Wheel running reduces ethanol seeking by increasing neuronal activation and reducing oligodendroglial/neuroinflammatory factors in the medial prefrontal cortex

The therapeutic effects of wheel running (WR) during abstinence on reinstatement of ethanol seeking behaviors in rats that self-administered ethanol only (ethanol drinking, ED) or ED with concurrent chronic intermittent ethanol vapor experience (CIE-ED) were investigated. Neuronal activation as well as oligodendroglial and neuroinflammatory factors were measured in the medial prefrontal cortex (mPFC) tissue to determine cellular correlates associated with enhanced ethanol seeking. CIE-ED rats demonstrated escalated and unregulated intake of ethanol and maintained higher drinking than ED rats during abstinence. CIE-ED rats were more resistant to extinction from ethanol self-administration, however, demonstrated similar ethanol seeking triggered by ethanol contextual cues compared to ED rats. Enhanced seeking was associated with reduced neuronal activation, and increased number of myelinating oligodendrocyte progenitors and PECAM-1 expression in the mPFC, indicating enhanced oligodendroglial and neuroinflammatory response during abstinence. WR during abstinence enhanced self-administration in ED rats, indicating a deprivation effect. WR reduced reinstatement of ethanol seeking in CIE-ED and ED rats, indicating protection against relapse. The reduced ethanol seeking was associated with enhanced neuronal activation, reduced number of myelinating oligodendrocyte progenitors, and reduced PECAM-1 expression. The current findings demonstrate a protective role of WR during abstinence in reducing ethanol seeking triggered by ethanol contextual cues and establish a role for oligodendroglia-neuroinflammatory response in ethanol seeking. Taken together, enhanced oligodendroglia-neuroinflammatory response during abstinence may contribute to brain trauma in chronic alcohol drinking subjects and be a risk factor for enhanced propensity for alcohol relapse.     

Toxicology of 3-epi-deoxynivalenol,a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC₅₀ values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F₁ mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.     

Cytogenesis in the adult monkey motor cortex: Perivascular NG2 cells are the major adult born cell type

We used confocal microscopy and immunohistochemistry (IHC) to look for new cells in the motor cortex of adult macaque monkeys that might form the cellular bases of improved brain function from exercise. Twenty‐four female Macaca fascicularis monkeys divided into groups by age (10–12 years, 15–17 years), postexercise survival periods, and controls, received 10 weekly injections of the thymidine analog, bromodeoxyuridine (BrdU) to mark new cells. Sixteen monkeys survived 15 weeks (5 weeks postexercise) and 8 monkeys survived 27 weeks (12 weeks postexercise) after initial BrdU injections. Additionally, five Macaca mulatta female monkeys (～5.5–7 years) received single injections of BrdU and survived 2 days, 2 weeks, and 6 weeks after BrdU injections. Neural and glial antibodies were used to identify new cell phenotypes and to look for changes in proportions of these cells with respect to time and experimental conditions. No BrdU⁺/DCx⁺ cells were found but about 7.5% of new cells were calretinin‐positive (Cr⁺). BrdU⁺/GABA⁺ (gamma‐aminobutyric acid) cells were also found but no new Cr⁺ or GABA⁺ cells colabeled with a mature neuron marker, NeuN or chondroitin sulfate antibody, NG2. The proportion of new cells that were NG2⁺ was about 85% for short and long survival monkeys of which two, newly described perivascular phenotypes (Pldv and Elu) and a small percentage of pericytes (2.5%) comprised 44% and 51% of the new NG2⁺ cells, respectively. Proportions of NG2⁺ phenotypes were affected by post‐BrdU survival periods, monkey age, and possibly a postexercise sedentary period but no direct effect of exercise was found. J. Comp. Neurol. 523:849–868, 2015. © 2014 Wiley Periodicals, Inc.     

rdentification of label-retaining cells in human gastric ：ancer xenograft in nude mice

Objective： This study has investigated the existence of label-retaining cell and its distribution in gastric cancer, in the hope that this information will assist investigations on gastric cancer stem cells. Methods： The gastric carcinoma cell line BGC-823 was labeled with BrdU in vitro and then engrafted into the right axilla of nude mice, which developed tumors. Label-retaining cells were quantified by immunohistochemical methods. Results： BrdU positive cells constituted about 96% of the cells in xenograft tumors after 10 days. Subsequently, BrdU positive cells gradually decreased, at the 80th day, label- retaining cells steadily occupied about 0.5%. This set of population cell localized in the margin of cancer nests, which had no difference in cellular morpha. Conclusion： The study demonstrates the presence of label-retaining cells in human gastric cancer xenografts in nude mice and the label-retaining cells may be related with cancer stem cells, which are most likely the cause for spread, metastasis and recurrence.     

In vivo hair growth-stimulating effect of medicinal plant extract on BALB/c nude mice

Abstract

Context: Chrysanthemum zawadskii var. latilobum (Asteraceae) (CZ) and Polygonum multiflorum Thunb. (Polygonaceae) (PM) have been used traditionally to treat different systemic diseases and acclaimed for various biological activities including hair growth.

Objective: This study investigates the hair restoration efficacy of selected medicinal plant extracts on nude mice.

Materials and methods: Nude mice genetically predisposed to pattern balding were used in this study. Topical methanol extracts of CZ and PM (10?mg/mouse/d) with standardized vehicle formulation, only vehicle (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v) and Minoxidil (2%) were applied daily for 40 consecutive days.

Results: In our study, the maximum hair score (2.5?±?0.29) was obtained in the CZ-treated group. Histological observation revealed a significant increase (p?<?0.001) in the number of hair follicles (HF) in CZ-treated mice (58.66?±?3.72) and Minoxidil-treated mice (40?±?2.71). Subsequently, immunohistochemical analysis also confirmed the follicular keratinocyte proliferation by detection of BrdU-labeling, S-phase cells in Minoxidil and CZ-treated mouse follicular bulb and outer root sheaths.

Conclusion: Our study revealed the underlying mechanism of stimulating hair growth in athymic nude mice by repair the nu/nu follicular keratin differentiation defect. Thus, the topical application of CZ may represent a novel strategy for the management and therapy of certain forms of alopecia.     

Automatic counting and positioning of 5-bromo-2-deoxyuridine (BrdU) positive cells in cortical layers of rat brain slices

5-Bromo-2-deoxyuridine (BrdU) staining is often used to evaluate cortical layer formation during mammalian brain development. This method allows the quantification of newly generated cells and therefore the study of the effects of xenobiotics or genetic factors on proliferation, cell death and migration behavior in a quantitative manner. However, these endpoints are generally assessed by time-consuming manual evaluation. In the present work, we introduce a novel procedure to identify and quantify BrdU⁺ cells within cortical layers, using the commercially available vHCS-Scan V.6.3.1 software to identify BrdU⁺ cell coordinates and the novel program ‘BrdeLuxe’ to define cortical layers and quantitatively assign BrdU⁺ cells to them. This procedure is compared to BrdU⁺ cell counting with the freeware ‘ImageJ’ in respect to the manual evaluation, all by two different researchers. BrdeLuxe shows high accuracy and precision for the determination of total number of BrdU⁺ cells compared to the manual counting, while ImageJ does not reach such results. Accuracy and precision are also higher for employing the BrdeLuxe program to evaluate the percentage of BrdU⁺ cells per brain layer compared to ImageJ. In terms of running time, BrdeLuxe is the fastest method of the three making it more suitable for multiple brain slices analyses.     

Reduction of gap junction protein connexin 32 in rat atrophic gastric mucosa as an early event in carcinogenesis

This study was conducted to examine the possible relationship among connexin 32 (Cx32) expression, cell proliferation and differentiation in the normal stomach, N-methyl-N′-nitro-nitrosoguanidine (MNNG)-induced atrophic gastritis, and carcinoma in rats. Atrophic gastritis and adenocarcinoma were induced by the administration of MNNG for 8 and 30 weeks, respectively. Cell proliferation was detected by staining with 5-bromo-2′-deoxyuridine (BrdU). The proliferative zone (BrdU-positive zone), located in the lower third of the gastric gland in controls, was elongated in atrophic gastritis. In adenocarcinoma, BrdU-positive cells were distributed diffusely. Cx32 expression was investigated by an indirect immunofluorecence method. In both control and atrophic gastritis specimens, Cx32 fluorescence was abundant in the surface epithelium, but was rarely detected in the glandular portion or the proliferative zone. The length of the Cx32-positive mucosa was significantly less than the control value in atrophic gastritis and no such positive mucosa was visible in adenocarcinoma. The results of this study indicate that the loss of cell-cell communication through the gap junction, associated with elongation of the proliferative cell zone, may be manifested much earlier than carcinoma. We regard this model as useful for investigating the development of atrophic gastritis into gastric carcinoma.     

Persistent impairment of hippocampal neurogenesis in young adult rats following early postnatal alcohol exposure

Background: Prenatal alcohol exposure can cause damage to the developing fetus with outcomes including growth deficiency, facial dysmorphology, brain damage, and cognitive and behavioral deficits. Smaller brains in children with FASD have been linked both with reduced cell proliferation in the developing CNS and with apoptotic cell loss of postmitotic neurons. Prenatal alcohol exposure in rodents during the period of brain development comparable to that of the first and second trimesters of human pregnancy persistently alters adult neurogenesis. Long‐term effects of alcohol exposure during the third trimester equivalent, which occurs postnatally in the rat, on adult neurogenesis have not been previously reported. The goal of this study was to examine the effect of postnatal binge‐like alcohol exposure on cell proliferation and neurogenesis in hippocampal dentate gyrus during adolescence and young adulthood. Methods: Male Long‐Evans rat pups were assigned to 3 groups: alcohol‐exposed (AE), sham‐intubated (SI) or suckle control (SC). AE pups received ethanol in a milk formula in a binge manner (2 feedings, 2 hours apart, total dose 5.25 g/kg/day) on postnatal days (PD) 4–9. BrdU was injected every other day on PD30–50. Animals were perfused either on PD50 to examine cytogenesis and neurogenesis in hippocampal dentate gyrus at the end of BrdU injections or on PD80 to evaluate new cell survival. Dorsal hippocampal sections were immunostained for BrdU, a marker for proliferating cells, Ki67, endogenous marker of proliferation, and NeuN, a marker for mature neurons. Results: Binge‐like alcohol exposure on PD4–9 significantly reduced the number of mature neurons in adult hippocampal dentate gyrus (DG) both on PD50 and PD80, without altering cumulative cytogenesis on PD50. In addition, the number of new neurons, that were generated between PD30 and 50, was further reduced after 30 days of survival in all 3 groups (SC, SI, and AE). Conclusions: These observations suggest that early postnatal binge alcohol exposure results in long‐term deficits of adult hippocampal neurogenesis, providing a potential basis for the deficits of hippocampus‐dependent behaviors reported for this model.     

Cell-kinetic analysis of pancreatic cancers in syrian golden hamsters

Cell-kinetic analysis of pancreatic cancers using bromodeoxyuridine (BrdU) was performed on four transplantable pancreatic ductal adenocarcinomas, which were experimentally induced by givingN-nitrosobis (2-hydroxypropyl) amine (BHP) to Syrian golden hamsters. The volume-doubling time (Td)of the tumor was 0.43, 1.03, 2.30, and 3.50 d for tumor lines J, D, B, and F, respectively. In the pulse-labeling study, the labeling index was 15.8, 23.3, 27.9, and 31.7, respectively. The most rapidly growing tumor line, J, unexpectedly showed the lowest BrdU-labeling index (LI). The further study of tumor using the cumulative-labeling method revealed that the duration of S phase (Ts), the generation time (Tg) and the growth fraction (GF) were 2 h, 14 h, and 90% for tumor J and 12 h, 37 h, and 85% for slowly growing tumor B, respectively. Cells of limited life span (CLLS) of these lines did not differ from each other. These results conclude that the marked difference ofTg between tumor lines J and B resulted in a distinction of their growth rates. The disagreement ofTs/Tg also lead to a reversion of LI between the two lines. Furthermore, LI of BrdU with one-point pulse labeling did not seem to be a sufficient indicator of the growth characteristics, at least, in the present experiment.     

Time course of mucosal cell proliferation following acute aspirin injury in rat stomach

The time course of replicating cell proliferation in the gastric fundic mucosa following acute aspirin-induced injury was determined by BrdU labeling. Gastric erosions were produced in adult rats by gastric gavage using aspirin (200 mg/kg) suspended in 0.15 M HCl. Lesion scores indicated significant gross injury in the aspirin-treated rats at all times measured (from 2 to 48 hr). BrdU labeling was not elevated at 2 or 8 hr after gavage. A significant increase in labeling was observed at 15 hr, reached a maximum at 16 hr, and declined with a slight, but significant increase still present at 48 hr. Elevations in BrdU labeling were uniform and seen in areas adjacent to and distant from the gross injury. The BrdU labeling in the fasted control rats decreased during this same time period. The height of the proliferative zone was not altered from control in the aspirin-treated rats despite the marked differences in proliferation activity. This study demonstrates the importance of the time course in the assessment of mucosal cell proliferation following injury.