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Jul 2020

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Activity-based Anorexia for Modeling Vulnerability and Resilience in Mice
基于活动的厌食症小鼠脆弱性和恢复力模型的建立   

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Abstract

Activity-based anorexia (ABA) is a widely used rodent model of anorexia nervosa. It involves combining limited access to food with unlimited access to a running wheel, leading to a paradoxical decrease in food intake, hyperactivity, and life-threatening weight loss. Although initially characterized in rats, ABA has been tested in mice with results that vary based on strain, sex, age, the amount of time food is available, and the number of days of food restriction. Here, we present our ABA protocol for modeling both vulnerability and resilience to diet and exercise in C57BL/6 female mice. While vulnerable mice exhibit the expected increase in running, reduction in food intake, and excessive weight loss, resilient mice exhibit an adaptive increase in food intake, decrease in total wheel running, and weight stabilization. In contrast to previous ABA studies in which resilience is defined by the relative rate of weight loss, our protocol leads to a resilient phenotype that more closely resembles the maintenance of a stable bodyweight exhibited by most humans who diet and exercise without developing anorexia nervosa. This protocol will be useful for future studies aimed at identifying the physiological and neural adaptations underlying both resilience and vulnerability to this eating disorder.

Keywords: Anorexia nervosa (神经性厌食症), Animal model (动物模型), Activity-based anorexia (基于活动的厌食症), Mouse (小鼠), Food restriction (限制性进食), Resilience (恢复力), Vulnerability (易损性), Hyperactivity (极度活跃)

Background

In the 1950s, Hall and Hanford first reported that rats placed on a restricted feeding schedule and given access to a running wheel exhibit a paradoxical increase in running and a decrease in food intake, leading to extreme weight loss (Hall et al., 1953; Hall and Hanford, 1954). The extent to which these responses are life-threatening was established in a later study, in which all the tested rats effectively ran themselves to death (Routtenberg and Kuznesof, 1967). Collectively, these findings formed the basis for the most commonly used animal model of anorexia nervosa, activity-based anorexia (ABA). ABA shares several characteristics with the human disorder, including extreme weight loss, increased voluntary physical activity, self-starvation, loss of estrus (Dixon et al., 2003), and greater vulnerability during adolescence (Woods and Routtenberg, 1971). As an animal model, it enables examination of how caloric restriction in combination with physical activity leads to neural and endocrine adaptations that may contribute to the development of the disorder. While most ABA studies have used rats, an increasing number are using mice, with results that vary based on strain, sex, age, the amount of time food is available, the number of days of food restriction, and how wheel-running is analyzed (Gelegen et al., 2007 and 2008; Klenotich et al., 2012). Given the number of variables affecting susceptibility to ABA, consistency across studies with respect to the parameters used and how the data are analyzed would enhance utility of the model.


A critical question in the field has been what factors promote vulnerability or resilience to anorexia nervosa. This has been difficult to model in ABA since most studies restrict food for 5 days or less, during which all animals show significant weight loss. Weight loss is particularly rapid in adolescent animals. Some investigators categorize animals as ‘vulnerable’ or ‘resilient’ based on how quickly they lose weight during this time period, either by evaluating individual differences within a group (Barbarich-Marsteller et al., 2013; Chowdhury et al., 2013) or by making strain comparisons (Gelegen et al., 2007). However, until recently no implementation of ABA has demonstrated a truly resilient phenotype, defined as adaptation and weight stabilization in the face of food restriction and exercise. We recently published our ABA study in which roughly half of the animals exhibit resilience by decreasing total running, increasing food intake, and reaching a stable bodyweight (Beeler et al., 2020). The other half of the animals are vulnerable to ABA, as demonstrated by increased wheel running, a failure to adapt food intake to increased energy expenditure, and excessive weight loss. Notably, we also detected vulnerability and resilience in food-restricted mice housed with a locked running wheel; although, wheel running does appear to accelerate weight loss. Here, we present the detailed protocol that we used to obtain distinct vulnerable and resilient phenotypes, which involves the use of young adult C57/BL6 female mice instead of adolescent mice, extending the number of days of food restriction from 5 to 10, and conducting a thorough analysis of individual differences.


This protocol will be useful for future studies aimed at characterizing vulnerability and resilience to anorexia nervosa. By generating both phenotypes, this protocol allows studies to examine the physiological, neural, and endocrine factors that differentiate the two, facilitating greater insight into the biological basis of this eating disorder. This protocol also provides an opportunity to evaluate the extent to which interventions decrease vulnerability and favor adaptation and resilience.


Materials and Reagents

  1. Mouse cages: 19.56 cm (width) × 30.91 cm (length) × 14.94 cm (height) (Model #9, Thoren Caging Systems, Inc., Hazleton, PA)

  2. Wire bar lids (WBL7115SMD-AMG, Allentown Inc., Allentown, NJ)

  3. Beta Chip Hardwood bedding (Northeastern Products, WF Fisher and Sons, Somerville, NJ)

  4. Prolab IsoPro rodent diet (WF Fisher & Son, Inc., catalog number: LD5P75) stored at 4°C

  5. Mouse igloo (Bio-Serv, catalog number: K3327)

  6. Clorox Fresh Scent Disinfecting Wipes

  7. Paper towels

  8. 8-week-old female C57BL/6N mice (Taconic Biosciences, Germantown, NY)

Equipment

  1. Low-profile wireless running wheels for mice (Med Associates, catalog number: ENV 047)

  2. USB interface hub for wireless running wheels (Med Associates, catalog number: DIG-807)

  3. PC computer

    Green features turned ‘off’ so that the display does not go off, the PC does not hibernate or go to sleep, and the hard drive does not turn off

  4. Analytical balance scale (1 mg)

  5. Compact scale (0.1 g) (Fisher Scientific, catalog number: 0191933)

  6. White noise machine (Marpac Dohm-Dual Speed)

  7. Digital thermometer (VWR, catalog number: 35519-047)

  8. External lights (GE Fluorescent Under Cabinet Light Fixtures)

  9. Light timer (Indoor 24 Hour Mechanical Outlet Timer with 2-Outlets)

  10. Red headlamp (Energizer headlamp with one red LED)

Software

  1. Wheel Manager (SOF-860, Med Associates, Inc., St. Albans, VT)

Procedure

  1. Order 8-week-old female C57BL/6N mice from Taconic Biosciences.

  2. Upon arrival, group house mice (3-4/cage) with beta chip bedding, Isopro food, a water bottle, and an igloo. Place cages in a temperature-controlled room dedicated exclusively to ABA testing, with the white noise machine turned ‘on’ high to minimize exposure to animal facility noise. Set the light timers to the 12-h light/dark cycle that will be used throughout the duration of the experiment. If the overhead lights cannot be controlled by a timer, turn them ‘off’ and control the light cycle with external fluorescent light fixtures (18-22 inch) plugged into a mechanical timer. The number of lights required will be determined by the size of the room. Two fluorescent lights are adequate for a room with 2 racks containing a total of 30 cages.

  3. Allow animals one week to acclimate with unlimited access to food and water.

  4. Start the experiment (one week after arrival)

    1. Weigh and allocate mice to one of four groups, counterbalancing weights across the groups. Information gained by testing each group is outlined in the Notes on Data Analysis section.

      1. Activity-based anorexia (ABA, freely moving running wheel, food restriction)

      2. Food-restricted control group (FR, locked wheel, food restriction)

      3. Wheel control group (WH, freely moving running wheel, unlimited food)

      4. Homecage control group (HC, locked wheel, unlimited food)

    2. Individually/singly house mice with either a freely moving running wheel (ABA and WH groups) or a wheel locked with a pin (FR and HC groups). Provide food and water to all groups ad libitum.

    3. Cage set-up: Remove all scents by wiping down each piece of the running wheel with a Clorox® wipe and drying with a paper towel. Place the base of the wheel flat against the bottom of the cage and cover with bedding. Orient the wire lid so that mice can access food or water without sitting on the wheel (Figure 1A). Verify that the wheel does not touch the wire lid and can freely turn (Figure 1B and Video 1). Limit the amount of bedding used so that a build-up of bedding underneath the wheel will not prevent it from turning.



      Figure 1. Overhead (A) and side (B) view of a running wheel inside a cage. Arrow indicates the space between the wire lid and the tip of the wheel.


      Video 1. Representative video of a mouse running on the wheel. Note that the water bottle and food were removed for illustrative purposes.


    4. Wheel set-up: Identify mice on the computer (Wheel Manager software) one at a time. Turn on one running wheel (top switch), enter the corresponding mouse ID into the computer, place the wheel in the cage, and then place the mouse in the cage with Isopro food and water before turning on the next wheel. After all IDs have been entered, click ‘Start Acquisition’ under the File menu.

  5. Baseline Data Collection (3 days):

    1. Baseline Day 1: Two days after individually housing the mice (Figure 2A), weigh the mice and food immediately prior to onset of the dark cycle. Different containers should be used (mouse only and food only) so that faeces and urine do not come into contact with the food. To minimize the number of times that each cage is moved, weigh the mouse and then the food from the same cage. When removing the mouse from the cage, place a finger on the running wheel to prevent it from rotating.

    2. Baseline Day 2: Follow the same procedure as the previous day.

    3. Baseline Day 3: Follow the same procedure as the previous day. Subsequently, remove the food from the ABA and FR groups 2 h after onset of the dark cycle. If the room does not contain an overhead red light, enter the room wearing a red headlamp so that the light/dark cycle is not disturbed.

  6. ABA Data Collection (10 days):

    1. Preparation: Each day, record the weight of large pellets of food (9-11 g) to the nearest 0.01 g for each ABA and FR mouse. Use fresh pellets in the event that a scent was left behind the previous day. Store food at room temperature in cups/containers labeled with the mouse ID and cover with foil.

    2. Immediately prior to onset of the dark cycle, weigh all the mice and food in HC and WH groups.

    3. Backup data without interrupting data collection by clicking ‘Export Data’ under the File menu.

    4. Place the labeled food containers in front of the corresponding cages.

    5. Once the lights have turned off, place the pre-weighed pellets in the overhead food bin of the ABA and FR mice and then leave the room for 2 h. Wear a red headlamp while administering the food, if necessary.

    6. Calculate the % baseline (%B) bodyweight by dividing the current weight by the bodyweight recorded on baseline day 3 and multiplying by 100. Any mouse that is at 75% of the baseline weight or lower is at risk of death and must be removed from the experiment.

    7. After 2 h of food availability (first 2-h of the dark cycle), remove the food pellets from the mice in the ABA and FR groups, place in the labeled containers, and cover with foil. Do not turn on the overhead lights when entering the ABA room; wear a red headlamp, if necessary. While in the room, fill the food bin (~40 g food) of mice that require removal from the experiment (≥ 25% bodyweight loss).

    8. Record the post-intake weights of food pellets to 0.01 g for the ABA and FR groups and calculate the intake during the 2-h period of food availability for each mouse. Throw away partially eaten food and weigh fresh food the next day.

    9. Repeat the steps above every day for 10 consecutive days.

    10. On ABA day 10, remove all remaining mice from the experiment after 2 h of unlimited access to food.

Data analysis

  1. Bodyweight data

    1. Conduct a survival analysis (e.g., Kaplan-Meier) of the ABA and FR groups across all 10 days of food restriction (Figure 2B). This is a way of comparing groups based on when they lost 25% or more of their baseline bodyweight and were removed from the experiment. Percent survival indicates the percentage of animals within each group that remained in the experiment (i.e., not removed) on each day. Earlier removal from the experiment indicates a higher vulnerability to ABA.

    2. For each day of food restriction, calculate the average percent baseline (%B) bodyweight for each group (Figure 2C). Given that mice are removed from the experiment on different days, use the linear mixed-effects model to analyze the data.



      Figure 2. Experimental timeline (A), survival curve (B), and bodyweight data (C). Numbers inside the survival curve indicate the number of surviving mice on each day of food restriction. Error bars indicate the SEM; n = 19 (ABA), n = 19 (FR), n = 12 (homecage), n = 14 (wheel control).


    3. Identification of Vulnerable and Resilient Phenotypes: Within the ABA and FR groups, characterize mice that were removed from the experiment as ‘vulnerable’ and mice that remained throughout the duration of the experiment as ‘resilient.’ A comparison of bodyweight across the days of food restriction reveals rapid weight loss in vulnerable mice and maintenance of bodyweight in resilient mice (Figure 3).



      Figure 3. Bodyweight of individual mice (light traces) across the days of food restriction in (A) activity-based anorexia (ABA) and (B) food-restricted control (FR) groups. Group averages for resilient (dashed line) and vulnerable (solid line) mice are shown in bold. n = 10 vulnerable ABA, n = 9 resilient ABA, n = 6 vulnerable FR, n = 13 resilient FR.


  2. Food intake data

    1. Average the food intake across all 10 days of the experiment and use a one-way ANOVA for group comparisons (Figure 4A).

    2. Compare the average intake of vulnerable and resilient ABA and FR mice across all 10 days of the experiment using a two-way ANOVA (Figure 4B).

    3. Calculate the average intake before each animal’s last day in the experiment and the intake on the day of removal. Compare vulnerable and resilient ABA and FR mice using a two-way ANOVA (Figure 4C).



    Figure 4. Food intake data. A. Food intake for all mice in each group averaged across 10 days of restriction. B. Average food intake of vulnerable (solid) and resilient (hatched) mice in the ABA and FR groups. C. Average food intake of vulnerable (triangle) and resilient (square) mice in the ABA and FR groups prior to removal vs. the day of removal. *P < 0.05; ***P < 0.001; †P < 0.01 vs. vulnerable groups before removal. Error bars indicate the SEM.


  3. Running wheel data

    1. Use the Wheel Manager software (SOF-860, Med Associates, Inc., St. Albans, VT) to extract the running wheel data. Calculate the average total running across all 10 days of the experiment for wheel control, vulnerable ABA, and resilient ABA mice. Use a one-way ANOVA for group comparisons (Figure 5A).

    2. Separately calculate the average light cycle (12-h) and dark cycle (12-h) running for each group and use a one-way ANOVA for group comparisons (Figure 5B, 5C).

    3. Optional: Calculate the maximum increase in light cycle and dark cycle running across 2 consecutive days of the experiment. Our work indicates that vulnerability to ABA is associated with an abrupt increase in light cycle running, as indicated by a significantly larger increase in running over a 2-day period (Figure 5D). This abrupt increase in light cycle running can occur on any day of food restriction but precedes removal from the experiment by 1-3 days for most mice.



      Figure 5. Wheel running data. (A) Total, (B) light cycle, and (C) dark cycle running averaged across the days of food restriction. (D) Maximum increase in wheel running across 2 consecutive days (averaged by group) during the light and dark cycles. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate the SEM.


    4. Optional: Collate the running wheel data into 5-min bins for each mouse on each day of the experiment (baseline day 1 to ABA day 10). Graph the data in a dot plot, with day-time on the x-axis and wheel revolutions on the y-axis; color code by group and indicate light and dark cycles (Figure 6A). Additionally, collate the running wheel data into 1-h blocks and then average across all baseline days (Figure 6B) and experimental/food restriction days (Figure 6C) for each group. The resulting graphs demonstrate group differences in running across the 24-h circadian cycle and can be used to characterize the extent of light cycle running. Running that precedes presentation of the food is referred to as food anticipatory activity.



      Figure 6. Analysis of the running wheel data across the 24-h circadian cycle. A. Dot plot of wheel running across the experimental days for all mice in 5-min bins. Black and gray bars indicate dark and light cycles, respectively. The red arrow indicates the start of food restriction (FR). B-C. Group averages across a 24-h period for (B) baseline days and (C) experimental days. The dotted lines mark the light cycle between 6 (lights on) and 18 (lights off) hours. Error bars indicate the SEM.

Notes

Notes on the Procedure:

  1. Mice have been seen eating Bed-o’Cob bedding when hungry, which is why this protocol uses beta chip bedding.

  2. Noise provokes activity in mice, including running behavior. To generate reproducible data, use a colony room that is dedicated solely to ABA testing so that the experimenter is the only person entering the room each day and noise is minimized.

  3. When the lights are on, the experimenter should only enter the room shortly before the onset of the dark cycle to weigh the mice and food. Entering the room earlier may wake the mice and cause them to run, confounding the results. It can be challenging to weigh a large cohort of mice within this limited time period. We suggest entering the room no sooner than 45 min before the lights go off, which we find is enough time to weigh 20-30 mice, depending on whether they have unlimited access to food.

  4. Room temperature has been shown to affect vulnerability to activity-based anorexia (Gutierrez et al., 2002). ABA should be tested in a room where the temperature is tightly controlled. In our experiments, the room temperature is monitored daily and ranges from 70-75°F.

  5. Vulnerability to ABA varies based on the age and strain of the mouse (Gelegen et al., 2007; Klenotich and Dulawa, 2012; Klenotich et al., 2012). Using a strain and/or age group that differs from that specified in this protocol will likely affect the distribution of vulnerable and resilient mice, such that all mice may only exhibit one phenotype.

  6. This protocol uses female mice since anorexia nervosa is more prevalent in women than men. It is possible that using male mice will lead to different results.

  7. The vast majority of mice that have lost 25% of their baseline weight and are removed from the experiment will recover overnight; however, this is not true for all mice. If a mouse appears particularly weak, we recommend placing a moistened pellet of food on the floor of the cage (in addition to filling the food bin) during the recovery period.

  8. Give food-restricted mice the largest pellets of food. This will reduce the possibility that a small piece of food will fall into the cage during feeding.


Notes on Data analysis:

  1. A comparison of food intake in the ABA and FR groups reveals how much wheel running affects consumption when food is only available for 2 h. A comparison of wheel running in the ABA and WH groups reveals how much food restriction affects the level of activity. The HC control group provides a measure of bodyweight and food intake in the absence of food restriction and/or wheel running.

  2. All data shown in Figures 1-6 have previously been published (Beeler et al., 2020).

Acknowledgments

This work was supported by grants from the National Institute on Drug Abuse, DA046058 (JAB), the National Institute of Mental Health, R21MH114182 (NSB), the National Institute on Minority Health and Health Disparities of the NIH, G12MD007599 (NSB), the PSC-CUNY Awards program jointly funded by the Professional Staff Congress and the City University of New York (JAB & NSB), and the Klarman Family Foundation Eating Disorders Research Grants Program (JAB & NSB). The described protocol was adapted from previous studies in which activity-based anorexia was tested in other mouse strains (Klenotich and Dulawa, 2012; Klenotich et al., 2012).

Competing interests

The authors have no financial or non-financial competing interests to report.

Ethics

This protocol was approved by the Institutional Animal Care and Use Committees of Hunter College (NB-Anorexia, 9/1/20-8/31/23) and Queens College (ACUP# 164, 2/26/20-2/25/23), USA.

References

  1. Barbarich-Marsteller, N. C., Underwood, M. D., Foltin, R. W., Myers, M. M., Walsh, B. T., Barrett, J. S. and Marsteller, D. A. (2013). Identifying novel phenotypes of vulnerability and resistance to activity-based anorexia in adolescent female rats. Int J Eat Disord 46(7): 737-746.
  2. Beeler, J. A., Mourra, D., Zanca, R. M., Kalmbach, A., Gellman, C., Klein, B. Y., Ravenelle, R., Serrano, P., Moore, H., Rayport, S., Mingote, S. and Burghardt, N. S. (2020). Vulnerable and Resilient Phenotypes in a Mouse Model of Anorexia Nervosa. Biol Psychiatry doi: 10.1016/j.biopsych.2020.06.030.
  3. Chowdhury, T. G., Wable, G. S., Sabaliauskas, N. A. and Aoki, C. (2013). Adolescent female C57BL/6 mice with vulnerability to activity-based anorexia exhibit weak inhibitory input onto hippocampal CA1 pyramidal cells. Neuroscience 241: 250-267.
  4. Dixon, D. P., Ackert, A. M. and Eckel, L. A. (2003). Development of, and recovery from, activity-based anorexia in female rats. Physiol Behav 80(2-3): 273-279.
  5. Gelegen, C., Collier, D. A., Campbell, I. C., Oppelaar, H., van den Heuvel, J., Adan, R. A. and Kas, M. J. (2007). Difference in susceptibility to activity-based anorexia in two inbred strains of mice. Eur Neuropsychopharmacol 17(3): 199-205.
  6. Gelegen, C., van den Heuvel, J., Collier, D. A., Campbell, I. C., Oppelaar, H., Hessel, E. and Kas, M. J. (2008). Dopaminergic and brain-derived neurotrophic factor signalling in inbred mice exposed to a restricted feeding schedule. Genes Brain Behav 7(5): 552-559.
  7. Gutierrez, E., Vazquez, R. and Boakes, R. A. (2002). Activity-based anorexia: ambient temperature has been a neglected factor. Psychon Bull Rev 9(2): 239-249.
  8. Hall, J. F. and Hanford, P. V. (1954). Activity as a function of a restricted feeding schedule. J Comp Physiol Psychol 47(5): 362-363.
  9. Hall, J. F., Smith, K., Schnitzer, S. B. and Hanford, P. V. (1953). Elevation of activity level in the rat following transition from ad libitum to restricted feeding. J Comp Physiol Psychol 46(6): 429-433.
  10. Klenotich, S. J. and Dulawa, S. C. (2012). The activity-based anorexia mouse model. Methods Mol Biol 829: 377-393.
  11. Klenotich, S. J., Seiglie, M. P., McMurray, M. S., Roitman, J. D., Le Grange, D., Dugad, P. and Dulawa, S. C. (2012). Olanzapine, but not fluoxetine, treatment increases survival in activity-based anorexia in mice. Neuropsychopharmacology 37(7): 1620-1631.
  12. Routtenberg, A. and Kuznesof, A. W. (1967). Self-starvation of rats living in activity wheels on a restricted feeding schedule. J Comp Physiol Psychol 64(3): 414-421.
  13. Woods, D. J. and Routtenberg, A. (1971). "Self-starvation" in activity wheels: developmental and chlorpromazine interactions. J Comp Physiol Psychol 76(1): 84-93.

简介

[摘要]活动性厌食症(ABA)是一种广泛应用的神经性厌食症啮齿动物模型。它将有限的食物和无限的轮子结合起来,导致食物摄入量的减少,过度活跃和威胁生命的体重减轻。虽然ABA最初是在大鼠身上发现的,但在小鼠身上进行了试验,结果因品种、性别、年龄、食物供应时间和禁食天数而异。在这里,我们提出了我们的ABA协议,用于模拟C57BL/6雌性小鼠对饮食和运动的脆弱性和恢复力。当易受伤害的小鼠表现出预期的跑步增加、食物摄入减少和过度体重减轻时,有弹性的小鼠表现出适应性的食物摄入增加、轮动减少和体重稳定。与以前的ABA研究不同,在ABA研究中,恢复力是由相对的体重减轻率来定义的,我们的研究方案导致了一种恢复力表型,这种表型更接近于大多数饮食和运动没有发展成神经性厌食症的人所表现出的维持稳定体重。这一方案将有助于今后的研究,旨在确定潜在的生理和神经适应的韧性和脆弱性,这种饮食失调。

[背景]20世纪50年代,Hall和Hanford首次报道说,限制喂食并允许老鼠使用跑步机的老鼠表现出自相矛盾的跑步增加和食物摄入减少,导致体重极度减轻(Hall等人,1953年;霍尔和汉福德,1954年)。这些反应对生命的威胁程度在后来的一项研究中得到了证实,在这项研究中,所有受试大鼠都有效地自行死亡(Routtenberg和Kuznesof,1967)。这些发现共同构成了神经性厌食症最常用的动物模型,即基于活动的厌食症(ABA)的基础。ABA与人类疾病有几个共同的特征,包括极度的体重减轻、自愿的体力活动增加、自我饥饿、发情期减少(Dixon等人,2003年),以及青春期更易受伤害(Woods和Routtenberg,1971年)。作为一种动物模型,它可以检查热量限制和体力活动是如何导致神经和内分泌适应的,而神经和内分泌适应可能导致疾病的发展。虽然大多数ABA研究使用的是老鼠,但越来越多的研究使用的是老鼠,研究结果因压力、性别、年龄、食物供应时间、禁食天数以及如何分析车轮转动而有所不同(Gelegen等人,2007年和2008年;Klenotich等人,2012年)。考虑到影响ABA易感性的变量数量,研究中所用参数的一致性以及数据分析的方式将提高模型的实用性。

该领域的一个关键问题是什么因素促进神经性厌食症的脆弱性或恢复力。这在ABA中很难建模,因为大多数研究限制进食5天或更少,在此期间所有动物都表现出显著的体重减轻。青春期动物的体重减轻尤其迅速。一些研究人员根据动物在这段时间内减肥的速度,将动物分为“易受伤害”和“有弹性”两类,或者通过评估群体内的个体差异(BarbarichMarsteller等人,2013;Chowdhury等人,2013)或进行菌株比较(Gelegen等人,2007)。然而,直到最近,ABA还没有表现出真正的弹性表型,即面对食物限制和运动时的适应和体重稳定。我们最近发表了我们的ABA研究,其中大约一半的动物通过减少总跑步量、增加食物摄入量和达到稳定体重表现出韧性(Beeler et al.,2020)。另一半的动物很容易受到ABA的影响,表现为车轮转动增加,食物摄入不能适应增加的能量消耗,体重过度减轻。值得注意的是,我们还检测到了禁食老鼠的脆弱性和弹性,这些老鼠被关在一个锁着的轮子里;尽管如此,跑轮子似乎确实能加速减肥。在这里,我们提出了我们用来获得不同的脆弱和弹性表型的详细方案,包括使用年轻成年C57/Bl6雌性小鼠而不是青春期小鼠,将禁食天数从5天延长到10天,并对个体差异进行彻底分析。

该方案将有助于今后的研究,旨在表征神经性厌食症的脆弱性和恢复力。通过产生这两种表型,这一方案允许研究检验区分这两种表型的生理、神经和内分泌因素,有助于更深入地了解这种饮食失调的生物学基础。该议定书还提供了一个机会,评估干预措施在多大程度上减少脆弱性,有利于适应和恢复能力。

关键字:神经性厌食症, 动物模型, 基于活动的厌食症, 小鼠, 限制性进食, 恢复力, 易损性, 极度活跃

材料和试剂

1.    鼠笼:19.56厘米(宽)× 30.91厘米(长)× 14.94厘米(高)(型号#9,Thoren Caging Systems,Inc.,Hazleton,PA)

2.    线材盖(WBL7115SMD-AMG,Allentown Inc.,Allentown,NJ)

3.    贝塔片式硬木床上用品(Northeast Products,WF Fisher and Sons,Somerville,NJ)

4.    Prolab异丙醇啮齿动物饲料(WF Fisher&Son,Inc.,目录号:LD5P75)储存于4°C级

5.    老鼠冰屋(Bio Serv,目录号:K3327)

6.    Clorox清香消毒湿巾

7.    纸巾

8.    8周龄雌性C57BL/6N小鼠(Taconic Biosciences,纽约州日耳曼敦)



设备


1.     轻巧的无线鼠标滚轮(Med Associates,目录号:ENV 047)

2.     用于无线车轮的USB接口集线器(Med Associates,目录号:DIG-807)

3.     PC计算机

绿色功能已关闭,因此显示器不会关闭,电脑不会休眠或进入睡眠状态,硬盘也不会关闭

4.     分析天平(1毫克)

5.     小型天平(0.1 g)(Fisher Scientific,目录号:0191933)

6.     白噪声机(Marpac Dohm双速)

7.     数字温度计(VWR,目录号:35519-047)

8.     外部灯(GE荧光灯柜下灯具)

9.     灯光定时器(室内24小时机械插座定时器,带2个插座)10。红色前照灯(带一个红色LED的电源前照灯)



软件软件



1车轮经理(悉尼威立雅州圣奥尔本市医疗联合公司SOF-860)



程序


1.     从Taconic Biosciences订购8周龄雌性C57BL/6N小鼠。

2.     到达后,用贝塔芯片床上用品、异丙醇食品、水瓶和冰屋将家鼠(3-4只/笼)分组。将笼子放在专门用于ABA测试的温度控制室中,白噪声机高开,以尽量减少动物设施噪声的暴露。将光定时器设置为在整个实验期间使用的12小时光/暗循环。如果顶灯不能由计时器控制,请将其“关闭”,并使用插入机械计时器的外部荧光灯(18-22英寸)控制灯光循环。所需的灯光数量将由房间大小决定。两个荧光灯足够一个有两个架子的房间,总共有30个笼子。

3.     允许动物在一周内适应环境,不受限制地获取食物和水。

4.     开始实验(到达后一周)

答。称量小鼠体重并将其分配到四组中的一组,在各组间平衡重量。

通过测试每组获得的信息在数据分析部分的注释中概述。一。基于活动的厌食症(ABA,自由运动的转轮,食物限制)。限食对照组(FR,锁轮,限食)iii.轮控组(WH,自由行走轮,不限食)iv.家笼对照组(HC,锁轮,不限食)

b。单独/单独饲养小鼠,带有自由移动的轮子(ABA和WH组)或用销钉锁定的轮子(FR和HC组)。向所有团体随意提供食物和水。

c。笼子的设置:用Clorox擦拭运转轮上的每一块,去除所有气味® 用纸巾擦干。将车轮底座平放在保持架底部,并用垫层覆盖。调整电线盖的方向,使老鼠不用坐在轮子上就可以接触到食物或水(图1A)。确认车轮没有接触导线盖并且可以自由转动(图1B和视频1)。限制所用垫层的数量,使垫层在车轮下方的堆积不会阻止车轮转动。





图1。笼内运行轮的俯视图(A)和侧视图(B)。箭头表示导线盖和车轮尖端之间的空间。





视频1。鼠标在轮子上运行的代表性视频。请注意,为了便于说明,已将水瓶和食物取下。



d。轮子设置:一次识别一个电脑上的鼠标(轮子管理软件)。打开一个滚轮(顶部开关),将相应的鼠标ID输入计算机,将滚轮放入笼子中,然后将鼠标放入装有异丙醇食物和水的笼子中,然后再打开下一个滚轮。输入所有ID后,单击“开始”

“文件”菜单下的“采集”。

5.     基线数据收集(3天):

答。基线第1天:单独饲养小鼠两天后(图2A),在黑暗周期开始前立即称量小鼠和食物的重量。应使用不同的容器(仅限老鼠和食物),以免粪便和尿液与食物接触。为了尽量减少每个笼子被移动的次数,先称量老鼠的重量,然后再称量同一笼子里的食物。将鼠标从笼中取出时,请将手指放在运行的滚轮上,以防其旋转。

b。基线检查第2天:遵循与前一天相同的程序。

c。基线检查第3天:按照与前一天相同的程序进行。随后,在黑暗周期开始后2小时,从ABA和FR组中移除食物。如果房间没有上方的红灯,请戴上红色前照灯进入房间,以免干扰明暗循环。

6ABA数据收集(10天):

答。制备:每天记录大颗粒食物(9-11克)的重量,精确到每只ABA和FR小鼠的0.01克。使用新鲜的颗粒,以防前一天留下香味。在室温下将食物储存在标有鼠标ID的杯子/容器中,并用箔纸覆盖。

b。在黑暗周期开始前,对HC和WH组的所有小鼠和食物进行称重。

c。通过单击“文件”菜单下的“导出数据”,在不中断数据收集的情况下备份数据。

d。将贴有标签的食品容器放在相应的笼子前面。

e。一旦灯关闭,将预先称重的颗粒放在ABA和FR小鼠头顶的食物箱中,然后离开房间2小时。如有必要,在进食时戴上红色前照灯。

f。将当前体重除以基线检查第3天记录的体重,再乘以100,计算出基线检查时的体重百分比(%B)。任何体重为基线体重75%或更低的老鼠都有死亡的危险,必须从实验中移除。

g。食物供应2小时后(黑暗循环的前2小时),从ABA和FR组的小鼠中取出食物颗粒,放置在标记的容器中,并用铝箔覆盖。进入ABA房间时不要打开顶灯;必要时佩戴红色前照灯。在房间里,装满需要从实验中移除的老鼠的食物箱(约40克食物)(≥ 25%体重减轻)。

h。记录ABA组和FR组的食物丸摄入后重量为0.01 g,并计算每只小鼠在2小时食物供应期内的摄入量。扔掉部分吃过的食物,第二天称新鲜食物的重量。

一。每天重复上述步骤,连续10天。

j。在ABA第10天,在2小时无限量进食后,将所有剩余的小鼠从实验中移除。



数据分析


1.     体重数据

答。在所有10天的食物限制期间,对ABA和FR组进行生存分析(如Kaplan-Meier)(图2B)。这是一种比较各组的方法,基于他们何时减掉25%或更多的基线体重并从实验中移除。存活率表示各组中每天仍在实验中(即未移除)的动物百分比。早期从实验中移除表明对ABA有更高的脆弱性。

b。对于每天的食物限制,计算各组的平均基线百分比(%B)体重(图2C)。假设小鼠在不同的时间被移出实验,使用线性混合效应模型来分析数据。





图2。实验时间线(A)、存活曲线(B)和体重数据(C)。存活曲线内的数字表示每天禁食时存活的老鼠数量。误差线表示扫描电镜;n=19(ABA),n=19(FR),n=12(homecage),n=14(车轮控制)。



c。识别脆弱和弹性表型:在ABA和FR组内,将从实验中移除的小鼠描述为“易受伤害的”,而在整个实验过程中保持的小鼠描述为“有弹性的”。通过对禁食天数内的体重进行比较,可以发现易受伤害小鼠的体重迅速下降,而有弹性小鼠的体重保持不变(图3)。





图3。(A)活动性厌食症(ABA)和(B)限食对照组(FR)禁食天数内小鼠个体体重(轻痕)。弹性(虚线)和脆弱(实线)小鼠的组平均值以粗体显示。n=10脆弱ABA,n=9弹性ABA,n=6脆弱FR,n=13弹性FR。



2.     食物摄入数据

答。在整个10天的实验中平均食物摄入量,并使用单因素方差分析进行组间比较(图4A)。

b。使用双向方差分析(图4B)比较整个10天实验中脆弱和有弹性的ABA和FR小鼠的平均摄入量。

c。计算实验中每只动物最后一天之前的平均摄入量和移除当天的摄入量。使用双向方差分析比较脆弱和有弹性的ABA和FR小鼠(图4C)。





图4。食物摄入数据。答。各组小鼠在禁食10天内的平均摄食量。B。ABA和FR组中脆弱(固体)和弹性(孵化)小鼠的平均食物摄入量。C。ABA组和FR组中脆弱(三角形)和弹性(方形)小鼠在移除前与移除当天的平均食物摄入量*P<0.05***P<0.001;†P<

0.01与移除前的弱势群体相比。误差线表示扫描电镜。



3.     行走轮数据

答。使用车轮管理器软件(SOF-860,Med Associates,Inc.,St.Albans,VT)提取行驶车轮数据。计算轮控、脆弱ABA和有弹性ABA小鼠在整个10天实验中的平均总跑步量。使用单因素方差分析进行分组比较(图5A)。

b。分别计算各组的平均光周期(12小时)和暗周期(12小时),并使用单因素方差分析进行组间比较(图5B、5C)。

c。可选项:计算连续两天实验中光周期和暗周期的最大增量。我们的研究表明,ABA的脆弱性与光周期运行的突然增加有关,如2天内运行的显著增加所示(图5D)。这种光周期运行的突然增加可以发生在任何一天的食物限制,但在实验前1-3天从大多数小鼠。





图5。车轮运行数据。(A) 总的,(B)光周期,和(C)黑暗周期运行的平均数横跨食物限制的日子(D) 在明暗循环中,连续两天(按组平均)车轮运行的最大增加量*P<0.05**P<0.01***P<0.001。误差线表示扫描电镜。



d。可选项:在实验的每一天(基线第1天到ABA第10天),为每只老鼠将转轮数据整理成5分钟的箱子。在点图中绘制数据,x轴上显示白天时间,y轴上显示车轮转数;按组显示颜色代码并指示明暗周期(图6A)。此外,将转轮数据整理成1小时的数据块,然后对所有基线日(图6B)和实验/禁食日(图6B)进行平均

6C)每组。得到的图表显示了在跑步中的群体差异

24小时的昼夜节律周期可以用来表征光周期运行的程度。

在呈现食物之前的跑步被称为食物预期活动。





图6。分析24小时昼夜节律周期的转轮数据。答。所有小鼠在5分钟箱中的轮在实验日内运行的点图。黑色和灰色条分别表示暗周期和光周期。红色箭头表示开始限食(FR)。B-C。(B)基线日和(C)实验日的24小时组平均值。虚线表示6小时(开灯)到18小时(熄灯)之间的光周期。误差线表示扫描电镜。



笔记


程序说明:

1.     有人看到老鼠饿了就吃Bed-o'Cob床上用品,这就是为什么这个方案使用beta芯片床上用品。

2.     噪音会刺激老鼠的活动,包括跑步行为。为了产生可复制的数据,使用专门用于ABA测试的菌落室,以便实验者是每天唯一进入房间的人,并且噪音最小化。

3.     当灯亮起时,实验者只应在黑暗周期开始前不久进入房间,称量老鼠和食物的重量。提前进入房间可能会吵醒老鼠,导致它们奔跑,混淆结果。在这个有限的时间段内给一大群老鼠称重是一个挑战。我们建议在灯熄灭前45分钟内进入房间,我们发现这足够让老鼠体重达到20-30只,这取决于它们是否能无限获取食物。

4.     室温已被证明会影响对基于活动的厌食症的脆弱性(Gutierrez等人,2002年)。ABA应在温度严格控制的室内进行检测。在我们的实验中,室内温度每天监测,范围为70-75℃°F。

5.     对ABA的脆弱性因小鼠的年龄和品系而异(Gelegen等人,2007;Klenotich和Dulawa,2012年;Klenotich等人,2012年)。使用与本方案中规定的不同的品系和/或年龄组可能会影响脆弱和有弹性小鼠的分布,因此所有小鼠可能只表现出一种表型。

6.     由于神经性厌食症在女性中比男性更为普遍,因此该方案使用雌性小鼠。使用雄性小鼠可能会导致不同的结果。

7.     绝大多数已经减掉了25%的基线体重的老鼠会在一夜之间康复;然而,并非所有的老鼠都是这样。如果老鼠显得特别虚弱,我们建议在恢复期将湿润的食物颗粒放在笼子的地板上(除了装满食物箱)。

8.     给禁食的老鼠吃最大粒的食物。这将减少一小块食物在喂食过程中落入笼子的可能性。



数据分析说明:

1.     比较ABA组和FR组的食物摄入量可以发现,当食物只能供应2小时时,车轮转动对消耗的影响有多大。比较ABA组和WH组的车轮运动,可以发现食物限制对活动水平的影响有多大。HC对照组在没有食物限制和/或车轮转动的情况下提供体重和食物摄入量的测量。

2.     图1-6所示的所有数据之前都已公布(Beeler等人,2020年)。



致谢


这项工作得到了国家药物滥用研究所DA046058(JAB)、国家精神卫生研究所R21MH114182(NSB)、国家卫生院少数民族健康和健康差异研究所G12MD007599(NSB)的资助,PSC-CUNY奖励计划由专业人员大会和纽约城市大学(JAB&NSB)以及Klarman家庭基金会饮食失调研究资助计划(JAB&NSB)共同资助。所述方案改编自先前的研究,其中在其他小鼠品系中测试了基于活动的厌食症(Klenotich和Dulawa,2012;Klenotich等人,2012年)。



相互竞争的利益相互竞争的利益



提交人没有财务或非财务利益可供报告。





伦理学


该方案由美国亨特学院(NB厌食症,9/1/20-8/31/23)和皇后学院(ACUP#164,2/26/20-2/25/23)的机构动物护理和使用委员会批准。



参考文献


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Serrano,P.,Moore,H.,Rayport,S.,Mingote,S。伯格哈特,N。S(2020). 神经性厌食症小鼠模型的脆弱和弹性表型。生物精神病学doi:10.1016/j.biopsych.2020.06.030。

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卡斯,M。J(2007). 两种近交系小鼠对活动性厌食症易感性的差异。欧洲神经精神药理学17(3):199-205。

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引用:Beeler, J. A. and Burghardt, N. S. (2021). Activity-based Anorexia for Modeling Vulnerability and Resilience in Mice. Bio-protocol 11(9): e4009. DOI: 10.21769/BioProtoc.4009.
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