ANIMALS BREEDING AND HOUSING CONDITIONS
Given the number of available running-wheels, the experiment will be organized in three successive identical sub-experiments, whose collected data will be pooled for analysis. Each sub- experiment will first involve 64 females and 32 males C57BL/6Jmice (experimentally-naïve) age 6 weeks. The choice of C57BL/6J strain is based on its wide use in addiction research and previous exercise-related experiments performed in our lab (Geuzaine and Tirelli, 2014; Lespine and Tirelli, 2015). Mice will be housed upon arrival in groups of 8 for one week (same-sex groups) in large transparent polycarbonate cages (38.2 x 22 cm surface x 15 cm height; TECHNIPLAST, Milano, Italy). After this acclimation period, mice will be individually housed in smaller TECHNIPLAST transparent polycarbonate cages (32.5 x 17 cm surface x 14 cm height) with pine sawdust bedding, between-animal visual, olfactory and acoustic interactions remaining possible.
Forty-eight male C57BL/6Jmice fed a high-fat diet (60% energy from fat) starting at the age of 5 weeks and 12 male C57BL/6Jmice fed a low-fat diet all the time were purchased from Jackson Laboratories (Jackson, FL, USA) at the age of 10 weeks. After arrival, they were housed individually in regular mouse cages with a 12:12 h dark/light cycle and free access to water and the same high-fat (D12492) and low-fat (D12450B) diets (Research Diets Inc., New Brunswick, NJ, USA), respectively. After a week of acclimation, mice on the high-fat diet were weighed and randomly divided into four groups. One group continued on the high-fat diet and was used as the HFC, and the other three were fed a modified high-fat diet where 15%, 35%, or 70% casein was replaced by an equivalent amount of protein derived from HPH. The treatments lasted for 10 weeks. The fat contained in HPH was adjusted by reducing the equivalent amount of fat in the form of lard. The mice fed the low-fat diet were used as the LFC. The diet composition is provided in the Supplementary Material, Table S1. Food consumption was monitored daily, and mice were weighed weekly. Semi-fasting (4–6 h) blood glucose was measured every week with a glucometer (ACCU-CHECK Aviva; Roche, Basel, Switzerland) using blood via tail snip. Oral glucose tolerance testing was performed during Weeks 5 and 8 of the treatment. At the end of experiment, mice were fasted overnight, and blood was collected by cardiac puncture after anesthesia with inhalation of isoflurane (Pharmaceutical Partners of Canada Inc., Toronto, ON, Canada) into serum collection tubes. Serum was obtained by centrifugation at 1500× g for 20 min and stored in cryogenic vials at −80 ◦
D ISCUSSION D ISCUSSION
Our results reproduce those previously obtained in our laboratory (Geuzaine and Tirelli, 2014) by showing that wheel-running exercise is ineffective at significantly altering CPP to 10 mg/kg cocaine in male C57BL/6Jmice in comparison to a control housing. However, the results from the two other studies that have investigated the effects of wheel-running on CPP induced by cocaine differ with ours by reporting accentuated CPP after such exercise (Smith et al., 2008; Mustroph et al., 2011). This exercise-induced increase in CPP - somewhat contrasting with the protective effects of aerobic exercise on vulnerability to rewarding properties of drugs of abuse - has been hypothetically ascribed to an enhancement of associative learning capabilities resulting from neuroplastic changes induced by chronic wheel-running.
Acetaldehyde, the first ethanol metabolite, was recently suggested to play a major role in many behavioral effects of ethanol. However, no studies have directly investigated the behavioral effects of acetaldehyde after acute administration. Therefore, the aim of the present study was to characterize the locomotor, hypnotic and ataxic effects of acetaldehyde in C57BL/6Jmice. Various acetaldehyde doses (0-300 mg/kg) were injected intraperitoneally and their effects were investigated with several classical behavioral tests. The locomotor effects of acetaldehyde were measured in standard activity boxes. In addition, the loss of righting reflex was used to assess the hypnotic effects of acetaldehyde. Finally, the ataxic effects of acetaldehyde were studied with the horizontal wire test. The results show that acetaldehyde induced a significant hypolocomotor effect at 170 mg/kg and higher doses. In addition, the hypnotic effects of acetaldehyde were evidenced by a loss of righting reflex in doses between 170 and 300 mg/kg. However, the locomotor and hypnotic effects of acetaldehyde were very brief relative to what is observed after ethanol
Amphetamine-induced rotation. On D+25, mice were injected with d-
amphetamine(5 mg/kg i.p.). Twenty minutes later, they were placed in a 50 x 50 cm white arena. 360° rotation behaviors were analyzed with a videotracking software (Viewpoint). Serial Order Learning. After acquisition of the operant response, food-deprived mice underwent 12 sessions of SOL in operant boxes equipped with two levers and a food-tray (Meds-Associates). At each trials, both right and left levers were available. Only the left – right lever-presses sequences were reinforced by a food reward (20 mg Noyes pellet). Other sequences were not reinforced. An 8 s inter-trial interval separated two trials. Sessions stopped once 40 rewards have been delivered or after an hour, whichever came first. Anti-NeuN immunohistochemistry. 50 µm thick brain sections were incubated overnight in primary mouse-anti- NeuN IgG (Chemicon), then in secondary Horse-Anti-Mouse Biotinylated IgG (Chemicon). 3,3’-diaminobenzidine (Sigma) was used as a chromogen. Infarct volume was then calculated by multiplying the average of two successive infarct areas by the distance between them.
Figure 4 Effect of C5 status on different measures of susceptibility to candidiasis. (a) Heart fungal load was measured in 64 F 2 mice 48 h after
low dose infection with C. albicans. (b) Survival was monitored in 122 F 2 mice for up to 28 days after infection with a high dose of C. albicans.
(c) Serum TNF-a levels were measured 48 h after infection with a low dose of C. albicans in 56 F 2 mice. For all phenotypes there was a
significant linkage between C5-deficiency and an increased susceptibility to infection (***Po0.001). The response of A/J and B6 parents are included for comparison. C5 alleles are identified as ‘a’ and ‘b’, for A/J and B6, respectively, and were identified by genotyping (see Materials and methods). Horizontal bars indicate the mean value for each of the traits analyzed for each group.
The preliminary objective of our study was to define experimental conditions to study the combination of TCDD treatment and an obesogenic diet in mouse liver. We performed dose-response experiments to establish that 5 μg/kg TCDD is the threshold dose that leads, after repeated exposure during 6 weeks, to the first signs of liver impairment in C57BL/6Jmice. Physiologically based pharmacokinetic modeling of a subchronic exposure of mice to 5 μg/kg TCDD predicted final concentrations of 67 ppt TCDD in blood and 57,000 ppt in liver (wet weight), which are consistent with the concentrations previ- ously described in mouse liver (Boverhof et al. 2005; Vezina et al. 2004). Even if caution must be applied when extrapolating results from mice to humans due to species differ- ences, the concentrations predicted by the model in the blood of our mice (11,551 ppt lipid adjusted) are within the same range as those measured in the blood of the population close to the Seveso industrial accident (zone A, range 15–56,000 ppt) (Eskenazi et al. 2004) or of the Ranch Hand cohort of U.S. veterans exposed to the herbicide Agent Orange (range 318–40,376 ppt) at the time of discharge from Vietnam (Emond et al. 2005). These concentrations range far above the background TCDD levels found in the general popula- tion. A large study of dioxin blood levels in the United States [the University of Michigan Dioxin Exposure Studies (https://sph.umich. edu/dioxin/)] reported average TEQ blood levels of 23.9 ppt (lipid adjusted) in adults who were 18 years old or older. In contrast to blood, the concentration of TCDD has been measured only rarely in human liver (Leung et al. 1990) due to a limited access to biopsies. A physiologically based pharma- cokinetic model of the Ranch Hand cohort predicts TCDD levels of 5,535 ppt in liver for individuals with 162 ppt TCDD in the blood (Emond et al. 2005). These concentra- tions suggest that, for highly exposed Seveso residents or U.S. veterans with TCDD concen- trations above 40,000 ppt in their blood, their level of TCDD in the liver might be within the same range or even higher than the ones predicted in our rodent model (1,360,000 ppt lipid adjusted). Considering that the
The AhR-/- mice suffer from a nystagmus-like ocular instability
Because a great deal is known about the anatomy and physiology of the circuits responsible for gaze control, spontaneous and reflexive eye movements can be used to assess the integrity of many sensorimotor functions . Thus, to assess potential neurological brain dysfunctions, spontaneous and reflexive eye movements were monitored in the AhR-/- mice. First, video-oculography was performed in dark . Figure 1A presents the eye position in horizontal and in vertical plans in the absence of head movements (spontaneous eye movements). As animals are quietly seated in dark, the eyes of wild-type mice (AhR+/+) and heterozygous mice (AhR+/-, not shown) remain stable. In contrast, the gaze of AhR-/- mice is unstable at rest, as revealed by the presence of a spontaneous horizontal movement observed in all animals (n=12) (Figure 1B). This ocular instability affects both eyes and is also observed in the light. It consists in rhythmic sinusoidal, purely horizontal eye movements. The frequency of the ocular instability varies from 0.5 to 5 Hz depending on each individual AhR-/- mouse (mean in dark=1.84±0.74 Hz; figure 1B with two examples: 0.5 Hz, upper panel to 2.8 Hz, lower panel; note absence of vertical movement) and varies with the lighting condition: indeed, we systematically measured the frequency of the eye oscillation for each mice (n=12) in the dark and in the light (Fig 1C). For a majority of individuals (n=8; equation: y=0.94+0.34x, r 2 =0.5089; ANCOVA F(1,22)=9.41, p<0.01), the frequency of the nystagmus in the light is higher. This last observation is commonly observed with congenital nystagmus in humans, as the nystagmus increases when the patient tries to fixate an object.
Figure 6. Behavioral characterization of four mouse strains. (a) Average total resting time for each of the four strains of mice over 24 hours (n=7 animals for each strain). (b) Average duration of resting bouts (defined as a continuous duration with one single behavior). While all strains tend to spend roughly the same total amount of time resting (a), the CAST/EiJ tends to rest for longer stretches. Mean +/- SEM are shown, *pE\$129$ZLWK7XNH\¶VSRVWWHVt. (c) Total time spent grooming exhibited by the BTBR strain as compared to the C57BL/6J strain within 10 th -20 th minute after placing the animals in a novel cage. Here we show that using the computer system we were able to match manual scoring by two experimenters and reproduce previously published results 4 demonstrating the propensity of the BTBR strain to groom more than a control C57BL/6J. Mean +/- SEM are shown, *p<0.05 by 6WXGHQW¶V7WHVWRQH-tailed. p = 0.04 for System and p =0.0254 for human µ+¶S = 0.0273 IRU KXPDQ µ$¶). (d-e) Characterizing the genotype of individual animals based on the patterns of behavior measured by the computer system. The pattern of behaviors for each animal is a 32- dimensional vector, corresponding to the relative frequency of each of the eight behaviors of interest, as predicted by the system, over a 24-hour period. (d) To visualize the similarities/dissimilarities between patterns of behaviors exhibited by all twenty-eight individual animals (seven mice for each of the × four strains) used in our behavioral study, we performed a Principal Component Analysis (PCA) on the patterns of behaviors. The result shows that animals tend to cluster by strain (with the exception of 2 BTBR mice that tended to behave more like DBA/2J). (e) Confusion matrix for an SVM classifier trained on the patterns of behavior using a leave-one-animal-out procedure. The SVM classifier is able to predict the genotype of individual animals with an accuracy of 90% (chance level is 25% for this 4- class classification problem). The confusion matrix shown here indicates the probability for an input strain (along the rows) to be classified, based on its pattern of behavior, as each of the four alternative strains (along the columns). The higher probabilities along the diagonal and the lower off-diagonal values indicate successful classification for all categories. For example, the value of 1.0 for the C57BL/6J strain means that all C57BL/6J animals were correctly classified as such.
Figure 5: (A) Average total resting time for the four strains of mice. (B) Average duration of
resting bouts. While all strains tend to spend roughly the same total amount of time sleeping, the CAST/EiJ tends to sleep fewer longer stretches. Mean +/- SEM are shown, *P<0.01 by ANOVA with Tukey’s post test. (C) Average grooming duration exhibited by the BTBR strain as compared to the C57Bl/6J strain over one hour. Here we show that using the computer system we were able to match manual scoring by an experimenter and reproduce the key result from the study by McFarlane et al (McFarlane, Kusek et al. 2008) demonstrating the propensity of the BTBR strain to groom more than a control C57Bl/6J. Mean +/- SEM are shown, *P<0.05 by Student’s T test, one-tailed. (P = 0.04 for System and P =0.0254 for Human). Characterizing the genotype of individual animals based on the patterns of behavior measured by the computer system. (D) Multi-Dimensional Scaling (MDS) analysis performed on the distributions of behavior types measured over 4 (6 hour-long) windows. The MDS analysis reveals that animals tend to cluster into strains (with the exception of 2 BTBR mice that tended to behave more like DBA/2J). (E) Pattern classification analysis performed on the distributions of behavior types measured over 4 (6 hour-long) windows. Using an SVM classifier on the patterns of behavior we were able to predict the genotype of individual animals with accuracy of 90% (chance level is 25% for this 4-class classification problem). The confusion matrix shown here indicates the probability with which an input pattern of behavior (along the rows) was classified as each of the 4 alternative strains (along the columns). The higher probabilities along the diagonal and the lower off-diagonal values indicate successful classification for all categories. For example, the value of 1 for C57Bl/6J means that this strain was perfectly classified.
Khoa T. Phan, Tuan T. Tran, Duc D. Nguyen, and Nam Thoai
Abstract Congestion control is a distributed algorithm to share network bandwidth
among competing users on the Internet. In the common case, quick response time for mice traffic (HTTP traffic) is desired when mixed with elephant traffic (FTP traffic). The current approach using loss-based with Additive Increase, Multiplicative Decrease (AIMD) is too greedy and eventually, most of the network bandwidth would be consumed by elephant traffic. As a result, it causes longer response time for mice traffic because there is no room left at the routers. MaxNet is a new TCP congestion control architecture using an explicit signal to control transmission rate at the source node. In this paper, we show that MaxNet can control well the queue length at routers and therefore the response time to HTTP traffic is several times faster than with TCP Reno/RED.
Animal housing and handling
Four-week-old female mice (Elevages Janvier, France) were housed in groups of four under pathogen-free conditions at the Experimental Therapy Unit (Medi- cal School, Nantes, France) in accordance with French institutional guidelines (CEEA.PdL.06, authorization no 8405 and 8449). Mice were given access to food and water ad libitum. This report adheres to the EU direc- tive 2010/63/EU and the ARRIVE Guidelines for report- ing animal research [ 22 ], and a completed checklist is included in Additional file 1 .
(tables 3 – 6 and figures 1 – 2).
CYP450 mRNA levels
Isolation of RNA and preparation of cDNA. For each organ tested, total RNA was extracted from a pool of 3 mice to maximize tissue material. About 100 mg of tissue was homogenized in 1 mL of Trizol and incubated 5 minutes at room temperature. Chloroform (200 µL) was added, the mixture shaken for 15 seconds and then, centrifuged at 16,000 g for 30 minutes at 4°C. The aqueous supernatant (500 µL) was transferred to a new tube and ethanol 70% was added (1,1 volume). RNA was extracted using the Qiagen kit (RNeasy Mini kit; Qiagen Sciences, MD, USA) according to the manufacturer’s recommendations. RNA concentration and quality was assessed by spectrometry. Total RNA (2 µg) from each sample was used for reverse transcription. RNA, random primers (6 µg) and dNTP (25 mM) were preheated for 5 minutes at 65°C. Then, 5X-first strand buffer, 80 units of RNAse inhibitor, DTT (0.01 M) and 400 units of Superscript II (Invitrogen, Carlsband, CA, USA) were added to a final volume of 40 µL. Reverse transcription was carried out for 50 minutes at 42°C and stopped by heating to 70°C for 15 minutes (final RNA concentration 50 ng/µL). The resulting cDNA was frozen at -80°C until analyzed.
A growing body of evidence supports the hypothesis that autophagy in ﬂuences virus replication as well as IFN-I and cytokine responses upon RSV infection in macrophages, DCs and epithelial cells. 50 – 52 Thus, Pokharel et al. showed that autophagy is necessary for IFN- β production via the activation of TGF-β— SMAD2/3 signaling pathway in RSV-infected BMDMs. 53 By using transgenic mice expressing a recombinant LC3 protein coupled to the green ﬂuorescent protein GFP, it has been shown that autophagy is particularly strong in the lungs, heart and other neonatal organs during the ﬁrst hours of life. 54 Autophagy plays also a critical role in the acquisition of age-dependent features in macrophages. Indeed, BMDMs from aged mice (>100 weeks) exhibit reduced autophagic process compared to young mice (6–8 weeks). 55
characterize several human inflammatory chronic diseases. 24, 25 Thus, fibroblasts from DMD patients show a low capacity for glutathione synthesis. 26 In contrast, a noticeable feature of
mdx mice dystrophic muscles is the up-regulation of glutathione peroxidase and glutathione
reductase, which respectively utilizes and recycles reduced glutathione. 27 However, this protective mechanism does not entirely compensate the chronic oxidative challenge since glutathione level in 6-8-week-old mdx mice tibialis anterior muscles is decreased by 20% compared to control mice. 27 Accordingly, green tea polyphenols, which beyond their inherent antioxidant properties promote glutathione synthesis, reduce muscle damage and necrosis in mdx mice. 16, 17 This does not rule out that the intrinsic increased turnover of glutathione in
Der f skin sensitization and intranasal challenge induce type 1 respiratory allergy with increased airways resistances and lung inflammation
In order to follow as closely as possible the atopic march, asthma was induced by percutaneous sensitization and in- tranasal challenge (Figure 2A). Total and specific serum IgE levels significantly increased in Der f-allergic mice on day 35, one day after the 2 nd challenge (0.36 vs 1.14 μg/ml and 0.44 vs 1.6 A.U respectively) (Figure 2B), but not on day 26, before challenges (not shown), suggesting that skin exposure is not sufficient for inducing a full allergic re- sponse and that bronchial exposure is essential. Regarding respiratory function, Der f-allergic mice displayed airway hyperresponsiveness (AHR) one and three days (day 28 and day 30) after the first challenge (Figure 2C). A second challenge on day 35 increased AHR dramatically, a result confirmed using flexivent® on day 37 (Figure 2D). Overall
Univariate sensitivity analysis showed that parameter estimates remained largely stable as long as c was larger than 2 d -1 and k was smaller than 20 d -1 (Table 3).
We provided the first mathematical description of viral kinetics during EBOV infection in vivo in a small animal model. The model could well reproduce the viral load data and provided estimates of the elimination rate of infected cells and the basic reproductive number. Furthermore the dramatic decline in viremia in mice receiving oral favipiravir could also be modeled, revealing a strong antiviral effectiveness in blocking virion production in mice treated at 6 dpi. In mice treated at 8 dpi, the model predicted that death occurred before favipiravir achieved its maximal antiviral effectiveness.