No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification.Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited  Despite stringent governmental regulations, lead exposure continues to be a major public health problem, with blood levels being elevated in a bimodal distribution predominantly affecting young children 1–5 years of age and individuals > 50 years of age . The CenIn addition to children, many adults maintain elevated blood levels due to past occupational or environmental exposure. With the reduction of Pb in fuel and soldered cans as well as increased awareness and vigilance, acute environmental Pb exposure has decreased dramatically . HoweverIn vivo models have demonstrated a decrease in bone density with Pb exposure , for intramembranous ossification, and undifferentiated mesenchymal cells, for endochondral bone formation . Fracturinvolved .Given the known effects on cells involved in bone formation and remodeling and the absence of literature on Pb in skeletal repair, the purpose of the present study was to evaluate the effects of elevated blood Pb levels on fracture healing. We employed an established murine tibia fracture model  to charaAll Pb solutions were made using Pb acetate  dissolved in distilled water. All mice had unrestricted access to the normal rodent diet supplied by the vivarium staff, and drinking water was replenished at least one time per week by the investigative staff to ensure continuous Pb exposure. All Pb waste solutions were disposed of appropriately. Mice were housed in groups of ≤5 animals per microisolator cage in the vivarium and maintained on a 12-hr light/dark cycle. All procedures were carried out in accordance with the regulations of and following the approval of the University of Rochester Animal Use and Care Committee.n = 32) at 6 weeks of age were divided into eight groups of four mice each. Each group was exposed to one of the following doses: 0, 55, 230, 580, 1,160, 1,750, 2,300, or 5,800 ppm Pb in the drinking water. This preliminary exposure was carried out to determine the whole-blood Pb (BPb) concentrations needed to approximate environmentally relevant levels of human exposure. This cohort is referred to as group A. Pb exposures of 0, 55, and 230 ppm Pb in the drinking water were selected as most environmentally relevant. A second cohort (group B) of female C57/Bl6 mice (n = 54) was divided into three groups of 18 animals and housed under conditions as outlined above. All experiments related to the analysis of the fracture healing process were performed with group B except one, which documented chronic nonunions in mice exposed to 2,300 ppm Pb.Female C57/Bl6 mice . This method was adapted by Parsons from the Department of Environmental Health and Toxicology, SUNY at Albany . All ins3, and the total volume was brought to 10 mL with distilled water and analyzed.After 6 weeks, four animals from each of the 0, 55, 230, 2,300, and 5,800 ppm Pb groups were sacrificed. The femora and tibiae were removed, the epiphyses and soft tissues were discarded, and the bone marrow was flushed out with a 25-gauge 5/8-inch needle . The remaining diaphy-seal bones were washed with phosphate-buffered saline (PBS) to remove all marrow elements and blood cells. The bones were then processed as described previously . Briefly6 cells per 6-cm plate for nodule formation assays, and medium was changed every 3 days. After 17 days in culture, triplicate cultures were fixed with 10% formalin, rinsed in distilled water, and stained by von Kossa’s method . Total nodular area was quantified by histomorphometry.Group B animals were sacrificed after 6 weeks of Pb exposure. The femora and unfractured tibiae were excised, and the soft tissues were removed. Primary bone marrow cells were isolated and prepared as described previously . Bone maOsteoclast progenitor cells (OCPs) were prepared from splenocytes as described previously . Animals5 cells/well in a 96-well plate in α-MEM supplemented with macrophage-colony–stimulating factor  and receptor activator nuclear factor κB ligand . Fifty percent medium was added the next day, and medium was changed every other day thereafter. Cultures were incubated at 37°C for 6–7 days and then fixed and stained for tartrate-resistant acid phosphatase (TRAP) using the leukocyte acid phosphatase kit (Sigma). The number of TRAP+ multinucleated cells was then counted to quantify osteoclast formation as described previously  supplemented with M-CSF (30 ng/mL) for 12 days. Individual colonies, defined as > 40 cells, were then quantified under an inverted microscope. The total number of colonies represents the original number of monocyte/macrophage and osteoclast precursors , RANKL (100 ng/mL), 1% l-glutamine, 1% penicillin/streptomycin, and 1% nonessential amino acids (Gibco). Fifty percent medium was added the next day, and medium was changed every other day thereafter. After 12 days, the wafers were scraped, dried, stained with toluidine blue, and examined under a 40× objective. Wafer images were captured, and resorption pits on the wafer surface were traced to determine the total pitted area using Osteometrics software  as described previously  was placed into the tibia. The pin was trimmed proximally at the level of the tibial plateau. The wound was then closed with 4–0 nylon sutures. The left tibiae were placed in a modification of the guillotine three point-bending device described by Fractured tibiae were harvested at 7, 14, and 21 days. The tissues were fixed in 10% formalin for 24 hr and decalcified in 10% EDTA at pH 7.2 for 2 weeks. Samples were then processed, embedded in paraffin, and cut in 3-μm sagittal sections. Three contiguous sections (100 μm apart) for each specimen were stained with Alcian blue and counterstained with hematoxylin, orange G, and eosin (ABH/OG) as described previously . Additio+ osteoclasts was then expressed per total area of fracture callus.Quantitative analysis was performed on the sagittal ABH/OG- and TRAP-stained sections by histomorphometric analysis using Osteometrics software. Bone and cartilage formation was quantified on ABH/OG-stained slides by outlining the perimeter of the fracture callus under the 2× objective. Areas of new woven bone and cartilage were then traced. This procedure was repeated until the entire fracture callus had been evaluated. The areas of woven bone, cartilage, and total fracture callus were obtained directly in square millimeters from the software. The amount of mesenchymal tissue was then calculated by subtracting bone and cartilage area from the total callus area. Bone, cartilage, and mesenchymal tissue areas each were expressed as a percentage of total fracture callus. Osteoclast quantification was performed on the TRAP-stained slides. Using the 10× objective, the perimeter of fracture callus was outlined and individual TRAP positive osteoclasts were identified and counted. The entire fracture callus was evaluated. The number of TRAP35S-labeled sense and anti-sense riboprobes as described previously , and collagen type X (Col X) were used to synthesize eviously . Cut seceviously . Emulsiot-test and analysis of variance (ANOVA) where appropriate. A value of p ≤0.05 was accepted as statistically significant.Data are expressed as mean ± SEM. Statistical significance was determined using the Student in vivo. Additionally, mice appear to be very tolerant to these Pb exposures. No gross physical or behavioral changes were noted in animals with BPb levels three to four times greater than a lethal human exposure, in preliminary experiments. There was no statistically significant difference by ANOVA testing in body weight among the various dose groups in unpublished preliminary experiments (data not shown).We established a reproducible murine Pb dosing protocol by introducing various concentrations of Pb into the drinking water of group A animals and determining BPb levels, similar to our rat exposure protocol . We noteBy 14 days after fracture, all animals showed radiographic evidence of fracture callus formation . HoweverConsistent with our radiographic findings, histologic analysis revealed no remarkable differences at 7 and 21 days because all fracture sites consisted of undifferentiated mesenchyme and fibrocartilage at the early time point and remodeling bone at the latter time point . In our In situ hybridization and TRAP staining in the 14-day fracture group helped assess phenotypic gene expression and quantify osteoclasts in the fracture callus, respectively  were exposed to 2,300 ppm Pb and analyzed radiographically and histologically. In contrast to the lower-dose treatment groups (group B), the radiolucency in the day-14 X rays was not accompanied by surrounding fracture callus  healed. Because it is well known that mice have an extremely robust ability to heal fractures, we speculate that mice healed fractures at Pb exposures at which humans could not.In this study, remarkable effects of Pb on fracture healing were clearly apparent, even at the lowest dose –5. The pin vitro studies (in situ osteoclast numbers (in vivo Pb exposure renders this possibility less likely.Although the precise mechanism by which Pb inhibits fracture-healing remains a topic for future investigation, the phenotype is very reproducible and is somewhat reminiscent of phenotypes described in other mouse models. The increased chondrogenesis observed is similar to that seen in the fracture callus of parathyroid hormone and prostaglandin-treated mice, indicating that Pb may be an agonist of protein kinase A signaling in chondrocytes, as predicted in our  studies . The lit studies . The sec studies . The thi studies , which i studies . An addi numbers , 5 and O numbers  are unafThe overall clinical significance of Pb inhibition of fracture healing relates to persons with osteoporosis. We have argued that because of the high environmental Pb exposures from the 1940s to 1960s, women currently going through menopause are at an additional risk of osteoporosis . It is n


The aim of this study was to investigate the effects of prenatal alcohol exposure on radial-maze learning and hippocampal neuroanatomy, particularly the sizes of the intra- and infrapyramidal mossy fiber (IIPMF) terminal fields, in three inbred strains of mice .Although we anticipated a modification of both learning and IIPMF sizes, no such effects were detected. Prenatal alcohol exposure did, however, interfere with reproduction in C57BL/6J animals and decrease body and brain weight (in interaction with the genotype) at adult age.Prenatal alcohol exposure influenced neither radial maze performance nor the sizes of the IIPMF terminal fields. We believe that future research should be pointed either at different targets when using mouse models for Fetal Alcohol Syndrome  or involve different animal models. In 1968, Lemoine et al. published a paper, in French, that described "des anomalies dans les infants de parents alcooliques"  followedSince then literally thousands of studies have been done and substantial advances in the understanding of FAS have been made, not in the least because of animal models, among them the mouse ,5. TheseThe aim of this study was to determine whether different genotypes with varying hippocampal anatomy are oppositely affected by similar exposures to prenatal alcohol. To this end, pregnant females of three highly inbred mouse strains, C57BL/6J, BALB/cJ, and DBA/2J, which vary in hippocampal anatomy e.g. ,10), wer, wer10])2 = 9.5, df = 2; p < 0.01).Table F2,96 = 5.0; p < 0.01). C57BL/6J made fewer errors than BALB/c  and DBA/2 . Treatment had no effect, neither alone, nor in interaction with strain. Figure F2,96 = 13.9; p < 0.001) with again C57BL/6 males differing from the other two strains .Figure F2,96 = 104.3; p < 0.001; brain weight: F2,91 = 86.1; p < 0.001). BALB/c weighed more than C57BL/6  and DBA/2 . The latter strain, in turn, weighed less than C57BL/6 . C57BL/6 males had higher brain weights than BALB/c  and DBA/2 . In addition, BALB/c had higher brain weights than DBA/2 . Prenatal exposure to alcohol affected both variables. Body weights were affected independently of the origin of the genotype  with mice exposed to ethanol prenatally weighing less than control  and pair-fed  animals. The effect on brain weight depended on the background of the strain . This interaction effect was mainly caused by the BALB/c strain where prenatal alcohol decreased brain weight , whereas no significant effects were seen in the other two strains.Body and brain weights are presented in Figures F2,73 = 8.0; p < 0.001), stratum lacunosum-moleculare , stratum radiatum , suprapyramidal MF  and IIPMF . DBA/2 showed a smaller regio inferior than C57BL/6  and BALB/c . C57BL/6 showed a larger stratum lacunosum-moleculare than DBA/2  and BALB/c  while BALB/c males had a larger stratum lacunosum-moleculare than DBA/2 males . C57BL/6 exhibited smaller stratum radiatum and suprapyramidal MF than DBA/2  and BALB/c . C57BL/6 showed larger IIPMF sizes than DBA/2  and BALB/c . BALB/c had larger IIPMF sizes than DBA/2 .The results of the hippocampal data are presented in Table Prenatal alcohol exposure did not affect any of the hippocampal variables, neither as main factor, nor in interaction with the background.These data demonstrate that, in three inbred strains of mice , prenatal exposure to alcohol does neither affect spatial memory nor hippocampal neuroanatomy. Prenatal alcohol exposure did influence body and brain weight in some strains and dramatically reduced live birth rates in C57BL/6 animals.A first caveat is, of course, the observation that the low number (2) of C57BL/6 pups prenatally exposed to ethanol precludes any strong conclusions for that strain. We still decided to include these animals in the present report because simple visual inspection of the data shows that even for these two lone survivors there is not even a trend towards any differences in behavior or hippocampal morphology, just as is the case for the other two strains. Of course, sample sizes are much more adequate for strains BALB/c and DBA/2, so that the conclusions based on those strains are much stronger.In short, our results on both behavior and neuroanatomy appear not to be in line with those, for instance, summarized in Berman and Hannigan , who conAnother explanation for the lack of an effect on learning might be that this type of radial maze is not sufficiently demanding and that other spatial learning tests would be more appropriate to detect prenatal alcohol effects. However, Wainwright et al.  did not Another striking result of this study is the low delivery rate of C57BL/6J females. Out of 13 pregnancies only one female gave birth to a viable litter. The other twelve pregnancies resulted in still-born pups, or the mother died while giving birth, or the pups were eaten by their mothers immediately after birth. Whether this finding reflects a higher sensitivity in developing C57BL/6J embryos to alcohol or higher blood alcohol concentrations in the mother or a combination of both cannot be inferred from these experiments. Although the only litter born might not be a representative sample, males from this litter appeared to perform equally well in the radial maze as their pair-fed and control counterparts; neither were the sizes of the IIPMF terminal fields affected.Summarizing, in this experimental design, which used three distinct inbred strains of mice, prenatal alcohol exposure influenced neither radial maze performance nor the sizes of the IIPMF terminal fields. We believe that future research should be pointed either at different targets when using mouse models for FAS  or involve different animal models, including zebrafish  or guineSubjects were male mice from the inbred strains C57BL/6J, BALB/cJ, and DBA/2J that are known to differ in their sensitivity to ethanol (e.g. ). The miEight days before mating, female mice of all three stains were habituated to alcohol solutions as their only source of liquid . Females were then mated with experienced males from the same strain and, throughout gestation, exposed to a 12% ethanol solution as their only source of liquid. Two control groups were included: first, a pair-fed group which consumed an isocaloric solution of dextrin (Amisol) and a same amount of food as consumed by the alcohol-exposed dams and, second, a control group which received food and tap water ad libitum. Although food and liquid consumption were not specifically measured, no obvious differences in consumption were noticeable. It has been shown that voluntary drinking paradigms lead to significantly elevated blood alcohol levels in pregnant females . PregnanAt the age of 3–4 months animals were tested in the radial maze. The 8-arm radial maze used in these experiments was similar to the original one used by Schwegler and Crusio . TheTwenty-four hours prior to the experiment animals were moved to the test room. Animals were habituated for 1 day and subsequently trained for 5 days. The habituation consisted of a 15-min exploration trial with free access to all arms but without a food reward. Immediately afterwards companion females were removed from the home cages and all experimental animals, now single housed, were deprived of food. During the training sessions animals were weighed daily and kept at 80–90% of their original body weight. In between sessions the maze was cleaned with a dry cloth. On the first two days, trials were terminated after 15 minutes or after the animal had eaten all rewards, whichever came first. Thereafter, no time limit was imposed and trials were terminated when animals had found all 8 food rewards. The situation of animals not eating all rewards occurred frequently on the first two days, but never on days 3 to 5. For this reason, data from days 1 and 2 were not included in the analyses. Previous experiments have shown that significant learning occurs very rapidly and that strain or mutational effects can reliably be shown with this method Two variables were sampled, one representing learning performance and the other running speed. Learning was measured by the number of errors while activity was depicted as the mean distance traveled (cm) per second. An error was counted if an animal entered an arm previously visited or did not eat the reward. Average running speed was estimated by dividing the distance traveled by the amount of time needed to complete a trial.Histological treatments were performed as previously described by Schwegler and Lipp  see alsoMethods used for visualization and measurements of the hippocampal terminal fields were similar to those described previously (e.g. ). Sampli2-tests the number of breeding pairs used (pairings), pregnancies, and births were compared for each strain. Pregnancies were compared relative to the number of pairings, and births relative to the number of pregnancies. The radial maze data of days 3 to 5 were analyzed using two-way repeated measures ANOVAs with strain and treatment as main factors. Both between subjects factors consisted of three levels . The hippocampal data were analyzed by means of two-way ANOVAs, both factors being identical to the above-mentioned analysis. When necessary, pair-wise comparisons were made using least square means. All ANOVAs were performed using the SAS GLM procedure.Using χFS participated in the interpretation of the data and drafted the manuscript, LJ carried out the radial maze tests and performed the histology, JYB participated in the design of the study and carried out the morphometrical analyses, and WEC conceived of the study, participated in its design, carried out the statistical analyses, and participated in the interpretation of data and drafting of the manuscript. All authors read and approved the final manuscript.


Male C57BL/65 mice received a basal diet supplemented with 4% soya-bean oil, linseed oil or fish oil, in which the major polyunsaturated fatty acids were linoleic acid, alpha-linolenic acid and long chain omega-3 fatty acids, respectively. Groups of animals were injected into the right flank with EL4-lymphoma cells, others with thymoma cells. Tumour implantation caused a gradual decrease in food consumption with both types of tumour, while body weight increased, especially in the EL4-bearing animals receiving the soya-bean diet. The weight gain was due to body water accumulation and was accompanied by decreases in body fat and minor changes in carcass protein and ash contents. The dietary treatments did not produce significant differences in tumour incidence and mortality, but tumour size was decreased by diets supplying omega-3 fatty acids: in the EL4 mice tumour weight was markedly depressed by linseed oil, compared to soya-bean oil, whereas thymoma tumour weight was lowest in mice receiving fish oil and highest in the soya-bean oil group. Both types of tumour caused pronounced hypoglycaemia and hyperinsulinaemia in the hosts, and the effect was modulated by the diets in the EL4 but not in the thymoma animals: the plasma glucose level was especially low in the linseed oil group and relatively highest in the soya-bean oil treatment. The degree of hyperinsulinaemia depended on the diet only in the thymoma-bearing mice, with linseed and fish oils producing higher insulin levels than soya-bean oil. A slight hyperinsulinaemia was also observed in linseed and fish oil-fed control mice. Serum triglycerides were elevated in tumour-bearing animals, without consistent differences between dietary treatments. Although no clear pattern emerged concerning total cholesterol and LDL levels, HDL values were strongly affected by the type of oil: in the control animals linseed oil caused an increase in HDL-cholesterol compared to the other two oils. The thymoma-bearing mice responded to the linseed and fish oil diets with greatly elevated HDL-cholesterol levels. The results point to important differences in the responses of the two implanted tumours and hosts not only to the omega-6 and omega-3 fatty acids, but also to the type of dietary omega-3 fatty acids, namely alpha-linolenic acid and long chain fish oil polyunsaturated fatty acids.


Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blue-berry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects.The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from  Plants synthesize an array of chemical compounds that are not involved in their primary metabolism. These ‘secondary compounds’ instead serve a variety of ecological functions, ultimately to enhance the plant's survival during stress . In addiCaenorhabditis elegans, which has become a popular model for studying aging and longevity, due to its short 2- to 3-week lifespan, rapid generation time and experimental flexibility -enriched fraction from BB, could prolong adult lifespan and delay aging in Vaccinium angustifolium) or a C18 column fraction containing their bulk polyphenols, mean lifespan of wild-type animals was lengthened by 28% . Similar results were obtained using a crude extract of a different species of blueberry (Vaccinium asheii) (not shown).Adult wild-type animals grown under our standard laboratory conditions at 25 °C have a mean lifespan of 12.7 days and average maximum lifespan of 19.7 days. On media containing either crude BB extract . However, treatment with the PAC-enriched fraction increased lifespan to a similar extent as the starting BB polyphenol mixture or the remixed fractions , proanthocyanidins (PAC) or hydroxycinnamic esters, mainly chlorogenic acid (CA). Major components of each of these fractions have been shown to confer significant antioxidant activity and ATC can protect cells against oxidative stress in vitro . To deteongevity   animals, which have a mutation in the cytochrome b large subunit of mitochondrial complex II (mev-1(kn1) animals . Together, these gene expression analyses show that BB treatment did not cause a general induction of stress-response genes under normal growth conditions, but appeared to increase overall health, which may have promoted greater survival under thermal stress. However, we cannot rule out the possibility that BB polyphenol treatment may increase expression levels of stress-response genes during old age.We next considered the possibility that BB treatment acted as a mild stressor that induced expression of protective enzymes and this increased gene expression delayed aging  animals which lacked sir-2.1 gene activity, showing that BB polyphenols and resveratrol did not act through the same mechanism to increase C. elegans lifespan  animals, suggesting that BB polyphenols might act through osr-1 (sek-1(ag1) or unc-43(n1186) animals  and skn-1(zu67) animals, showing that BB may act independently of these genes (daf-16(mgDf50) animals . Interestingly, BB and ampicillin had additive effects on daf-16(mgDf50) lifespan, further supporting the conclusion that BB polyphenols do not act through simple antimicrobial effects  mutation, consistent with our finding that BB also did not protect wild-type animals against oxidative stress from paraquat or hydrogen peroxide treatments. In addition, we found that three stress-response pathways were dispensable for the beneficial effects of BB polyphenols upon lifespan. These pathways were represented by: (i) sir-2.1, which promotes longevity during calorie restriction, (ii) daf-16, which promotes longevity and stress resistance, and (iii) skn-1, which promotes oxidative stress resistance. Specifically, BB treatment was able to extend lifespan of animals lacking any of these three genes. In contrast, BB treatment did not prolong lifespan of animals with defects in the osr-1/unc-43/sek-1 pathway that promotes resistance to osmotic stress. One interpretation of this latter finding is that the effects of BB treatment are mediated, at least in part, through these genes. Alternatively, mutations in the osr-1 pathway could place animals under some stress that is not affected by BB treatment. To date, osr-1 pathway mutants have not been thought to suffer from different causes of death than wild-type animals, suggesting that the latter explanation is less likely. Therefore, we propose that BB exerts beneficial effects through interactions with the osr-1 pathway.By using in vitro. These findings may either show that (i) the antioxidant effects of these compounds in vitro are not relevant to their in vivo effects in this model, or (ii) differential bioavailability of these compounds is a major determinant of their effects in whole animals. Due to the structural diversity in these fractions, we did not attempt to directly measure the levels of polyphenols inside the animals. However, we did observe the accumulation of BB pigments (primarily ATC) inside the intestines of treated animals, leading us to conclude that components of BB extracts could enter the body of these animals. Our analysis showed that BB did not protect animals against oxidative stress from paraquat, hydrogen peroxide or genetic mutation [mev-1(kn1)], which increases superoxide production in vivo ; GR1307, daf-16(mgDf50); TK22, mev-1(kn1); VC199, sir-2.1(ok434); EU1, skn-1(zu67)/nT1; AM1, osr-1(rm1); AU1, sek-1(ag1); and MT2605, unc-43(n498n1186). Before analysis, the sir-2.1(ok434) strain was backcrossed twice against wild type.All strains were maintained at 15 °C on nematode growth medium (NGM) as described . StrainsVaccinium angustifolium) was applied to a preconditioned C18 column . The C18 column was washed with water to remove fructose, glucose and organic acids, which are abundant in blueberries, then with 100% methanol to obtain the total polyphenolic fraction. Methanol was removed under vacuum using a rotary evaporator  at 30 °C. To obtain the proanthocyanidin fraction, the total polyphenol fraction was dissolved in 50% ethanol and applied to a column of Sephadex LH20 , preconditioned with 50% ethanol. The column was washed with 60% ethanol until the eluant was colorless, and then with 70% aqueous acetone to elute the blueberry proanthocyanidins. To obtain the anthocyanin fraction, the total polyphenol fraction was dissolved in acidified water and washed four times with three volumes of ethyl acetate. Anthocyanins were partitioned into the water fraction, which was subsequently freeze dried. Pure chlorogenic acid  was used instead of blueberry chlorogenic acid since the chlorogenic acid fraction obtained from blueberry fractionation is contaminated with minor flavonol, and flavonol glycoside components. These components are all contained in the ethyl acetate fraction obtained during anthocyanin isolation.Commercially prepared single strength wild blueberry juice (fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the nonpermissive temperature for fertility of fem-1(hc17). Lifespan scoring was initiated after hermaphrodites completed the final larval molt, on the first day of adulthood. For aging assays with BB extracts, treatments were added to NGM agar plates on the first day of the lifespan assay. For lifespan assays with fertile strains, hermaphrodites were transferred daily for the first 4 days of adulthood to avoid progeny overgrowth. In these cases, all treatment plates were prepared on day 0 of adulthood. Statistical analyses and survival plots of lifespan data were performed with JMP analysis software . Pharynx pumping rates were scored on adults at room temperature (24 °C) under a Nikon SMZ1500 stereomicroscope .For aging assays, synchronous populations were obtained by allowing 5–10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17) hermaphrodites at 25 °C as for aging assays, except paraquat was added to NGM medium to 10 mm final concentration . For hydrogen peroxide, we scored 5-h survival of adult day 5 fem-1(hc17) animals in S-basal medium with indicated concentrations of hydrogen peroxide.Thermotolerance assays were performed with hermaphrodites on adult day 5, after the majority of egg-laying had ceased. Animals were transferred onto 3-cm NGM agar plates supplemented as indicated and then incubated at 35 °C for 16 h. Survival was scored as the number of animals responsive to gentle touch as a fraction of the original number of animals on the plate. Animals that had died from dessication on the sides of the plate were censored. Paraquat-induced oxidative stress assays were performed with To determine lipofuscin levels, adult hermaphrodites were anesthetized in 0.2% sodium azide and mounted on 2% agarose pads for visualization of intestinal fluorescence on a Nikon E800 microscope using an Endow GFP filter with a mercury UV source (Nikon). Images were captured using a constant exposure time with a Hamamatsu ORCA digital CCD camera  using OpenLab software . Lipofuscin levels were measured using ImageJ software (NIH Image) by determining average pixel intensity in each animal's intestine.To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase , as described . Fixed afem-1(hc17) animals were grown at an approximate density of 200 animals/plate in the presence or absence of 200 µg mL−1 BB polyphenols. Animals were kept at 25 °C except for heat-stressed samples, which were transferred to 35 °C for 2 h before collection and processing. Animals were washed from plates with cold M9 buffer into Eppendorf tubes, and allowed to settle on ice. The worm pellet was resuspended in 300 µL Absolutely RNA lysis buffer  and stored frozen at −80 °C. RNA was prepared as per kit instructions. Complementary DNA was prepared with the ProStar Ultra HF RT-PCR kit (Stratagene). Real-time PCR was performed in an MJ Research Opticon thermal cycler , using SybrGreen 2x master mix  with 1 µm primers and 0.5 µL cDNA in a 25-µL reaction volume using the following gene-specific primers (T04C12.6), GTGTGACGACGAGGTTGCCGCTCTTGTTGTAGAC (F) and GGTAAGGATCTTCATGAGGTAATCAGTAAGATCAC (R); hsp-12.6(F38E11.2), ATGATGAGCGTTCCAGTGATGGCTGACG (F) and TTAATGCATTTTTCTTGCTTCAATGTGAAGAATTCC (R); hsp-16.1(T27E4.8), GTCACTTTACCACTATTTCCGTCCAGCTCATCAACGTTC (F) and CAACGGGCGCTTGCTGAATTGGAATAGATCTTCC (R); hsp-16.49(Y46H3 A2), GCTCATGCTCCGTTCTCCATATTCTGATTCAAATGC (F) and GCAACAAAATTGATCGGAATAGAACGTGATGAG (R); and hsp-70(F44E5.4), CGTTTCGAAGAACTGTGTGCTGATCTATTCCGG (F) and TTAATCA ACTTCCTCAACAGTAGGTCCTTGTGG (R).For reverse transcriptase PCR (RT-PCR) analysis of gene expression,


The role of circulating complement in host defense and immune disease is well established. Although a number of cells and tissues are capable of synthesizing complement components locally, the importance of such local synthesis in immune disease has been difficult to establish.We used bone marrow transplantation (BMT) between C3 knockout (C3KO) and wild type (WT) mice to construct animals that were discordant for systemic (hepatic) and local (monocytic) C3 synthetic capacity. An immune complex glomerulonephritis (GN) was then induced using intraperitoneal injections of horse spleen apoferritin (HSA) with a lipopolysaccharide (LPS) adjuvant. All HSA/LPS animals developed a proliferative GN with glomerular infiltration by monocytes. By sensitive ELISA, monocyte C3 synthesis could be detected in C3KO animals transplanted with WT bone marrow cells. Despite this, there were no significant differences among groups of mice in measures of clinical  or histologic  disease severity.In this model of GN, local synthesis of C3 by infiltrating cells does not appear to be of pathologic importance. The complement proteins are major effectors of inflammation in glomerulonephritis, both in humans and in animal models. The bulk of complement deposited in the glomerulus in acute glomerulonephritis is presumed to come from the circulation, and is synthesized in the liver. This constitutes “systemic” complement synthesis.A variety of other cells, however, are capable of synthesizing complement components. Most of these are epithelial; we have shown that renal proximal tubule epithelial cells (PTEC) synthesize a variety of components in humans and mice Many glomerulopathies are characterized by a mononuclear cell infiltrate. It has been suggested that complement synthesized by these infiltrating cells could be an important mediator of glomerular injury interstitiumglomerulus has not been addressed. In the experiments reported herein, we employ a novel strategy by which animals deficient in C3 undergo hematologic reconstitution with bone marrow from C3 replete animals. This strategy makes it possible to separate the contribution of systemic (liver-derived) complement from that potentially produced by monocytes.Although we have reported several studies addressing the role of locally synthesized complement by tubular epithelium in the renal C57BL/6 mice, either wild type (WT) or homozygous C3 knockout (C3KO), were purchased from The Jackson Labs . The protocol was reviewed and approved by the Committee for the Humane Use of Animals at SUNY Upstate Medical University.4CL, 10 mM KHCO3 and 0.1 mM EDTA, pH 7.35). The BMC were washed, resuspended in DMEM, counted, and injected intravenously via a tail vein using a 26-gauge needle.Eight week old male mice were irradiated with 11 Gray, a lethal dose which is split into two half doses delivered four hours apart. Such a dose regime avoids a substantial amount of gut induced radiation disease, and minimizes gastric distress. To prevent death from nasal pseudomonas, twenty-four hours prior to, and for one week after irradiation, the animals were given tetracycline . On the day following irradiation, each animal received approximately four million bone marrow cells (BMC) that had been harvested by flushing the marrow cavity of femurs and tibiae of donor WT or C3KO mice with DMEM  supplemented with 1% fetal calf serum . The BMC were washed several times with PBS and then contaminating red blood cells were lysed with AKC buffer  were delivered by the same route three days per week as adjuvant. Each group of transplanted animals was composed of six male mice who were approximately 12 weeks old at the time of induction of glomerulonephritis. Additional groups of six WT and six C3KO animals  served as vehicle control groups and received 0.2 ml injections of 150 mM NaCl, IP, five days per week. All six groups of mice were injected by this protocol for a total of six weeks.The irradiated and transplanted animals were allowed to recover for 4 weeks, at which time immune complex glomerulonephritis was induced using a method we have described previously 2 narcosis. For 24 hours prior to euthanasia, they were placed in metabolic cages for collection of urine. Immediately after death, blood was obtained by vena cava puncture, and tissue was obtained from kidney and spleen. Some tissue samples were place in 10% buffered formalin. American Histolabs, Inc., , performed embedding, sectioning and staining with either hematoxylin and eosin, or periodic acid-Schiff (PAS) stain for histology. Additional tissue samples were quick frozen in M-1 embedding matrix  for immunohistochemistry.Nine weeks after starting injection, animals were euthanized by COA series of digital images representing the complete cortex from a periodic acid-Schiff-stained kidney section from each animal was collected with a SPOT Diagnostics camera , as we have previously described ▪ Glomerular cellularity, defined as the total cells in ten representative glomerular cross sections.▪ Glomerular crescents, defined as two or more cell layers surrounding the glomerular tuft, in 50 glomeruli.o-phenylenediamine in PBS-glycerol.Frozen sections of kidneys were thawed slowly and washed with PBS, followed by incubation with either 10% normal goat serum (for C3c slides) or 10% normal rat serum (for CD14 slides) for 30 minutes at 37°C. Excess serum was blotted and the slides were incubated for one hour at 37°C with either goat anti-mouse C3c with a FITC label , or rat anti-mouse CD14 , both antibodies diluted in PBS. Slides were again washed with PBS, and the CD14 slides were incubated with goat anti-rat IgG–FITC  for an additional 30 minutes at 37°C. Following incubation with antibodies, slides were washed with PBS and then mounted with 0.1% The concentration of C3 in the plasma was measured by Mouse High-Sensitive Complement C3 ELISA , following manufacturer's directions. For this double antibody sandwich ELISA, samples from WT background mice were diluted 1∶50,000, and samples from C3KO background mice were diluted 1∶50.The bone marrow transplantation process created chimeras that survived to undergo the injection protocol. Examination of peripheral blood smears and reticuloendothelial structures (spleen) of selected animals confirmed hematologic reconstitution .All animals receiving HSA/LPS developed proteinuria . There wIR/WT and WTIR/C3KO; All animals receiving HSA/LPS injections developed a proliferative glomerulonephritis (HSA/LPS treated mice are shown in IR/C3KO and WTIR/WT). On the other hand, when C3KO animals were reconstituted with WT bone marrow cells, detectable C3 was present in plasma, albeit at only a fraction (0.67%) of that found in WT animals. C3KO animals and C3KOIR/C3KO chimeras had C3 concentrations below 0.001 mg/ml, approaching the lower limit of the assay.Plasma C3 concentrations in the experimental animals are shown in Several lines of evidence support a role for the complement system in many forms of glomerulonephritis The liver is the source of most circulating complement. For several years, however, it has been appreciated that there is extrahepatic synthesis of a variety of components, both in humans and in experimental animals A number of published studies of extrahepatic complement synthesis have dealt with the kidney and monocytes. Renal complement synthesis was first demonstrated in lupus-prone (NZB) mice by Passwell and colleagues The role of this tubular complement synthesis has been examined in experimental animals, based upon observations in human disease. Chronic glomerulonephritis that progresses to ESRD is always accompanied by tubulointerstitial disease: peritubular edema, fibrosis, inflammatory cell infiltration, and tubular atrophy. We have employed experimental animals models to support an important role for such tubular complement component synthesis in tubulointerstitial disease IR/WT chimeras, similar to those we have employed, have suggested a role for local (presumably monocyte/macrophage-derived) C3 in the reticuloendothelial system in the immune response to viruses The in vivo role of monocyte-derived complement, specifically C3, has been studied much less. Studies in C3KOThe synthesis of monocyte C3 and other complement proteins could theoretically be important in the glomerular component of glomerulonephritis. Many glomerulonephritides, both in humans and in animal models, are characterized by monocytic infiltrate. In vitro, monocyte C3 synthesis is enhanced by stimuli such as cytokines Testing this hypothesis in intact animals would be difficult. The presence of circulating liver-derived C3 in plasma would make it impossible to establish the presence of what would likely be quantitatively smaller amounts of locally synthesized protein. Moreover, the known strong in situ hybridization signal from tubular C3 mRNA could overwhelm a weaker glomerular signal To overcome this problem, we employed the construction of “chimeras”, similar to work done by Verschoor et al. who studied the immune response to herpes virus IR/WT chimeric mice, they did not have immunohistochemical evidence of glomerular C3 deposition.As we have noted previously, the glomerulonephritis model we have employed resulted in marked glomerular hypercellularity. Using immunofluorescent techniques, we demonstrated that much of this hypercellularity is derived from infiltrating monocytes. Additionally, animals with wild type background had evidence of C3 deposition in their glomeruli. Our previous studies have also shown such deposits of C3 in the glomerulus, beginning at about three weeks after induction of glomerulonephritis IR/WT chimeric mice confirms this, although the concentration is unlikely to be physiologically important. Some workers have suggested a role for C3 synthesis by monocytes in local inflammatory conditions such as glomerulonephritis and transplant rejection As would be expected from the work of Drouin and colleagues First of all, immunohistochemistry for C3 in C3KO animals reconstituted with WT bone marrow cells shows no C3 glomerular deposition, despite active inflammation and trace C3 protein in infiltrating cells. Such experiments are critical to establishing this, since the strong C3 deposition in C3 replete (i.e. WT) mice would overcome any locally synthesized proteins.The other important observations relate to the severity of the histologic and clinical disease. There was no difference in two histologic markers of severity (glomerular cell counts and crescents) between C3KO and WT animals, regardless of whether they were reconstituted with WT or C3KO bone marrow cells. Furthermore, there was no difference among groups in proteinuria or renal function. The glomerular histology in these animals did not differ from our previously published observations in untransplanted animals It could be argued that, despite glomerular deposition in WT animals, C3 is not critical to the glomerular component of this model. As our previous studies have shown The rationale of these studies, however, was to develop a model that would permit differentiating the effect of systemic synthesis, activation and deposition of a complement component from that which is produced locally, in much smaller quantities and mediates its effects in the immediate microenvironment. Thus, our results suggest that in our specific model of immune complex glomerulonephritis, neither systemic nor locally synthesized C3 is critical to the glomerular inflammatory process. A similar bone marrow transplant strategy could be employed in other models in which the glomerular role of C3 appeared more critical.glomerular and monocytic C3. Our previous studies have suggested that the importance of locally synthesized complement is most likely in the tubulointerstitial component of inflammatory renal disease, where it is synthesized by the renal tubular epithelium Finally, it should be noted that the focus of these experiments was exclusively on These studies demonstrate the importance of examining intact animal models of disease, in addition to in vitro work, when considering the role of locally synthesized inflammatory mediators. Although monocytes clearly are capable of synthesizing the central protein of the complement cascade in response to inflammation, this ability does not appear to have an important role in vivo.


The Cognitive Vulnerability Model holds that both clinical and sub-clinical manifestations of animal fears are a result of how an animal is perceived, and can be used to explain both individual differences in fear acquisition and the uneven distribution of fears in the population. This study looked at the association between fear of a number of animals and perceptions of the animals as uncontrollable, unpredictable, dangerous and disgusting. Also assessed were the perceived loomingness, prior familiarity, and negative evaluation of the animals as well as possible conditioning experiences.162 first-year University students rated their fear and perceptions of four high-fear and four low-fear animals.Perceptions of the animals as dangerous, disgusting and uncontrollable were significantly associated with fear of both high- and low-fear animals while perceptions of unpredictability were significantly associated with fear of high-fear animals. Conditioning experiences were unrelated to fear of any animals. In multiple regression analyses, loomingness did not account for a significant amount of the variance in fear beyond that accounted for by the cognitive vulnerability variables. However, the vulnerability variables accounted for between 20% and 51% of the variance in all animals fears beyond that accounted for by perceptions of the animals as looming. Perceptions of dangerousness, uncontrollability and unpredictability were highly predictive of the uneven distribution of animal fears.This study provides support for the Cognitive Vulnerability Model of the etiology of specific fears and phobias and brings into question the utility of the harm-looming model in explaining animal fear. Fear of animals is prevalent in many countries. In Australia, studies have found up to 40% of children suffer from fear of animals such as spiders and snakes  while inAlthough fears and phobias of dangerous animals may be readily understandable, fear of non-dangerous animals is poorly accounted for in the literature. However, Armfield  has arguThe Cognitive Vulnerability Model neatly explains two traditionally vexing questions regarding the characteristics of fears and specific phobias. First, why is it that some people fear a given stimulus when others do not, despite apparently similar learning histories? According to the Cognitive Vulnerability Model, it is the perceptions of a stimulus as uncontrollable, unpredictable, dangerous and disgusting which directly determine fear of that stimulus. While learning experiences may help shape these vulnerability-related perceptions they are not causal per se.The second question addressed by the Cognitive Vulnerability Model is what causes the uneven distribution of feared stimuli and situations in the population? Although the idea of biological preparedness , an inbuet al.[et al.[Some support for the Cognitive Vulnerability Model of the etiology of fear comes from a study by Armfield and Mattiske  which foet al. combinedl.[et al. found scet al.[Despite the consistency of the findings of Riskind et al.,20 with et al., the stuet al.. This anet al.,20. It iet al.[There is now an extensive body of research investigating the harm-looming model and the more recent extension dealing with looming maladaptive style ,22. Overet al., the finDemand characteristics may also affect the study by Riskind and Maddux , which aet al.[Another study taken to support the harm-looming model has been reported by Riskind and Maddux . Howeveret al. where thWhile the harm-looming model continues to attract considerable attention in the literature on the etiology of fear, several other theories and models are also prominent. Neo-conditioning theories of fear acquisition assume an important place for the concept of latent inhibition ,26 and hLatent inhibition is well documented in the animal conditioning literature, having been demonstrated with a variety of animals using a number of indices of conditioning . HoweverNon-experimental studies have also failed to find a strong relationship between prior familiarity and fear. Doogan and Thomas , for exaOne variable which is rarely studied in relation to fear is negative evaluation. It is possible that some animals are perceived negatively whereas other animals are seen more positively. The possible dimensions of evaluation are many. Animals might be seen as more or less ugly, intelligent, useless, soft, aggressive, desirable, spiteful, etc. Negative evaluation is a potentially important variable in fear research due to the possibility that many of the relationships found between fear and other variables are in fact spurious. If there is a relationship between fear and negative perceptions  of the feared stimulus, the finding of a relationship between, for example, fear and unpredictability may occur merely because unpredictability is perceived as a negative characteristic. For this reason there is a need to examine the relationship between fear and other variables after controlling for the potentially confounding effect of negative evaluation.Armfield and Mattiske  found stThe current study tested several hypotheses. First, it was predicted that each of the vulnerability variables  would be significantly positively correlated with subjective fear of a number of different types of animals. Second, it was hypothesised that the four vulnerability variables would account for a significant amount of the variance in fear of each of the animals beyond that accounted for by perceived loomingness and after controlling for gender, negative evaluation, and prior familiarity. Finally, it was proposed that perceptions of unpredictability, uncontrollability, dangerousness, and disgustingness across a number of animals would exhibit significant positive correlations with fear of those animals. That is, the Cognitive Vulnerability Model would be able to explain the uneven distribution of fear across a number of animals.SD = 7.6).A total of 162 first-year university students in Adelaide volunteered for participation in the study. Sixty-five subjects were male and 96 were female, with one subject not identifying his/her gender. The ages of all participants ranged from 17 to 47 with a mean of 22.9 years . One version investigated spiders, ducks, rabbits, and cockroaches while the other version used cats, snakes, rats, and guinea pigs. The two versions were used in order to reduce the amount of time required for completing the questionnaire. Participants were randomly allocated one of the two questionnaire versions.The variables measured were subjective fear, perceptions of dangerousness, disgustingness, uncontrollability, and unpredictability, loomingness, negative evaluation, familiarity, and learning history. All the variables were measured using self-endorsing items with possible response scores ranging from 1 to 7. Each item required a rating for all four animals used in each questionnaire. The internal reliability coefficients for each scale for each animal are given in Table None, and the values 2 to 7 assigned respectively to points on the scale described as Very Little, A Little, Some, Much, Very Much, and Terror. This scale has been extensively cited [Following Geer , the parly cited  and has ly cited .Perceptions of dangerousness were assessed using four questions. Two of these questions referred to the potential dangerousness of the animal while the other two questions measured the subject's perceived likelihood of actually being harmed. In an attempt to obtain a general measure of perceived dangerousness for each animal the questions either referred to situations where the specific breed or genus of the animal was unknown or were related to thoughts concerning the majority of these animals. The four items used to measure disgust in this study consisted of a short version of an eight-item scale employed by Armfield and Mattiske . This meet al.[et al.[Loomingness was measured using the five-item scale developed and employed by Riskind et al.. An examl.[et al. found a Participants were asked four questions designed to measure negative evaluations of each animal. These evaluation items were obtained from the Semantic Differential Scale developed by Osgood, Suci, and Tannenbaum . The fouConsiderable experience to No personal experience.Familiarity was assessed by asking the participants how much personal experience they had had with each animal, with responses ranging from None inflicted to Extremely serious) or degree of fear (No fear to Terrified). An option of Unknown was provided for ratings of mother's and father's fear.Six types of learning events were assessed, which may be classifiable as classical, vicarious, and informational conditioning experiences. These items required ratings of the worst harm/pain/illness ever inflicted on oneself, seen or heard about being inflicted upon another, the degree of mother's and father's fear, and the most fear ever seen being demonstrated by another person. This method of eliciting historical and experiential factors in the development of animal fears has been commonly used ,48. UnliEthical approval was obtained for the study and informed consent was obtained from the participants who were told that participation was voluntary, that they would not be individually identifiable and that they were free to both discontinue their participation at any time and to decline to answer any particular question. Participants were tested in a group format in a university setting and were given brief information regarding the questionnaire, being told that it concerned beliefs that may or may not be related to animals. In an attempt to reduce demand characteristics, the participants were not informed that the questionnaire related to fears of animals. In addition, items relating to uncontrollability, unpredictability, dangerousness, disgustingness, loomingness, negative evaluation, and familiarity were presented in a mixed order prior to the questions relating to fear and learning history.R correlations were used to assess the association between fear of each animal and the Independent Variables (IVs), including possible conditioning experiences. Two series of hierarchical multivariate regression models were constructed for fear of each animal. For one series, the cognitive vulnerability variables were entered as the last step, after controlling for perceived loomingness and other possible confounders. In the other series, perceived loomingness was entered as the last step after controlling for the cognitive vulnerability variables and other possible confounders. Statistical significance was assessed via the change in R2. Finally, aggregate data were used to determine the linear associations between the fear of animals and the various IVs. Because of the large number of analyses, the criterion alpha for rejecting the null hypothesis was set at 0.01 to reduce the risk of Type 1 error.Descriptive statistics were calculated for the fear, negative evaluation, perceptions of dangerousness, disgustingness, uncontrollability, unpredictability, loomingness, and familiarity associated with each of the eight animals. Pearson The descriptive statistics for the high-fear animals on all the measures are presented in Table None and Very Little on average. Of the low-fear animals, ducks were the most feared (mean = 1.67) while rabbits were the least feared. Guinea pigs were perceived as the most disgusting and unpredictable of the low-fear animals and were evaluated the most negatively. Ducks were rated as the most uncontrollable of the low-fear animals and cats obtained the highest rating of loomingness and dangerousness.As expected, all the low-fear animals were feared less than the high-fear animals Table . All feaAll the low-fear animals were rated as less dangerous, disgusting, uncontrollable, and unpredictable than the high-fear animals and were evaluated less negatively. Although the low-fear animals were rated as less looming than the high-fear animals there was little difference between cats  and cockroaches . Finally, although cats were rated as the most familiar of any of the eight animals, spiders  were rated as more familiar than the remaining low-fear animals, and cockroaches were rated as more familiar to participants than were guinea pigs.All of the vulnerability variables were significantly correlated with fear of each of the high-fear animals Table . UncontrPerceptions of loomingness were significantly, albeit moderately, correlated with fear of each of the high-fear animals Table . HoweverR correlations between fear and potential conditioning experiences for each animal. Overall, there were very few statistically significant associations. Only one association between fear of high-fear animals and possible learning experiences reached statistical significance, while no significant associations were evident for low-fear animals.Table In order to test the effects of the vulnerability variables on fear after controlling for the other variables, a series of hierarchical multiple linear regression analyses were conducted. For each animal, the vulnerability variables were entered as a block at Step 3, following the forced entry of gender and negative evaluation at Step 1 and loomingness at Step 2. Due to its non-significant effect on fear of most of the animals used in this study, Familiarity was not used in the regression equations. In addition to testing for the independent effects of the vulnerability variables on each animal, the independent effect of loomingness was also determined. Once again, a series of hierarchical multiple linear regression analyses were conducted, this time with gender and negative evaluation at Step 1, uncontrollability, unpredictability, dangerousness, and disgustingness at Step 2, and loomingness at Step 3. These analyses were done for each animal using fear as the DV. Because of the correlations between many of the variables, checks for multicollinearity were carried out as recommended by Tabachnick and Fidell . HoweverA summary of the full series of multiple regressions is given in Table R2 = 0.98) and uncontrollability (R2 = 0.94) while dangerousness and loomingness were also good predicators of fear  declare that they have no competing interests.JMA conceived, researched, performed the statistical analysis, and wrote this manuscript.1. I think that _____ would feel pleasant to touch.2. If I touched a _____ it would be important for me to wash my hands afterwards.3. I think that _____ are dirty or unclean animals.4. I would be revolted or disgusted if a _____ came into contact with my skin.1. I believe that I would be able to deal effectively with a _____ by myself when encountered.2. If a _____ was nearby I would not feel in control of the encounter.3. I do not believe that I would lose control over my actions in any way (e.g. panic or freeze) if a _____ came as rapidly as it could towards me.1. I find most _____ to be predictable in their movements.2. I think that the movement of _____ can be guessed in advance.3. I never know what a _____ is going to do.1. How potentially dangerous do you think that most _____ are to you?2. I believe that if I came into contact with an unknown _____ I would be harmed.3. I think that the majority of _____ are harmless to me.4. I think that if I encountered an unknown _____ I would not be harmed in any way.The pre-publication history for this paper can be accessed here:


This research was conducted on ten glass slides selected from the histopathology evaluation chickens. Five slides of control's chickens healthy and five slides of chickens infected experimentally with chicken anemia virus (CAV slide) between one and twenty-one days post infection (PI), they were analyzed in magnifications of 200× and 400×. Histopathology showed severe bone marrow hypoplasia to complete aplasia, fully depletion of the erythrocytic and granulocytic series, both accompanied by space occupying adipocytic replacement. Foci of erythropoietic hyperplasia with intense mielopoietic activity, some hemocytoblast increased of size, with large nucleus. A quantitative analytical technique by Positive Pixel Count Algorithm was applied. It demonstrated that measures area stained of control slides were higher than CAV slides . So, the control slides showed strong positivity, due to the presence of bigger quantity of cells of erythrocytic and granulocytic series. The CAV slides of seven days PI had high positivity (average: 94%), it was explained because the chicken anemia virus takes place severe lesions between ten to seventeen days PI, after 21 days PI the cellular regeneration is observed that is evidenced by means of focuses of erythroblastoid cells hyperplasia. This technique demonstrates in a quantitative way the severe decrease of the cellular components of the bone marrow in presence of the infection for CAV, supporting with numeric data the histology image evaluated by the pathologist. Therefore, it can be used as support to the histopathology of field samples to evaluate the presence of lesions taken place by CAV and this way to improve the quality and efficiency of the veterinary pathology services. Chicken anemia virus (CAV) is a small, non-enveloped, single-stranded DNA virus, first isolated by Yuasa et al. 1979), a Gyrovirus, within the family Circoviridae 979, a Gy. The vir10 glass slides stained with H&E, selected of the histopathology evaluations of chickens. Five slides coming from chickens healthy controls and five slides coming from chickens infected experimentally with chicken anemia virus (CAV slide) between one and twenty one days PI, they were analyzed in m agnifications of 200× and 400×.Positive Pixel Count is a multipurpose algorithm that measures area and intensity of staining for two-colored slides. Pixels are classified according to colour and intensity of staining. Pixel counts (area) are calculated for strong positive (Red), positive (Orange), weak positive (Yellow), and negative (Blue) pixels. This is useful for measuring positivity as a fraction of total stained area.Histopathology showed bone marrow with abundant cells of the erythrocytic and granulocytic series from chickens healthy controls Figure  and seveThe results demonstrated that the number of Strong Positive and Positivity (%) of control slides were higher than CAV slides . The control slides showed strong positivity, due to the presence of bigger quantity of erythroblastoid cells. The CAV slides of 7 days PI had high positivity (average: 94%), because the CAV produces the lesions but severe starting from the 10 days PI up 21 days PI, after the cellular regeneration is observed, being able to be evidenced by means of focuses of erythroblastoid cells hyperplasia.This technique demonstrated in a quantitative way the severe decrease of the cellular components of the bone marrow in presence of the infection for CAV, supporting with numeric data the histology image evaluated by the pathologist. Therefore, it can be used as support to the histopathology of field samples to evaluate the presence of lesions taken place by CAV in the farms and this way to improve the quality and efficiency of the veterinary pathology services.


We have studied the motor abilities and associative learning capabilities of adult mice placed in different enriched environments. Three-month-old animals were maintained for a month alone (AL), alone in a physically enriched environment (PHY), and, finally, in groups in the absence (SO) or presence (SOPHY) of an enriched environment. The animals' capabilities were subsequently checked in the rotarod test, and for classical and instrumental learning. The PHY and SOPHY groups presented better performances in the rotarod test and in the acquisition of the instrumental learning task. In contrast, no significant differences between groups were observed for classical eyeblink conditioning. The four groups presented similar increases in the strength of field EPSPs (fEPSPs) evoked at the hippocampal CA3-CA1 synapse across classical conditioning sessions, with no significant differences between groups. These trained animals were pulse-injected with bromodeoxyuridine (BrdU) to determine hippocampal neurogenesis. No significant differences were found in the number of NeuN/BrdU double-labeled neurons. We repeated the same BrdU study in one-month-old mice raised for an additional month in the above-mentioned four different environments. These animals were not submitted to rotarod or conditioned tests. Non-trained PHY and SOPHY groups presented more neurogenesis than the other two groups. Thus, neurogenesis seems to be related to physical enrichment at early ages, but not to learning acquisition in adult mice. Interactions between an organism and its environment can lead to important neurobehavioral changes, and for several decades environmental enrichment  has been used to induce these changes in both intact and injured central nervous systems. The term “enriched environment” as an experimental process was introduced in the late 1940s by Donald Hebb The positive effects of environmental stimulation are not restricted to the cellular level, but also reach brain functioning. Behavioral studies have shown that exposure to enriched environments enhances memory capabilities in various tasks, particularly in spatial learning tests Although it is assumed that environmental enrichment enhances learning and memory, this is a general assumption taken from the observation that these housing conditions also improve spatial memory performance. Nevertheless, little is known from a quantitative point of view regarding the effects of environmental stimulation on other associative learning tests in which the hippocampus is also involved. To determine whether an enriched environment has consequences on associative learning tests similar to those previously described for spatial learning, we studied here the effects of different living conditions on classical eyeblink conditioning as well as on instrumental conditioning, two associative learning paradigms in which the hippocampus seem to be involved One week after arrival, 3-month-old animals were assigned randomly to one of the following four experimental groups: alone (AL), physical (PHY), social (SO), and social-physical (SOPHY). For the AL group, animals were placed in individual cages provided exclusively with the regular soft-wood mouse bedding. For the PHY group, animals were also placed in individual cages, but provided with a wheel, a tunnel, a ladder, a dummy mouse, and nesting material. For the SO group, animals were caged together , provided exclusively with mouse bedding. Finally, for the SOPHY group, animals were caged together (n = 3 per cage), but in an enriched environment, as for the PHY group. Animals were maintained in this situation for 30 days before the beginning of the experimental tests. For experiments, a total of 20 animals per group were selected at random for the rotarod test. Those animals were further divided for classical (n = 10 per group) and instrumental (n = 10 per group) conditioning. At the end of the behavioral study, animals were prepared for the immunohistochemical study see .In addition, 1-month-old animals were divided into the same four groups (n = 10 per group) and maintained for 30 days in the four types of environmental conditions. These animals were not used in any motor or learning test . After the 30-day period, these animals were directly prepared for the immunohistochemical study, without any additional manipulation or training.F = 13.487; P<0.001; P = 0.678) or between the AL and SO groups (P = 0.246). By the 5th session, all PHY and SOPHY animals had already reached the selected criterion, whilst 42±12.5% of AL and 54±16.4% of SO mice reached criterion only during this session.The rotarod test is a behavioral task assessing motor coordination performance. F = 13.487; P<0.01] larger (261±45.3 s and 267±54 s) than those collected from the AL and SO (79±10.1 s and 114±25.9 s) during the first session. The differences were still significant (P<0.01) during the 5th session between the PHY (380±20.2 s) and the SOPHY (305±60.5 s) versus the AL (255±39.6 s) and the SO (270±46.5 s) groups.With regard to the mean time spent on the rod during a session per group, the PHY and SOPHY groups presented values significantly  larger from the 2nd to the 10th conditioning sessions for the AL and PHY groups, and from the 5th to the 10th sessions for the SO and SOPHY groups, as compared with values collected during habituation sessions.In a second series of experiments, we carried out the classical conditioning of eyelid responses in the four  experimental groups  following the design illustrated in F = 2.533; P = 0.78]. Thus, neither physical nor social enrichments had any beneficial effect on animal performance during the classical conditioning test.Although the SO and SOPHY groups showed learning curves with lower values than those reached by the AL and PHY groups , no signF = 138.551; P≤0.002] larger from the 4th to the 10th sessions for the AL and PHY groups, and from the 7th to the 10th sessions for the SO and SOPHY groups, compared with values (considered as 100%) collected during the two habituation sessions.F = 1.219; P = 0.198] with respect to the slopes of fEPSPs evoked across conditioning sessions. According to these results, physical and social enrichments have no significant effects on the activity-dependent synaptic plasticity evoked at the CA3-CA1 synapse by classical eyeblink conditioning.In coincidence with what was described above for the percentage of CRs, the SO and SOPHY groups again yielded fEPSP curves with values slightly lower than those achieved by the AL and PHY groups. However, there were no significant differences between the four experimental groups . No significant differences were observed between the PHY and SOPHY groups (P = 0.182) or between the AL and SO groups (P = 0.517). In summary, physical, but not social, enrichment had a positive effect on animal capabilities of acquiring an instrumental task using a fixed-ratio schedule.F = 3.495; P = 0.016] difference between these four groups of animals 20 days after the BrdU labeling. Furthermore, the mean number of double-labeled NeuN-BrdU neurons was rather low: AL, 6.41±0.59; PHY, 5.53±0.88; SO, 3.35±0.85; and SOPHY, 5.12±0.82 (not illustrated). A possible explanation for this low number of double-labeled neurons, and for the lack of a significant effect of physical and/or social enrichment on dentate gyrus proliferation and survival of newborn neurons, is that these mice were too old at the time of the test (≈6 months at the time of the immunohistochemistry).To determine putative changes in neurogenesis in the dentate gyrus of rotarod-trained and instrumental-conditioned animals , we developed an accumulative BrdU-pulse labeling . These animals were <3 months old at the time of immunohistochemistry. In this case, we obtained significant differences between groups. Firstly, no pycnotic nuclei were seen in BrdU-labeled cells in any of the experimental groups after the immunohistochemical processing. After DAB-Ni development, numerous BrdU-positive cells were observed in the subgranular zone in the PHY-n  and — esThe present study was designed to determine the effects of an enriched environment on motor performance and on different forms of hippocampal-dependent learning. We also studied whether there is a causal relationship between these types of learning and hippocampal neurogenesis. The results show that motor performance and instrumental conditioning are improved by the physical factors of the enrichment, but the retention of classically conditioned memories is not. Furthermore, the improvement of some forms of learning does not seem directly related with the increase of newly generated neurons in the dentate gyrus of the hippocampus, since increasing social interaction is able to modify the number of hippocampal new neurons without affecting the learning paradigms assessed here.Enriched environment usually includes increasing motor stimulation through access to running wheels. Several studies even indicate that this increased physical activity is a key factor of the enrichment, since the use of a wheel, without any other enriching element, is sufficient for regulating both learning and hippocampal neurogenesis Besides motor coordination performance, we also assessed two associative learning tests: trace classical eyeblink and instrumental conditionings. Although both paradigms are hippocampal-dependent, we found that physical enrichment has clear effects on operant conditioning abilities, without affecting the acquisition of classical conditioning of eyelid responses. Again, the social factor did not influence the learning processes. Taken together, spatial learning, rotarod, and instrumental conditioning are the learning paradigms in which acquisition is improved after exposure to enriched environments. According to these results, it can be hypothesized that only those forms of learning which require precise motor abilities are improved by an enriched environment. This is not the case of the classical conditioning of the eyelid responses, for which no special motor capacity is necessary.The question of whether newly born neurons in the dentate gyrus of the adult hippocampus participate in learning and memory has been discussed for years However, differences in the number of adult-born dentate gyrus granule cells became evident when the immunohistochemical study was carried out in younger, non-conditioned mice. In fact, it is well known that neurogenesis declines with age Enrichment can also include increased social stimulation through larger numbers of animals per cage. Although it was claimed that social interaction alone cannot elicit the cerebral effects of enriched environments ad libitum. Electrophysiological and behavioral studies were carried out in accordance with the guidelines of the European Union Council (2003/65/European Union) and current Spanish regulations  for the use of laboratory animals in chronic experiments. Experiments were also approved by the ethical committee of the local institution .Experiments were carried out on male C57Bl/6 mice obtained from an official supplier . Animals were 1 or 3 months old at the moment of arrival, weighing 17±3 and 25±2 g respectively. Before experimental manipulations, animals were housed in separate cages (n = 10 per cage). All mice were kept on a 12/12 h light/dark cycle with constant ambient temperature (21±1°C) and humidity (50±7%). Food and water were available In this study, we used an accelerating rotarod treadmill for mice  were prepared as follows. Firstly, they were anesthetized with 0.8–1.5% isoflurane, supplied from a calibrated Fluotec 5  vaporizer, at a flow rate of 1–4 L/min oxygen  and delivered by a mouse anesthesia mask . Once anesthetized, animals were implanted with bipolar recording electrodes in the left orbicularis oculi muscle and with stimulating electrodes on the ipsilateral supraorbital nerve. Electrodes were made of 50 µm, Teflon-coated, annealed stainless steel wire . Electrode tips were bared of the isolating cover for 0.5 mm and bent as a hook to allow a stable insertion in the upper eyelid. In the same surgical step, mice were also implanted with bipolar stimulating electrodes in the contralateral (right) Schaffer collateral/commissural pathway of the dorsal hippocampus  was used for fEPSP recordings.Experimental sessions were carried out with three animals at a time. Animals were placed in separate small (5×5×10 cm) plastic chambers located inside a larger (30×30×20 cm) Faraday box. Both the electromyographic (EMG) activity of the orbicularis oculi muscle and fEPSPs were recorded with Grass P511 differential amplifiers , at a bandwidth of 0.1 Hz-10 kHz. A high-impedance probe  was presented as a conditioned stimulus (CS), whilst the unconditioned stimulus (US) consisted of a 500 µs, 3× threshold, square, cathodal pulse applied to the supraorbital nerve 500 ms after the end of the CS . A totalDuring habituation and conditioning sessions, fEPSPs were evoked in the CA1 area by single 100 µs, square, biphasic (negative-positive) pulses applied to Schaffer collaterals 300 ms after CS presentation . Pulse iAt the end of the experiments, and in order to determine the location of hippocampal electrodes, mice included in this test were deeply re-anesthetized  and perfused transcardially with saline and 4% phosphate-buffered paraformaldehyde. Brains were dissected out, postfixed overnight at 4°C, and cryoprotected in 30% sucrose in PBS. Brain sections were obtained in a microtome  at 50 µm. Selected sections including the dorsal hippocampus were mounted on gelatinized glass slides and stained using the Nissl technique with 0.1% toluidine blue.A total of 10 animals per group were assigned for operant conditioning. Training and testing took place in basic Skinner box modules (n = 3) measuring 12.5×13.5×18.5 cm . The operant chambers were housed within a sound-attenuating chamber (90×55×60 cm), which was constantly illuminated (19 W lamp) and exposed to a 45 dB white noise . Each Skinner box was equipped with a food dispenser from which pellets  could be delivered by pressing a lever. Before training, mice were handled daily for 7 days and food-deprived to 80% of their free-feeding weight. Shaping took place for 15 min during successive days, in which mice were shaped to press the lever to receive pellets from the food tray using a fixed-ratio (1∶1) schedule. Animals were maintained on this 1∶1 schedule until they reached the selected criterion — namely, until they were able to obtain ≥20 pellets for 2 successive sessions. Mice reached criterion after 4–7 days of training (see ref. 15 for details).Once criterion for the 1∶1 schedule was reached, conditioning was carried out for 10 days using a fixed-interval (FI30s) schedule. For this, the first lever press carried out by the mouse after each period of 30 s was rewarded with a pellet. Each session lasted for 15 min. The start and end of each session was indicated by a tone  provided by a loudspeaker located in the recording chamber. Conditioning programs, lever presses, and delivered reinforcements were controlled and recorded by a computer, using a MED-PC program . Those animals were further used for the immunohistochemical study (see below).et al. 2006). In a first set of experiments we injected animals  that received instrumental conditioning. In this case, BrdU injections were carried out during the four days following the end of the experiments. These animals were maintained for 20 additional days in the same physical and environmental situation comprising the survival period after proliferation when the newborn cells develop differentiated phenotypes To determine cell proliferation and neurogenesis in the hippocampus of trained mice, a daily BrdU  pulse by intraperitoneal injection was administered for four days , using a freezing microtome (Leica), and were further processed for immunocytochemistry. For BrdU immunohistochemistry, sections were processed essentially as described In the case of classical conditioning experiments, EMG and hippocampal activities and one-volt rectangular pulses corresponding to CS and US presentations were stored digitally on a computer through an analog/digital converter , at a sampling frequency of 11–22 kHz and an amplitude resolution of 12 bits. Commercial computer programs  were modified to represent EMG and fEPSP recordings. Data were analyzed off-line for quantification of CRs and fEPSP slopes with the help of homemade representation programs For the rotarod test, collected data  were collected by computer and stored for off-line analysis Computed results were processed for statistical analysis using the Sigma Stat for Windows package . Unless otherwise indicated, data are represented by the mean ± s.e.m. Collected data were analyzed using a two-way ANOVA test, with time or session as repeated measure, coupled with contrast analysis when appropriate. One-way ANOVA allowed checking the statistical differences between different groups. Regression analysis was used to study the relationship between the fEPSP slope and the percentage of CRs.