大鼠球囊巩膜外加压后眼压和虹膜血管的改变

目的　探讨巩膜外加压对大鼠加压后即刻和不同时段眼压（intraocular pressure,IOP）、角膜和虹膜血管的影响。方法　取正常大鼠18只。于每只大鼠右眼切开结膜,在外直肌下方放置球囊导管,充气加压4~5 ATM（1 ATM=101.325 kPa）。于加压、撤压前后不同时段分别测量大鼠的IOP,拍摄眼前节像。观察IOP、角膜和虹膜血管的改变。结果　加压后即刻IOP（40.66±10.55） mmHg （1 kPa=7.5 mmHg）迅速高出基线（8.45±1.23）mmHg（P=0.000）。加压后15 min、30 min、45 min、60 min后IOP逐渐下降,但仍高出基线水平,除60 min（P=0.929）外,其他3个时间点与基线水平差异均有统计学意义（均为P<0.05）。撤除巩膜外加压后即刻的IOP（6.09±0.49） mmHg迅速低于基线水平（P=0.000）。撤压后15 min、30 min后IOP逐渐恢复到基线水平。巩膜外加压后角膜水肿、虹膜血管扩张。撤除巩膜外加压后角膜出现皱褶,虹膜血管仍然扩张。结论　球囊巩膜外加压后即刻IOP迅速增高,以后逐渐下降。随着加压时间的延长对侧眼的IOP有逐渐增高的趋势。球囊巩膜外加压可能影响大鼠的葡萄膜巩膜房水流出途径。     

Nitric oxide and oxidative stress in placental explant cultures

Placental explant culture, and cellular cytolysis and cellular differentiation have been previously studied. However, oxidative stress and nitric oxide profiles have not been evaluated in these systems. The aim of this study was to determine the release of lipid peroxidation and nitric oxide from placental explants cultured over a seven day period. Placental explants were maintained for seven days in culture and the medium was changed every 24 hours. The response was assessed in terms of syncytiotrophoblast differentiation (human chorionic gonadotropin, hCG), cellular cytolysis (lactate dehydrogenase, LDH), oxidative stress (thiobarbituric acid reactive substances, TBARS), and nitric oxide (NO). Levels of hCG increased progressively from day two to attain its highest level on days four and five after which it decreased gradually. In contrast, the levels of LDH, TBARS, and NO were elevated in the early days of placental culture when new syncytiotrophoblast from cytotrophoblast were forming and also in the last days of culture when tissue was declining. In conclusion, the levels of NO and lipid peroxidation follow a pattern similar to LDH and contrary to hCG. Future placental explant studies to evaluate oxidative stress and NO should consider the physiological changes inherent during the time of culture.     

An in vitro model to assess the peripheral vestibulotoxicity of aromatic solvents

Epidemiological and experimental studies indicate that a number of aromatic solvents widely used in the industry can affect hearing and balance following chronic exposure. Animal studies demonstrated that long-term exposure to aromatic solvents directly damages the auditory receptor within the inner ear: the cochlea. However, no information is available on their effect on the vestibular receptor, which shares many structural features with the cochlea and is also localized in inner ear. The aim of this study was to use an in vitro approach to assess and compare the vestibular toxicity of different aromatic solvents (toluene, ethylbenzene, styrene and ortho-, meta-, para-xylene), all of which have well known cochleotoxic properties. We used a three-dimensional culture model of rat utricles (“cysts”) with preserved functional sensory and secretory epithelia, and containing a potassium-rich (K⁺) endolymph-like fluid for this study. Variations in K⁺ concentrations in this model were considered as biomarkers of toxicity of the substances tested. After 72 h exposure, o-xylene, ethylbenzene and styrene decreased the K⁺ concentration by 78 %, 37 % and 28 %, respectively. O- xylene and styrene both caused histopathological alterations in secretory and sensory epithelial areas after 72 h exposure, whereas no anomalies were observed in ethylbenzene-exposed samples.These in vitro results suggest that some widely used aromatic solvents might have vestibulotoxic properties (o-xylene, styrene and ethylbenzene), whereas others may not (p-xylene, m-xylene, toluene). Our results also indicate that variations in endolymphatic K⁺ concentration may be a more sensitive marker of vestibular toxicity than histopathological events. Finally, this study suggests that cochleotoxic solvents might not be necessarily vestibulotoxic, and vice versa.     

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP⁺) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.     

Experimental 70% hydrofluoric acid burns: histological observations in an established human skin explants ex vivo model

Background: Hydrofluoric acid (HF) is particularly dangerous due to the potential for systemic effects and induction of severe skin necrosis through two mechanisms: corrosiveness and local tissue toxicity. In addition, because it is only partially dissociated (pK_a 3.2), it is capable of penetrating deeply into tissues. There is a lack of experimental studies that objectively characterize the behavior of HF diffusion into human skin, specifically the kinetics of tissue penetration resulting in severe cellular lesions.

Methodology/principal findings: We describe the cutaneous effects of HF using an established ex vivo human skin model. The diffusion of 70% HF starts within the first minute of contact at the epidermal surface and after 2?min reaches the basal layer. In the subsequent minute, the epidermis is destroyed and lesions appear in the papillary dermis after 4?min. Soon after, damage appears in the upper reticular dermis. Thus, 70% HF needs only 5?min of contact to completely penetrate human skin explants. This experiment is reproducible and corroborates previous studies and clinical effects reported in accidental HF exposures.

Conclusion/significance: This study shows that the management of HF chemical skin exposure is a question of minutes, especially for initial decontamination. These experimental observations could be useful for objectively comparing skin decontamination methods. Further studies should help to confirm these preliminary results.     

Expression of Calcium‐Sensing Receptor in Quail Granulosa Explants: A Key to Survival During Folliculogenesis

This study investigated the potential role of the calcium‐sensing receptor (CaR) in mediating survival of granulosa cells (GCs) in follicles of the Japanese quail (Coturnix coturnix japonica). Immunoreactivity of CaR was shown in GCs of quail preovulatory follicles as well as in the remnants of the GC layer after ovulation. Conversely, the CaR could not be detected by immunocytochemistry in the granulosa of smaller undifferentiated follicles. The presence of CaR in follicles destined to ovulate was confirmed by immunoblot and the receptor was identified as a protein of 115–125 kDa. Addition of different CaR agonists to granulosa explants in culture for 24 hr caused inhibition of apoptosis elicited by gonadotropin withdrawal on its own or in combination with C₈‐ceramide addition. Furthermore, R‐568, a specific, positive allosteric modulator of CaR, not only inhibited apoptosis but also increased GC number per viewing field in cultured granulosa explants. This observation could be attributed not to a rise in GC proliferation but to a more compact tissue structure, including a distinct distribution pattern of connexin‐43 gap junction proteins. Incubation in the presence of LY294002, a specific phosphatidylinositol‐3 kinase inhibitor, increased GC apoptosis, indicating that this pathway is involved in GC survival signaling. However, LY294002‐induced apoptosis was considerably attenuated by incubation with R‐568, indicating that other pathways might be major contributors to the survival mediated by CaR agonists. We provide direct evidence for the presence of CaR in preovulatory granulosa explants and suggest a pivotal role for CaR in follicle selection. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Different effects of pioglitazone and rosiglitazone on lipid metabolism in mouse cultured liver explants

Background

Pioglitazone (PIO) and rosiglitazone (ROSI) are widely used as oral antidiabetic agents for treatment of type 2 diabetes. Although these medications exert similar effects on blood glucose, recent clinical studies indicated that PIO has a more pronounced beneficial effect on lipid parameters than ROSI. In order to get further insight into the lipid effects of both drugs, we tested whether PIO, compared to ROSI, could exert direct effects on lipid liver metabolism in relation with plasma lipids.

Methods

We performed in vitro studies using mice liver slices incubated 21 h either with ROSI (1 µmol/L) or PIO (7.5 µmol/L).

Results

We showed that both glitazones slightly reduced HMG‐CoA reductase mRNA levels at the same degree but only PIO reduced intracellular cholesterol content, suggesting an alteration of cholesterol uptake rather than an inhibition of cholesterol biosynthesis. This concept was supported by the reduction of scavenger receptor class B type I expression, hepatic lipase activity and high‐density lipoprotein cholesterol uptake in PIO‐treated liver explants. Conversely, hepatic lipase mRNA levels were increased 3.5‐fold. ROSI, but not PIO, induced acetyl‐CoA carboxylase and fatty acid synthase gene expression and increased apoB secretion suggesting a stimulation of lipogenesis. Concurrently, peroxisome proliferator‐activated receptor‐γ mRNA levels were induced by ROSI and not significantly changed by PIO. Besides, PIO appeared to be a more potent activator of AMP‐Activated Protein Kinase than ROSI.

Conclusions

PIO and ROSI exert specific direct effects on liver and extrapolating these data to humans could explain the significant improvements in plasma lipids observed in diabetic patients treated with PIO. Copyright © 2010 John Wiley & Sons, Ltd.     

Alpha-dystroglycan interactions affect cerebellar granule neuron migration

The interaction of alpha-dystroglycan (a-DG) with its extracellular binding partners requires glycans attached to its mucin core domain, and defects in the glycosylation of a-DG are associated with both muscular dystrophy and neuronal migration defects. The involvement of a-DG and one of its ligands, agrin, in cerebellar neuronal migration was investigated. Antibodies directed against glycosylated a-DG inhibited granule neuron migration in cerebellar slice cultures. a-DG interactions did not appear to influence neurite outgrowth in cerebellar explant cultures, but enhanced granule neuron binding was observed on cells transfected with a-DG. These results suggest that interactions involving a-DG influence the strength of attachment of granule neurons to the a-DG-expressing Bergmann glial cells that guide granule neuron migration in the cerebellum. Experiments using anti-agrin antibodies suggest that agrin is not involved in these interactions.     

The effects of ciliary neurotrophic factor on the hypothalamo-pituitary gonadal axis in vitro in female rats

Ciliary neurotrophic factor (CNTF) is a member of the neuropoietic family of cytokines. CNTF exerts its actions through activation of a receptor complex, which shows similarity of sequence, second messenger systems and distribution to the leptin receptor. Leptin has been demonstrated to exert profound effects on the hypothalamo-pituitary gonadal axis. This study examines the in vitro effects of CNTF on hypothalamic luteinizing hormone releasing hormone release (LHRH) and pituitary luteinizing hormone (LH) release compared to those of leptin in the female. We report that CNTF stimulates LHRH release from medial basal hypothalamic explants harvested from proestrous female rats and this effect is of similar magnitude to that seen with leptin. In contrast, CNTF suppresses LHRH-stimulated LH release from dispersed anterior pituitary cells harvested from proestrous female rats but has no effect on basal LH release. Leptin stimulates basal LH release but has no effect on LHRH-stimulated LH release. The suppressive effect of CNTF on LHRH-stimulated LH release has been confirmed in perifused anterior hemipituitaries. These results suggest a differential effect of CNTF on the hypothalamo-pituitary gonadal axis and a possible role in the modulation of pituitary gonadal function.