Schizandra oil extract depend on other components of the extract, but not lignans. Thus, Schizandra oil extract is a source not only of the well-known lignans, but of other active compounds with the fRSA. The fRSA of these substances causes of their pharmacological importance.

KS‐370G ameliorates renal fibrosis, oxidative stress and inflammation induced by unilateral ureteral obstruction

Renal fibrosis is a common morphological feature of progressive chronic renal diseases including unilateral ureteral obstruction (UUO). In this study, we evaluated the role of KS‐370G, a caffeamide derivative, in the kidney fibrosis induced by UUO. Adult ICR mice were subjected to UUO and then administered with either KS‐370G (10mg/kg/day, gavage) or vehicle from 1day after UUO. UUO was maintained for 14 days. KS‐370G administration daily for 13 days significantly decreased the UUO‐induced kidney fibrotic changes including collagen deposition and fibronectin protein expression. KS‐370G also attenuated renal weight loss which result from UUO. KS‐370G reduced iNOS expression that contribute to renal inflammation. KS‐370G attenuated the increases of oxidative stress by the prevention of UUO‐induced decreases of manganese superoxide dismutase (MnSOD). Furthermore, UUO‐induced AMP‐activated protein kinase (AMPK), p42/p44 mitogen‐activated protein kinase (MAPK), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (stat3) phosphorylation was markedly blocked by KS‐370G. In conclusion, KS‐370G prevented renal fibrosis following UUO by the inhibition of inflammation, oxidative stress and JAK2/Stat3 signaling pathway. The source of research support: National Science Council.

A high cholesterol diet ameliorates hippocampus-related cognitive and pathological deficits in ovariectomized mice

Both sex hormone deficiency and hypercholesterolemia are related to cognitive decline or Alzheimer's disease. However, their interactive influence on the neurodegenerative progress is not clear. This study was designed to assess the effects of ovarian hormone depletion and high cholesterol diet alone or in combination on hippocampus-related cognitive and pathological deficits in adult female ICR mice. Depletion of ovarian hormones by ovariectomy for 9 weeks resulted in significant spatial learning and memory deficits as revealed by the water maze testing. Such cognitive alteration was accompanied with increases in neuron death and decreases in choline acetyltransferase activity and synaptopysin expression in the hippocampus. On the other hand, the high cholesterol diet (3% cholesterol plus normal chow) did not exacerbate, but slightly alleviated cognitive decline and significantly attenuated hippocampal pathological changes in ovariectomized mice. Moreover, ovariectomized mice fed high cholesterol had increased serum estrogen levels compared with those fed a normal chow. These results indicate that high cholesterol intake increases the sex hormone synthesis and in turn partially attenuates hippocampus-related cognitive and pathological deficits caused by ovariectomy.

Gestational and Lactational Exposure to Bisphenol A Affects Anxiety-and Depression-Behaviors in Young Offspring Mice

Bisphenol A(BPA) is an environmental estrogenic disrupter widely used in the production of plastics,and ubiquitous human exposure to this chemical has been proposed to be a potential risk to public health.There has been a growing concern about the effect of early-life exposure to low doses of BPA.This study was to investigate the behavior difference of mice which exposed to BPA in gestation period or lactation period separately.After acclimatization for one week,adult female ICR mice were placed with males(two females∶one male) and vaginal smears were examined daily.A sperm-positive smear determined gestational day(GD) 1.Pregnant mice were divided into 2 groups randomly.One group was orally exposed to BPA dissolved in peanut oil(0.4 or 4 mg/kg·d) or only peanut oil as vehicle control from GD8 until offspring mice born.The other group was exposed to BPA with the same method from offspring mice born to postnatal day(PND) 14.At PND21 of age,open field,dark/light transition,mirror chamber,elevated plus-maze,forced swim and step-down were respectively used to test spontaneous activity,exploratory behavior,anxiety,depression,and passive avoidance memory in offspring mice.The results showed that gestational or lactational exposure to BPA differently affects behaviors in offspring mice.Gestational exposure to BPA weakened anxiety and increased spontaneous activity of young offspring,as well as enhanced exploratory behavior of male offspring and passive avoidance memory of female offspring.Lactational exposure to BPA decreased the spontaneous activity of young offspring,but no significant effect on anxiety was found in offspring mice.Both gestational and lactational exposure to BPA strengthened depression in male and female offspring mice.These results suggested that non-reproductive behaviors in offspring mice,such as anxiety,depression,passive avoidance memory,were affected by both gestational and lactational exposure to BPA,while the affection was more sensitive in pregnancy period.

Role of Oxidative Stress in Rabies Virus Infection of Adult Mouse Dorsal Root Ganglion Neurons

Rabies virus infection of dorsal root ganglia (DRG) was studied in vitro with cultured adult mouse DRG neurons. Recent in vivo studies of transgenic mice that express the yellow fluorescent protein indicate that neuronal process degeneration, involving both dendrites and axons, occurs in mice infected with the challenge virus standard (CVS) strain of rabies virus by footpad inoculation. Because of the similarities of the morphological changes in experimental rabies and in diabetic neuropathy and other diseases, we hypothesize that neuronal process degeneration occurs as a result of oxidative stress. DRG neurons were cultured from adult ICR mice. Two days after plating, they were infected with CVS. Immunostaining was evaluated with CVS- and mock-infected cultures for neuron specific beta-tubulin, rabies virus antigen, and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE) (marker of lipid peroxidation and hence oxidative stress). Neuronal viability (by trypan blue exclusion), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and axonal growth were also assessed with the cultures. CVS infected 33 to 54% of cultured DRG neurons. Levels of neuronal viability and TUNEL staining were similar in CVS- and mock-infected DRG neurons. There were significantly more 4-HNE-labeled puncta at 2 and 3 days postinfection in CVS-infected cultures than in mock-infected cultures, and axonal outgrowth was reduced at these time points in CVS infection. Axonal swellings with 4-HNE-labeled puncta were also associated with aggregations of actively respiring mitochondria. We have found evidence that rabies virus infection in vitro causes axonal injury of DRG neurons through oxidative stress. Oxidative stress may be important in vivo in rabies and may explain previous observations of the degeneration of neuronal processes.

Attenuated oocyte fertilization and embryo development associated with altered growth factor/signal transduction induced by endometriotic peritoneal fluid

To investigate whether the embryotoxic effect of peritoneal fluid (PF) from infertile women with mild endometriosis on mouse oocytes and embryos is associated with changes in embryonic epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and their receptors. Experimental animal study. University-based research laboratory. Adult ICR mice. Peritoneal fluid was obtained from fertile women with no endometriosis (PF-NE) and infertile women with mild endometriosis (PF-E). In vitro fertilization was performed, and mouse two-cell stage embryos were cultured in human tubal fluid medium with or without PF. Rates of fertilization, cleavage, and blastulation. The embryonic EGF and IGF-I levels in culture medium were analyzed by enzyme-linked immunosorbent assay, and EGF receptor, IGF-I receptor, and phosphorylated extracellular signal-regulated protein kinases (p-ERK) expression was determined by immunofluorescence and confocal microscopy. When oocytes and embryos were cultured in media with PF-E, the fertilization capability of oocytes and the development potential of embryos were decreased. The levels of embryonic EGF, IGF-I, and their receptors were increased. However, p-ERK of the postreceptor signal transduction pathway was down-regulated. Endometriotic PF may attenuate oocyte and embryo development by impairing embryonic growth factor/receptor/signal transduction, resulting in endometriotic infertility.

Downregulative Effects of Nitric Oxide on Oocyte Fertilization and Embryo Development: Possible Roles of Nitric Oxide in the Pathogenesis of Endometriosis-associated Infertility

Aims: To investigate the effects of elevated nitric oxide (NO) levels in peritoneal fluids (PF) on oocyte fertilization and pre-implantation embryo development, and the relation of those effects to endometriosis-associated infertility. Methods: PF from women undergoing laparoscopy for infertility of minor endometriosis, tubal blockage and operation for tubal ligation was aspired at the pouch of the cul-de-sac during surgery. Oocytes and embryos of adult ICR mice were cultured in vitro with or without endometriotic PF. The fertilization rate of oocyte and the cleavage rate of 2-cell embryos were examined. Also, the clinical indexes of IVF-ET of women with minor endometriosis and tubal infertility were analyzed. Results: Oocyte fertilization rate of endometriotic women with IVF-ET treatment was significantly lower than that of tubal block women. The dose-related adverse effects of endometriotic PF and SNP (NO donor) in culture medium on oocyte fertilization and embryos development were confirmed. Conclusion: Increased NO levels in PF play an important role in mediating the effects of endometriotic PF on oocyte fertilization and embryo development. IVF might serve as an alternative treatment for endometriosis-associated infertility.