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HomeNeuroscienceFrontiers | Male and Feminine C57BL/6 Mice Reply In a different way...

Frontiers | Male and Feminine C57BL/6 Mice Reply In a different way to Awake Magnetic Resonance Imaging Habituation



Anesthetics have lengthy been normal follow and a necessity in fundamental and preclinical neuroscience utilizing rodent magnetic resonance imaging (MRI) (Ferris et al., 2011). Throughout scanning, even minor head movement by the animal can distort the acquired photographs, thus corrupting the information set or at finest complicating the information evaluation and subsequent interpretation of the outcomes (Hajnal et al., 1994). Because of this, anesthetics have been used to offer animal immobility whereas concurrently making certain sign acquisition with low stress and excessive reproducibility (Hanusch et al., 2007). Lately, different methods primarily based on educated, awake animals have emerged (King et al., 2005; Yoshida et al., 2016; Han et al., 2019). This effort is partly pushed by the well-known incontrovertible fact that anesthesia modifies varied physiological parameters and processes corresponding to vascular reactivity, mind metabolism, and neuronal and glial actions (Gao et al., 2017). Regardless of these shortcomings, anesthesia utility in rodent MRI stays a normal, and far effort has additionally gone into optimizing anesthetic protocols to reduce physiological alterations attributable to totally different anesthetic brokers (Schlegel et al., 2015; Sharp et al., 2015; Petrinovic et al., 2016; Shim et al., 2018). Nonetheless, many varieties of experiments corresponding to task-based purposeful (f) MRI or comparisons of the awake with the sleep state can’t be carried out within the anesthetized animal. Subsequently, the transition to awake imaging is important to refine rodent MRI as a translational device. Thus, in recent times an array of recent MRI in vivo strategies have emerged for imaging each rats and mice within the awake state (Ferris et al., 2011). The principle benefit of transitioning to awake MRI is the flexibility to accumulate imaging knowledge that stay utterly unperturbed by the confounding results of anesthesia, thus bettering the examine of the mind in its pure working state (Peeters et al., 2001; Wang et al., 2010). One other consideration is the widespread use of awake animals in optical imaging of the mind. That is considerably simpler to implement as these microscopy strategies are largely silent compared to MRI and due to this fact much less annoying for the animal. To ensure that such optical strategies to totally complement preclinical MRI, the transition to awake MRI is required as knowledge are in any other case acquired in vastly totally different physiological states. As of right now, awake MRI in rodents has already been utilized in a broad number of neuroscience purposes, together with awake fMRI of the default mode community (Yoshida et al., 2016; Madularu et al., 2017) alone or mixture with optogenetic stimulation (Desai et al., 2011), chemogenetics (Giorgi et al., 2017), sensory stimulation (Harris et al., 2015; Chen et al., 2020), behavioral duties (Han et al., 2019), evaluation of cortical plasticity (Matsubayashi et al., 2018), drug response (Moore et al., 2016), glymphatic operate (Stanton et al., 2021), and varied illness fashions (Ferris et al., 2014). Some MRI research, nevertheless, require intensive scan protocols both because of the want to accumulate a number of knowledge units for evaluation (e.g., for quantitative MRI combining diffusion, perfusion, and relaxometry) or gated acquisitions the place closing scan time is determined by the animal respiration sample. For such research to be possible with awake animals a validated, prolonged coaching scheme because the one offered right here is required.

When conducting awake rodent MRI, different key technical and methodological challenges come up. One main problem is to reduce the awake animal’s physique and head movement ranges in the course of the stay imaging classes. In earlier awake rodent MRI research, minor and steady physique and head movement ranges have been completed by both surgically implanted head holders or by (Bergmann et al., 2016; Chang et al., 2016; Yoshida et al., 2016; Han et al., 2019; Chen et al., 2020) utilizing much less invasive restraining kits that make the most of pads securing the top and decrease physique (stomach) (Madularu et al., 2017) to restrain the animals. With out habituation, nevertheless, restraining can induce extreme stress that in flip may confound the acquired imaging knowledge (Asaad and Lee, 2018). Subsequently, animals must endure repeated habituation classes to be comfy not solely with being restrained but additionally with the loud acoustic noise attributable to the MR gradients throughout scans. The rodent habituation can both be carried out utilizing an obtainable MRI scanner, a mock MRI surroundings, or a mixture of each (Chen et al., 2020).

To make sure that animals are sufficiently habituated to the MRI surroundings, many various physiological and behavioral parameters can be utilized as proxy measurements of animal’s stress ranges. These parameters embody coronary heart fee (Ferris et al., 2011), respiratory fee (Ferris et al., 2011; Chang et al., 2016; Madularu et al., 2017), physique weight (Yoshida et al., 2016), quantity and weight of fecal boli (Yoshida et al., 2016; Madularu et al., 2017), corticosterone blood plasma ranges (Ferris et al., 2011), ultrasonic calls, and motion-associated fMRI variance (King et al., 2005; Madularu et al., 2017). Monitoring these parameters over the habituation interval is crucial as a result of it’s nonetheless not totally understood how rodents are affected (short-term and long-term) by the presently employed habituation procedures. This lack of perception holds again refinements to the habituation procedures and clouds potential variations that female and male rodents could exhibit. Limitations apart, steady MRI habituation is reported by a number of teams to lower rodent stress general and cut back stress-related conduct. Nonetheless, no consideration has been dedicated to investigating if intercourse variations in habituation response exist for neither rats nor mice. For lengthy, many researchers have relied completely on male rodents for animal experiments, primarily to keep away from the physiological variability linked with the estrous cycle of feminine rodents. Nonetheless, focusing solely on males severely holds again analysis, as organic outcomes, from a translational perspective, could solely be related to half of the inhabitants (Mauvais-Jarvis et al., 2017). With the regular improve in consideration to using feminine topics in science (Luine et al., 2017) and the rise in reputation of awake preclinical MRI (Ferris et al., 2011), it’s essential to elucidate sexual variations in response to habituation. It’s well-acknowledged that female and male rodents reply otherwise to stressors (Luine et al., 2017; Rincón-Cortés et al., 2019) and exhibit totally different anxiety-related behaviors (An et al., 2011). For instance, feminine rodents secrete a better focus of CORT in response to bodily and physiological stressors than do their male counterparts. As well as, feminine rodents additionally exhibit larger concentrations of corticosterone at baseline (Rincón-Cortés et al., 2019). Subsequently, it’s doubtless that female and male rodents will differ of their responses throughout habituation procedures. If true, such findings would require researchers to take these variations into consideration when habituating mice for awake MRI.

For these causes, this examine aimed to analyze the potential intercourse variations that mice exhibit in response to repeated MRI habituation. For this, we developed our setup for awake mouse MRI habituation counting on surgical implants beforehand established (Han et al., 2019; Chen et al., 2020) for head fixation. We monitored coronary heart fee, physique weight fluctuations, fecal boli weight and assessed corticosterone focus ranges in the course of the size of the MRI habituation protocol and in contrast knowledge between the sexes. Corticosterone measurements had been additionally carried out previous to animal dealing with and a pair of weeks after the MRI habituation had ended. The extent of the animal’s discomfort throughout MRI habituation session had been additionally assessed by scoring photographs from the facial video feed utilizing the mouse grimace scale (MGS). As well as, we employed the sunshine/darkish anxiousness check (LDT) to evaluate mouse anxiety-related and locomotor exercise responses to awake MRI habituation process and in contrast these parameters between men and women. Lastly, we repeated the LDT 14 days after the top of the MRI habituation protocol. On the whole, female and male mice reply to MRI habituation in methods per earlier awake mouse MRI research aiming for habituation for shorter scan classes. Habituation for prolonged awake MRI scan classes is due to this fact doable. Nonetheless, female and male mice present distinctly totally different responses by the course of our coaching protocol which means that 14-day habituation is required to ensure that each sexes to be prepared for awake MRI.

Supplies and Strategies


Naïve male (n = 20) and feminine (n = 22) 6-week-old C57BL/6 (Taconic Bioscience Inc., Ejby, Denmark) mice had been used throughout this examine. A subset of male (n = 9) and feminine (n = 9) mice had been chosen for fecal corticosterone metabolite evaluation and weren’t used for physiological recordings of coronary heart fee. Three of these males and three of these females had been utilized in management experiments and didn’t endure surgical procedure for head-holder implantation nor obtained MRI coaching. Upon arrival at our facility, mice got 2 weeks of acclimatization to the steady surroundings earlier than continuing with the experimental protocols. Mice had been housed sex-wise in cages of as much as three on a 12-h darkish/mild cycle (5:00 a.m. to five:00 p.m.) with advert libitum entry to meals and water.

Animals had been housed with a minimal of 1 cage-mate as single-housing of animals has been proven to disrupt sleep patterns and improve resting-state coronary heart fee in addition to improve blood plasma corticosterone ranges (Späni et al., 2003; Olsson and Westlund, 2007). Temperature and humidity had been managed at 21°C ± 2 and 45% ± 5, respectively. All cages had been equipped with bedding materials and cage enrichment (nesting materials, picket chew sticks, and two totally different hideouts). All mice had been dealt with by a single experimenter all through the experiment to reduce stress induced by a number of handlers. Physique weight was monitored post-surgery and all through the entire experimental interval. Physique weight reduction exceeding 20% of regular body weight was thought-about a humane endpoint.

After the experimental interval, all animals had been euthanized with barbiturate (Pentobarbital, 1,300 mg/kg) with a most injection quantity of 0.5 mL. All animal housing, dealing with, and experimental protocols had been carried out in keeping with the rules of the Danish Ministry of Justice and Animal Safety Committees beneath allow no. (2019-15-0201-00285). The experiment timeline and habituation protocol are outlined in Figures 1A,B, respectively.

Determine 1. Experiment timeline and awake MRI habituation protocol. (A) Timeline for all experimental procedures. LDT, mild/darkish check. FCM, fecal corticosterone metabolite evaluation. (B) Progressive awake MRI habituation protocol. Twenty minutes had been added to every session per day, as much as a complete of 180 min. MRI sound was performed from day 4 and onward.

Mock Magnetic Resonance Imaging Surroundings and Head-Holder

The animal mattress for awake utility and the head-holder used for fixation are custom-made 3D fashions, tailored from Han et al. (2019) to suit our present setup utilizing a rat-cryoprobe. The 3D fashions of the animal mattress, head-holder, and all parts for the mock MRI surroundings had been modified and designed utilizing Autodesk Fusion 360 software program. A library of our designs is offered on our GitHub repository:

The simulation surroundings consists of a habituation field (Determine 2A) closed with a lid throughout habituation, because the scanner bore can be darkish throughout scans. The habituation field can accommodate three chambers (Determine 2Bi) which emulate the within of the scanner bore with a cryo-probe the place we carry out the MRI scans. Contained in the habituation field, a mini-fan is put in for air flow (Determine 2Bii). Three audio sockets (Determine 2Ciii) are put in on the underside of the habituation field to suit every chamber’s audio connector (Determine 2Diii). Three viewports enable for webcam insertion, and infrared (IR) light-transmitting diodes (Determine 2Civ) are put in round every viewport to light up the rostral area of the mouse. The IR diodes are related in sequence and powered by a close-by energy provide (Elektro-Automatik, Artwork.Nr: 03100206). The chambers are outfitted with audio system (Determine 2Ev) on both facet of the animal’s head to play scanner sounds throughout habituation. The MRI audio sequences are performed utilizing Audacity software program (Audacity Group), and audio is transmitted through an auxiliary cable that connects to the audio sockets of the habituation field. Throughout every habituation session, the experimental MRI sound was performed at 110 dB. The chambers for one animal mattress and animal (Determine 2Evi). Webcam recordings allowed us to look at the animals whereas restrained contained in the habituation field (Determine 2F). Our habituation surroundings captures salient options of the scan state of affairs together with similar beds, mild situations, and sounds. Vibrations are usually not a priority in our scanner setup as our cryo-probe is suspended within the scanner bore so there isn’t a contact between the gradients and the animal mattress/receiver coil. Gradient vibrations are due to this fact not transferred to the animal mattress. Because of this, our simulation chamber doesn’t embody this impact.

Determine 2. Awake MRI setup and parts. (A) Awake MRI habituation field the place animals are educated in the course of the awake MRI habituation classes. (B) Overview of the within of the habituation field with connections for 3 chambers (i) with one animal inside every. A small fan offered air flow of the habituation field (ii) positioned together with the field. (C) Sideview of contained in the habituation field. The chambers had been related to the audio sockets (C,iii) positioned on the backside of the field. IR diodes offered illumination of the animals for video recordings. (D) Beneath the chamber. Every mock coil had an audio connector (D,iii) related to (C,iii). (E) Sideview of the mock MRI coil. The animal mattress (iv) was inserted into the chamber (i), and every chamber had audio system put in (v) on the left and proper ear of the mouse. (F) Webcam recording of the restrained animal on the MRI mattress contained in the habituation field. Black arrows: placement of the sunshine sensor (carotid arteries).

Head-Holder Implantation

All surgical procedures carried out on this examine had been primarily based on Han et al. (2019). Mice had been positioned in an hermetic field and briefly anesthetized with isoflurane (4%). The highest of the cranium was then rigorously shaved, and the mice had been positioned onto a stereotaxic body (RWD Life Science). Anesthesia was maintained with a mix of 1.2–1.5% isoflurane, 0.6 mg/l medical air and 0.4 mg/l oxygen. The shaven space was then sterilized with ethanol, and a drop-shaped piece of pores and skin was faraway from the top to uncover the cranium. Aponeurosis and periosteum had been rigorously eliminated alongside any muscle tissue on the interparietal bone posterior to lambda. The uncovered cranium was cleaned with saline, dried out with ethanol, after which the tissue edges had been mounted to the cranium utilizing a layer of tissue adhesive (3M, Vetbond). The uncovered a part of the cranium was then scraped with a surgical knife to provide a hatch sample (gridlines) to enlarge the floor space for higher adhesion. Subsequent, a skinny layer of light-curing self-etch (3M, ESPE Single Bond Common Adhesive) was utilized to the midline and on the interparietal bone space and subsequentially cured with blue mild. Then, a layer of light-curing flowable dental resin (Ionoseal, Voco, Germany) was utilized on the interparietal bone earlier than putting the {custom} head holder. A 1 mm in diameter alumina ceramic rod (KF Superior Ceramic, China) was then positioned down the midline and in-between the hole of the top holder to strengthen the construct. Lastly, the rod and holder had been secured by curing the resin and extra light-curing flowable resin was utilized across the holder. The rest of the uncovered cranium was coated with a skinny layer of dental cement, and the mouse was tagged for straightforward identification. After surgical procedure, all mice obtained a each day dose of antibiotics; 0.2 mg/g (STADA, Ampicillin, Vnr: 108259), anti-inflammatory medicine; 0.1 mg/g (ScanVet, Carprofen, Vnr: 027693) and painkillers; 0.12 mg/g (Indivior, Temgesic, Vnr: 521634) for no less than 4 days. Ampilicin and Carprofen had been administered IP whereas Temgesic was administered SC post-surgery and diluted (2 ml Temgesic/120 ml water) of their consuming water for the rest. In whole, mice got 10 days of restoration.

Habituation Protocol

Over the last 3 days of the restoration interval, all mice obtained 20 min of each day dealing with. This was accomplished to cut back stress ranges and be sure that animals had been accustomed to the caretaker earlier than the awake MRI habituation. A duplicate of the 3D-printed, experimental animal mattress was additionally positioned of their cage always to allow them to familiarize themselves with the part. After every session, mice got just a few droplets of condensed milk as a reward. On day one in all habituation, mice had been rigorously launched to the setup and restrained for 20 min in whole. Twenty minutes had been added within the following coaching session till reaching a complete of 180 min of habituation (Determine 1B). Solely on the fourth day and onward, the MRI sounds had been performed. Earlier than every awake MRI habituation session that included the MRI sound, animals got wax earplugs (Ohropax Basic). Animals had been educated sex-wise, 9 per day however had been divided into three totally different timeslots; morning (7:30–10:30), midday (11:00–14:00), and afternoon (14:30–17:30). Throughout your entire habituation interval, the female and male mice teams had been staggered to be equally divided into the three totally different timeslots. In whole, animals obtained 14 days of habituation (Determine 1). The animal experiments had been carried out in mixed-sex batches of 9 animals to make sure knowledge acquisition homogeneity throughout animal intercourse and decrease expertise bias. Nonetheless, to reduce the affect of various odors on mice conduct, animals’ intercourse was not combined in the course of the habituation classes.

Video Monitoring Throughout Awake Mice Habituation Protocol

Mouse physique motion was monitored for every habituation session utilizing webcams (LifeCam Studio, Microsoft) modified to permit for infrared mild detection. The webcams had been positioned by the viewports of the field (Determine 2A), so every animal was seen (Determine 2F). The video information had been saved to evaluate animal discomfort throughout habituation utilizing the mouse grimace scale (MGS). We used three frames; early (first minute), mid (±1 min), and late (final minute), for evaluation of the MGS. All photographs had been rated on the MGS whereas randomized and the experimenter blinded. The information was preprocessed in MATLAB (The Mathworks Inc., R0219a), and statistical evaluation was accomplished utilizing Prism 9 software program (GraphPad).

Peripheral Physiological Recordings and Fecal boli Assortment Throughout Awake Mice Habituation Protocol

Coronary heart fee and respiratory fee had been collected throughout every habituation session utilizing pulse oximetry (MouseOx, STARR Life Science, Oakmont, PA). The clamp of the light-sensor gadget (MouseOx, “Throat Sensor”) was lower brief to suit exactly on the carotid arteries after which secured within the backside of the animal mattress earlier than being rigorously positioned across the throat of the mice. When all three mice had been mounted on the animal beds and positioned within the habituation field, the MRI sequence sounds had been performed, and physiological and visible recordings had been commenced. After every habituation session, all fecal boli for every mouse had been collected and dried in a single day earlier than weighing. Physiological knowledge recordings had been accomplished with a sampling fee of 1 Hz and preprocessed utilizing an in-house MATLAB script whereas statistical analyses had been accomplished in Prism 9 software program.

Fecal Corticosterone Metabolite Evaluation

Fecal corticosterone metabolite (FCM) ranges had been assessed at a number of timepoints in the course of the experiment utilizing the subset of animals (n = 18). The timepoints for fecal pattern assortment had been (1) after 2 weeks of steady acclimatization and previous to any animal dealing with (pre dealing with) (2) 24 h after each third day of MRI habituation (3) 14 days after finish of MRI habituation (submit coaching) (Determine 1). We use FCM evaluation to non-invasively get hold of a direct measurement of the animal’s stress ranges throughout our awake MRI habituation protocol.

Fecal samples had been collected by rigorously putting the mice into single cages with clear bedding materials combined with a handful of bedding materials from their house cage. This was accomplished to reduce the novelty of the surroundings within the new cages. After a interval of roughly 1 h the animals had been gently transported again to their house cage. All of the excreted fecal samples from that interval had been collected with forceps and saved at –20°C. On the finish of the experimental interval all fecal samples had been ready in duplicates and in accordance with the producer’s directions FCM focus ranges had been analyzed utilizing a chemiluminescent immunoassay ELISA package as described within the producer’s directions (Arbor Assays, Ann Arbor, MI, United States). The chemiluminescence was learn with a microplate reader (Synergy HTX Multi-Mode Reader, BioTek, Santa Clara, CA, Unites States) and concentrations had been decided in pg/mL however later transformed to ng/mg. Statistical evaluation was carried out utilizing Prism 9 software program.

Automated Mild/Darkish Check Equipment

Behavioral knowledge had been collected utilizing the sunshine/darkish anxiousness transition check (LDT) to look at anxiety-like conduct in younger grownup mice. 4 particular person classes of LDT had been carried out for every mouse: After the surgical restoration interval ended (LD1)—baseline. Sooner or later after the primary habituation session (LD2), 1 day after the final habituation session (LD3), and a pair of weeks after the final habituation session (LD4) (Determine 1A). The automated setup consists of an IR-translucent mild/darkish field (Noldus, MMDLx-I001_V30) and an IR digital camera (Basler acA1300-60gmNIR, Basler) mounted within the ceiling alongside two oblique IR mild sources (Lux). The field measured 20 × 60 cm in whole (Mild compartment: 40 × 20 × 20 cm, Darkish compartment 20 × 20 × 20 cm). A small doorway within the center connects the 2 compartments. Earlier than every session, mice had been acclimatized to the room for no less than 30 min. Mice had been then positioned within the middle of the sunshine compartment, and recordings had been routinely initiated after 2 s of monitoring validation accomplished by the software program. Mice had been allowed to freely transfer and discover the 2 compartments for 10 min. Between classes, the field was rigorously cleaned with 70% ethanol and airdried for five min to make sure the odors of the earlier animal had been eliminated. The variety of transitions between the compartments, the entire motion, and whole time spent within the mild compartment had been extracted from EthoVisionXT software program (Noldus), preprocessed utilizing MATLAB-R2019a, whereas Prism 9 Software program was used for statistical evaluation.

Statistical Evaluation

All knowledge are offered as imply ± normal error of the imply (SEM) until specified in any other case. For statistical evaluation of single teams (males: females) we carried out a one-factor ANOVA utilizing a submit hoc Dunnett’s check for a number of comparability with Day 1 as management for each men and women until specified in any other case. Dunnett’s check was chosen as a result of we used Day 1 as management and weren’t concerned with comparisons with different days. Outcomes had been thought-about statistically vital at P< 0.05. For statistical evaluation of each teams (males vs. females) we carried out a two-factor combined ANOVA utilizing Holm-Sidak’s check for a number of comparability until specified in any other case. Right here, Holm-Sidak’s check was chosen as it’s extra highly effective than the Bonferroni check, and we didn’t must compute the boldness intervals. Outcomes had been thought-about statistically vital at P < 0.05.


Evaluation of Animal Discomfort Throughout Habituation

Our IR video recording setup permits us to look at the conduct and seize the facial expressions of the animals throughout every MRI acclimatization session (Determine 2F). From our observations, it’s clear that the animals had been transferring the decrease a part of their physique (this half was not fixated) when initially restrained however remained slightly calm for the rest of the session. In an effort to assess mouse discomfort in the course of the awake MRI habituation classes, we estimated their discomfort stage at three totally different time factors for every session utilizing the MGS. Photos from six mice had been omitted from evaluation on account of out-of-focus photographs. Mice exhibited low discomfort ranges in the course of the awake MRI habituation interval (imply worth for all days; males = 0.36 ± 0.014 SE, n = 8, females 0.36 ± 0.018 SE, n = 7) (Determine 3A). For day-to-day comparability, the MGS rating for all timepoints for every session was averaged for every animal, earlier than averaging throughout animals for each the female and male group. For each men and women, no distinction was discovered within the each day imply MGS rating fluctuations (males: P> 0.05; females = P> 0.05 in a Dunnett’s a number of t-test with Day 1 as management) (Determine 3A). A two-factor combined ANOVA didn’t reveal any variations when evaluating males vs. females [F(13,182) = 1.185, P> 0.05, main effect of days in a two-factor mixed ANOVA of mean MGS score × days] (Determine 3A). To analyze whether or not the mice exhibited totally different discomfort ranges throughout a single session, we in contrast the imply MGS rating between three totally different time factors (early, mid, and late) inside every session for men and women. We noticed a steady lower within the imply MGS rating throughout the timepoints for men and women. No distinction was discovered when evaluating males vs. females in a two-factor combined ANOVA fundamental impact (P> 0.05) (Determine 3B). Nonetheless, we discovered that the imply MGS rating for females decreased considerably from the early time level to the center and late time factors [F(2,39) = 3.672, P< 0.05, row effect of time point in session using a two-factor mixed ANOVA of mean MGS score × time point in the session, followed by multiple comparisons of row effects of time point in the session using Holm-Sidak’s test; *P< 0.05 vs. mean MGS score] (Determine 3B).

Determine 3. Each day imply MGS rating in the course of the awake MRI habituation interval and inside every MRI habituation session for male (n = 8) and feminine (n = 7) mice. (A) Imply MGS rating for every intercourse of the totally different days. (B) Imply MGS rating for every intercourse on the early, center, and late time factors inside all classes (14 days). Knowledge are offered as imply ± SEM.

Coronary heart Price Recordings

Imply coronary heart charges in the course of the acclimatization interval decreased considerably on the fourth day and from the tenth day onward of habituation for males (n = 11) and the 11, 12, and 14th day for females (n = 13) (*P < 0.05 in a Dunnett’s check, with day one as management) (Determine 4). Furthermore, we discovered a distinction in imply coronary heart fee for males vs. females [F(13,283) = 2.784, P< 0.001, main effect of days in a two-factor mixed ANOVA of acclimatization × days]. A number of comparisons of the principle results of days utilizing Holm-Sidak’s check vs. imply coronary heart fee between men and women on every day, revealed a distinction on day 13 of habituation (ΔP< 0.001) (Determine 4).

Determine 4. Results of awake MRI habituation on coronary heart fee for male (n = 11) and feminine (n = 13) mice. *P< 0.05 in a Dunnett’s check (day 1 as management). ΔP < 0.05 in Holm-Sidak’s a number of comparability utilizing two-factor combined ANOVA with intercourse × days of acclimatization. Knowledge are offered as imply ± SEM.

Physique Weight Fluctuations All through the Experimental Interval and Weight of Fecal boli Throughout Habituation Interval

The full weight of a head-holder, ceramic rod, and dental-cement was 0.89 g ± 0.08 SEM (n = 36). This weight was subtracted from the mouse weight post-surgery. We noticed post-surgery weight reduction in all animals. For males, physique weight decreased for six days after the surgical implantation of the head-holder (*P< 0.05 vs. physique weight after surgical procedure with day 1 as management in a Dunnett’s check), and it took 22 days earlier than they regained their preliminary weight. In the meantime, the physique weight of females decreased for five days (*P< 0.05 vs. physique weight after surgical procedure with day 1 as management in a Dunnett’s check), and preliminary weight was reached 9 days post-surgery (Determine 5A). In an effort to evaluate physique weight between sexes, relative change to baseline (Day 0) was calculated (Determine 5B). We discovered that the distinctive time course of the physique weight for men and women was considerably totally different [F(1,572) = 14.48, P< 0.001, column effect using a two-factor mixed ANOVA of sex × days]. Nonetheless, a number of comparisons utilizing Holm-Sidak’s technique didn’t reveal any days of significance. For the burden of fecal boli samples, we noticed a steady improve for each men and women because the size of the awake MRI habituation elevated, however not after the habituation size reached the utmost (180 min) (Figures 5C,D).

Determine 5. Results of surgical procedures and awake MRI habituation on mouse physique weight fluctuations and weight of fecal boli all through the awake MRI habituation. (A) Imply physique weight of men and women (*P< 0.05 vs. physique weight after surgical procedure with day 1 as a management in a Dunnett’s check). (B) Relative change in imply physique weight with Day 0 used for baseline correction. Notice that the vertical line on (A,B) signifies the beginning of awake MRI acclimatization. The horizontal strains on (A) point out the imply physique weight post-surgery for men and women, respectively, whereas the horizontal line on (B) signifies the corrected baseline (0%). (C) Imply weight of fecal boli throughout habituation interval. (D) Relative change in imply weight of fecal boli with Day 0 used for baseline correction. Notice that the vertical strains on (C,D) point out when awake MRI habituation size reached the utmost (180 min), whereas the horizontal line on (D) signifies corrected baseline (0%).

Mild-Darkish Check

Complete Distance Traveled

General, we noticed that each teams exhibited decreased locomotor exercise from LD1 by LD2 and LD3 with males exhibiting the largest lower. In LD4, we had been in a position to observe a slight improve in locomotor exercise from LD3 for each teams. Nonetheless, statistical evaluation for whole distance traveled (DIS) didn’t reveal any intercourse distinction [F(3,131) = 0.6230, P> 0.05, main effect using two-factor mixed ANOVA sex × LD]. Nonetheless, a DIS × LD distinction was noticed [F(3,131) = 4.481, P< 0.01, row effect using two-factor mixed ANOVA of distance × LD]. Put up hoc a number of comparability utilizing Dunnett’s check revealed statistical significance within the LD1 vs. LD3 comparability for males (**P< 0.01) indicating that mice displayed decreased locomotor exercise in LD3 when in comparison with baseline (Determine 6A).

Determine 6. Behavioral knowledge from light-dark check. (A) Complete distance traveled in the course of the light-dark check (10 min). (B) Complete time spent within the mild compartment (C). Complete quantity of transitions between the sunshine and darkish compartment. LD1 (light-dark check carried out earlier than surgical procedure; males, n = 15; females, n = 15). LD2 (light-dark check 1 day after first habituation day; males, n = 19; females, n = 20). LD3 (light-dark check carried out after final habituation day; males, n = 17; females, n = 19). LD4 (light-dark check carried out 14 days after habituation; males, n = 17; females, n = 17). *P< 0.05; **P< 0.01. Knowledge are offered as imply ± SEM.

Time in Mild Compartment

On the whole, we noticed that feminine mice spent much less time within the mild compartment as the sunshine darkish check progressed from LD1 by LD4. For male mice we noticed that the time within the mild compartment elevated from LD1 to LD2 whereafter time spent in mild decreased from LD2 by LD4. Statistical evaluation of time spent within the mild compartment (TSL) didn’t reveal any intercourse distinction [F(3,131) = 1.661, P> 0.05], fundamental impact utilizing two-factor combined ANOVA of intercourse vs. LD). A LD impact in TSL [F(3,131) = 9.176, P< 0.0001 row effect using a two-factor mixed ANOVA] adopted by Dunnett’s check revealed a distinction in LD1 vs. LD3 (*P< 0.05) and LD1 vs. LD4 for females (**P< 0.01) (Determine 6B).

Transitions Between Mild and Darkish Compartment

For transitions (TNS), we noticed that male mice had fewer transitions than feminine mice throughout all 4 LD checks besides LD3, nevertheless no intercourse distinction had been discovered [F(3,131) = 0.3166, P> 0.05] fundamental impact utilizing two-factor combined ANOVA of intercourse × LD). Nonetheless, vital was seen for TNS × LD [F(3,131) = 6.111, P< 0.001, using a two-factor mixed ANOVA]. Put up-hoc a number of comparability utilizing Dunnett’s check revealed statistical significance within the LD1 vs. LD3 (*P< 0.05) and LD1 vs. LD4 (*P< 0.05) for females and LD1 vs. LD4 (**P< 0.01) for males (Determine 6C).

Evaluation of Fecal Corticosterone Metabolites Ranges

Focus of fecal corticosterone metabolites had been assessed all through the experiment at eight totally different sampling timepoints, with one pattern level previous to animal dealing with, six pattern factors evenly spread-out in the course of the size of the awake MRI habituation and one pattern level 14 days after the MRI habituation had ended. As anticipated, we noticed that the imply FCM focus for females was typically increased than for males all through the entire pattern factors within the experiment. For each teams we noticed that day 1 of MRI habituation (D1) induced an elevated response in imply FCM focus when in comparison with the imply focus at pre dealing with stage. For male mice the imply FCM concentrations decreased constantly till D11 whereafter we noticed a slight improve for the D14 and Put up timepoints. An identical pattern may very well be noticed for the females besides D8 confirmed a short improve in imply FCM focus (Determine 7A). Statistical evaluation didn’t reveal any intercourse variations [F(7,79) = 0.2224, P> 0.05] fundamental impact utilizing two-factor combined ANOVA of intercourse × habituation). To check the impact of MRI habituation on FCM concentrations for every group, we used the D1 imply FCM focus as reference factors in six-paired two-sample two-sided t-tests. A major lower in imply FCM focus from the primary day (D1) of MRI habituation to the eleventh day (D11) FCM [t(5) ≥ 2.998, P≤ 0.0302] for each male and females mice (Determine 7A) coinciding with the primary time level the place animal coronary heart fee is considerably lowered and indicative that FCM concentrations decreased with steady awake MRI habituation. No vital variations had been present in both group on different days when utilizing D1 imply FCM focus as reference level.

Determine 7. Timeline illustration of fecal corticosterone metabolite (FCM) focus ranges on the sampling timepoints in the course of the experimental interval. Items are displayed in FCM ng/mg of fecal matter. (A) FCM focus for males (n = 6) and females (n = 6) at every sampling timepoint. (B) FCM focus for males (n = 6) and male controls (n = 3). (C) FCM focus for females (n = 6) and imply FCM focus for feminine controls (n = 3). The markers on x-axis signifies the totally different sampling timepoints (Pre = after steady acclimatization; D1–14 = in the course of the 14 days of MRI habituation; Put up = 14 days after final MRI habituation day). The horizontal strains for management on determine B, C point out imply FCM concentrations for the entire experimental interval. Knowledge factors are offered as imply ± SEM. *P < 0.05.

We additionally in contrast the animals in every group (n = 6) to a management group of their very own intercourse (n = 3) (Figures 7B,C). On the whole, we noticed very massive fluctuations of imply FCM focus ranges within the management teams for each men and women in the course of the awake MRI habituation and statistical evaluation didn’t reveal any distinction between male vs. management nor feminine vs. management.


Awake Mouse Magnetic Resonance Imaging Habituation Protocol

Our awake MRI habituation protocol had been designed to incorporate as most of the present finest practices with a purpose to current a protocol optimized for animal welfare and discount of animal stress. For instance, we applied a progressive awake MRI habituation protocol (Determine 1B) in comparable style to earlier stories utilizing rats and mice (Stenroos et al., 2018; Han et al., 2019; Chen et al., 2020; Cowl et al., 2021) to higher account for the potential brief and long-term ache and stress that non-progressive protocols may exert on the animals (Stenroos et al., 2018). As well as, we offered earplugs to the animals to assist decrease the stress and potential ear harm induced by the loud acoustic MRI noises. Our head-fixation protocol additionally allowed animals free motion of the decrease physique. It has beforehand been steered that researchers ought to embody behavioral end result measurements along with stress-related physiological measurements, as the conventional vary of physiological parameters alone doesn’t totally clarify the long-term results of awake MRI habituation on mice (Low et al., 2016). Subsequently, we applied the light-dark check to measure anxiety-like and locomotor behaviors along with recordings of coronary heart fee and FCM measurements. Lastly, our setup didn’t require sedation for head-fixation of the animals, which is per earlier stories (Yoshida et al., 2016; Han et al., 2019; Chen et al., 2020) and does away with the priority for the impact of lingering anesthetics. Our habituation protocol depends on the similarity between the scan simulation surroundings and the precise scan session. Earlier research have steered together with precise imaging classes within the habituation protocol as useful for decreasing animal stress (Chen et al., 2020) and to cut back the entire quantity of animal motion (Dinh et al., 2021). For many labs, nevertheless, scanner sources are too scarce to incorporate an precise MRI scanner within the habituation so we opted for a habituation protocol utilizing a simulated scanner surroundings solely to make sure our examine is related to as many labs as doable. For future work it might be useful to incorporate resting days as applied in comparable research (Russo et al., 2021). Nonetheless, our FCM knowledge reveals that if habituation will not be maintained stress of dealing with will increase once more (Put up time level in Determine 7A, each sexes). This have to be taken into consideration when incorporating relaxation days into an experiment timeline.

Results of Surgical Procedures and Awake Magnetic Resonance Imaging Habituation on Coronary heart Price, Physique-Weight, and Weight of Fecal boli in Male and Feminine Mice

We evaluated the time programs of a number of stress indicative physiological, behavioral, and biomarker parameters (coronary heart fee, physique weight, weight of fecal boli, and fecal corticosterone ranges). For coronary heart fee measurements, we noticed that each teams elicited decreased imply coronary heart charges after steady awake MRI habituation (10 days for males; 11 days for females) (Determine 4), which was longer than anticipated as earlier research report a lower in imply coronary heart fee after solely 7 days (Yoshida et al., 2016). Moreover, we noticed that males displayed decrease imply coronary heart charges ranges on the final 3 days of the awake habituation protocol than females. After we in contrast the distinct time course of imply coronary heart fee for men and women, we detected a major distinction (Determine 4), suggesting that awake MRI habituation impose extra stress on feminine than male mice, which is in concordance with stories that feminine rodents are extra delicate to physiological and bodily stressors (Rincón-Cortés et al., 2019). Lastly, it must be famous that our MouseOx pulse oximetry system was certainly able to offering different measurements corresponding to arterial saturation and breath fee, which could have been precious to incorporate. Nonetheless, because of the excessive sensitivity of the sensors to small motion, these measurements had been too noisy to incorporate.

The physique weight of each teams decreased quickly the times after surgical implantation of the head-holder. This impact was anticipated primarily based on different research that utilized surgical implants for head-fixation (Yoshida et al., 2016; Madularu et al., 2017; Stenroos et al., 2018). We noticed comparable each day fluctuations of imply physique weights of men and women; nevertheless, some distinct variations had been current (Figures 5A,B). Though each men and women recovered vital physique weight after solely 5 (females) and 6 (males) days, it took for much longer for males to regain their preliminary physique weight reduction post-surgery. Nonetheless, not one of the mice exceeded 20% of physique weight reduction post-surgery, and all had been in a position to regain their physique weight, indicative of a profitable postoperative restoration. Moreover, we noticed a gentle weight achieve all through the experimental interval for each teams, indicating that mice consuming conduct was not affected. Nonetheless, in future research, restoration could also be accelerated by administering isotonic glucose, or different sugar-based liquids, within the days following surgical procedure. Extreme excretion has been established as indicative of stress and feces weight used as a measure of it (Miyata, 1992). In our examine, the imply weight of fecal boli in each teams elevated because the habituation size elevated however stagnated after the habituation size reached 180 min (Determine 5C). This means that extended MRI habituation does induce elevated excretion within the animals. Nonetheless, the imply boli weight in the course of the habituation interval utilizing 180 min classes was within the vary of these present in an analogous examine (Yoshida et al., 2016), indicating that the excretion was not extreme. Taken collectively, these findings point out that the surgical procedure and habituation protocol doesn’t have an effect on the animals’ fundamental wellbeing (restoration, feeding, weight achieve, and weight stability) of the animals.

Results of Awake Magnetic Resonance Imaging Habituation on Mouse Grimace Scale and Mild-Darkish Check Outcomes in Male and Feminine Mice

Whereas the utmost care is taken to reduce animal discomfort throughout your entire habituation process, it’s unrealistic to keep away from discomfort and stress utterly. Because of this, we monitored animal discomfort and anxiousness response utilizing the MGS and LDT. The typical MGS rating for each teams was extraordinarily low (< 0.36) all through the experiment, indicating that mice solely expertise delicate discomfort throughout habituation. We did nevertheless observe that feminine mice displayed barely elevated discomfort ranges in the course of the preliminary minutes simply after being restrained (Determine 3B). This means that male mice exhibit larger stress/ache resilience than females. In just a few instances, for the longer (≥120 min) awake MRI classes, we noticed a layer of eye secretion that had developed across the eyes of the mice. In our expertise, this sometimes happens when mice are extremely aroused for longer intervals of time, indicating that some mice had been very alert in the course of the lengthy classes. Nonetheless, extra analysis is required to totally clarify this phenomenon. For instance, it will be attention-grabbing to correlate eye secretion occurrences with EEG knowledge.

The sunshine-dark check (LDT) was used to evaluate anxiety-like and locomotor exercise conduct throughout MRI habituation. The LDT check makes use of the inherent conduct of mice to keep away from shiny open areas (mild compartment) and choice for a protected surroundings (darkish compartment). Will increase in anxiety-like behaviors and reduces in locomotor exercise are adaptive illness behaviors in that they decrease danger taking. It was beforehand proven that extended restraining, just like these strategies utilized in awake MRI habituation, could cause short-term will increase within the stress hormone corticosterone and long-term alterations in physiological responses to painful stimuli (Low et al., 2016). As well as, it was proven that continual stress can induce modifications in anxiousness and modify locomotor exercise ranges (Naert et al., 2011). On the whole, we noticed that mice spent extra time in the dead of night compartment than the sunshine compartment per the light-dark check as a basic measure for anxiety-like conduct (Banasikowski et al., 2015). Each teams exhibited comparable locomotor exercise ranges in all LDT’s besides on LD3 the place we detected a lower in locomotor-activity for males. After the 14-day break, on LD4, we couldn’t detect this impact anymore (Determine 6A). This means that male mice could be extra vulnerable to lower their locomotor exercise when uncovered to annoying environments than females. Final, we assessed time spent within the mild compartment and whole transitions, that are typical measurements of anxiety-like responses. Though, statistical evaluation didn’t detect any distinction between men and women on time in mild, we did observe some particular person variations. Females spent much less time within the mild compartment on LD3 which is indicative of an elevated anxiety-like conduct. This improve remained even 2 weeks after the MRI habituation had ended on LD4 (Determine 6B). For frequency of transitions within the LDT’s we discovered no distinction between men and women, however did nevertheless detect particular person variations over the time course of the carried out LDT’s. For females we detected a lower in transitions on LD3 indicating a rise in anxiety-like conduct after 14 days of MRI habituation. This impact remained detectable for the females 14 days after the MRI habituation ended (LD4) indicating that our awake MRI habituation is able to inducing a long-term anxiousness impact. We additionally detected a major lower in transitions for males on LD4 however not on LD3. It’s unclear what the rationale for this statement can be. On the whole, primarily based on our knowledge from the facial video recordings and our behavioral knowledge from the LDT, we discovered that whereas general animal anxiousness and discomfort may be very low feminine mice do show increased discomfort and present extra anxiety-like behaviors than males, when uncovered to our 14-day awake MRI habituation protocol.

Discomfort, Stress, and Anxiousness in Male and Feminine Mice

Awake animal MRI is efficacious as a result of it avoids the physiological perturbations attributable to anesthesia. Nonetheless, stress and anxiousness are identified to change mind chemistry, physiology, and conduct (Luine et al., 2001; Vestergaard-Poulsen et al., 2011; Khan et al., 2016, 2018, 2019). For instance in research of continual stress utilizing rodent restraint fashions, physique weight has been proven to lower over time (Woo et al., 2018) and is accompanied by a rise within the plasma stage of the stress hormone corticosterone (Kim et al., 2013). Our awake MRI habituation protocol was developed to additionally enable for MRI research of prolonged length the place classes must last more than 2 h. We due to this fact applied a slightly lengthy MRI habituation protocol of 14 days together with awake MRI habituation classes lasting as much as 3 h (Figures 1A,B). Different MRI habituation protocols presently discovered within the literature, are sometimes using awake MRI habituation classes solely lasting ≤ 2 h (Bergmann et al., 2016; Yoshida et al., 2016; Madularu et al., 2017; Desjardins et al., 2019; Tsurugizawa et al., 2020). Subsequently, our MRI habituation protocol could be thought-about extra annoying. Nonetheless, our knowledge reveals that habituation for prolonged scan classes is feasible as imply coronary heart fee values in female and male mice had been similar to regular coronary heart charges present in unperturbed aware mice after 10 (male) and 11 (feminine) days of MRI habituation (Kass et al., 1998). As well as, we discovered that imply FCM ranges from the primary day of MRI habituation had been considerably decreased on the eleventh day of MRI habituation for each female and male mice (Determine 7A) coinciding with the time at which coronary heart fee has normalized (Determine 4). Moreover, imply FCM concentrations for each female and male on the final day of MRI habituation seem beneath that of pre dealing with ranges (Determine 7A). That is per different teams who reported decreased FCM focus ranges after a number of days of awake MRI coaching (King et al., 2005; Tsurugizawa et al., 2020; Russo et al., 2021). Lastly, throughout our MRI habituation interval, each female and male mice had been in a position to recuperate, keep and improve their physique weight. On the premise of our obtainable outcomes throughout the totally different stress-indicative parameters, it’s due to this fact evident that mice are in a position to habituate to the annoying MRI surroundings, even throughout utility of an prolonged MRI habituation protocol. Nonetheless, whereas each sexes are efficiently habituated in our examine, our large characterization does reveal lingering variations in anxiousness associated conduct exist post-training (Determine 6) and in corticosterone ranges (Determine 7). These variations are anticipated primarily based on the prevailing literature on stress response in female and male mice. Possible, these variations is not going to have an effect on MRI derived parameters or not less than their impact will likely be a lot lower than the impact of anesthesia. Nonetheless, these particulars have implications for research the place each awake MRI and conduct checks will likely be used to characterize an animal mannequin. Firstly, each sexes must be included as inherent physiological variations (e.g., stress hormone ranges) exist even after habituation. Secondly, separate teams must be used for awake MRI and conduct evaluation as in any other case the conduct outcomes could also be influenced by sex-specific, lingering results of the MRI habituation. These suggestions apply to each awake fMRI research and quantitative MRI research for which our protocol is aimed.


This examine is the primary to look at the results of surgical and restraint procedures related to awake MRI habituation on stress-related physiological, organic and behavioral parameters in each female and male mice. We discover that whereas intercourse variations exist, female and male mice may be efficiently habituated for prolonged awake MRI scan classes. Utilizing awake mice for MRI analysis functions requires very time-consuming habituation in comparison with experiments utilizing anesthetized animals. Nonetheless, many facets of mind physiology can’t be correctly investigated beneath anesthesia, and awake animal MRI strategies are due to this fact essential for contemporary neuroscience.

Knowledge Availability Assertion

The uncooked knowledge supporting the conclusions of this text will likely be made obtainable by the authors, with out undue reservation.

Ethics Assertion

The animal examine was reviewed and accredited by the Danish Animal Experimentation Council.

Writer Contributions

BH and TL designed the examine, analyzed the information and interpreted outcomes. BH acquired funding. TL constructed the coaching {hardware}, carried out the surgical procedures, animal coaching and knowledge acquisition, and wrote the preliminary manuscript draft. All authors edited the manuscript and contributed to the experimental setup.


This mission was supported by grants from the Sino-Danish Heart (SDC) and the Aarhus College Analysis Basis (AUFF NOVA).

Battle of Curiosity

The authors declare that the analysis was carried out within the absence of any business or monetary relationships that may very well be construed as a possible battle of curiosity.

Writer’s Notice

All claims expressed on this article are solely these of the authors and don’t essentially characterize these of their affiliated organizations, or these of the writer, the editors and the reviewers. Any product that could be evaluated on this article, or declare that could be made by its producer, will not be assured or endorsed by the writer.


We thank Xifan Chen and Zhe Han for his or her coaching of animal surgical procedure. As well as, we additionally thank Martin Snejbjerg for his assist with the design and technical help of constructing the MRI simulation surroundings. Final, we additionally thank Anna Bay Nielsen for her assist with fecal corticosterone stage evaluation.






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