Neuroinflammation, body temperature and behavioural changes in CD1 male mice undergoing acute restraint stress: An exploratory study.

BACKGROUND: Animal models used to study pathologies requiring rehabilitation therapy, such as cardiovascular and neurologic disorders or oncologic disease, must be as refined and translationally relevant as possible. Sometimes, however, experimental procedures such as those involving restraint may generate undesired effects which may act as a source of bias. However, the extent to which potentially confounding effects derive from such routine procedures is currently unknown. Our study was therefore aimed at exploring possible undesirable effects of acute restraint stress, whereby animals were exposed to a brightly lit enclosed chamber (R&L) similar to those that are commonly used for substance injection. We hypothesised that this would induce a range of unwanted physiological alterations [such as neuroinflammatory response and changes in body weight and in brown adipose tissue (BAT)] and behavioural modification, and that these might be mitigated via the use of non-aversive handling methods: Tunnel Handling (NAH-T) and Mechanoceptive Handling (NAH-M)) as compared to standard Tail Handling (TH).
METHODS: Two indicators of physiological alterations and three potentially stress sensitive behavioural parameters were assessed. Physiological alterations were recorded via body weight changes and assessing the temperature of Brown Adipose Tissue (BAT) using infra-red thermography (IRT), and at the end of the experiment we determined the concentration of cytokines CXCL12 and CCL2 in bone marrow (BM) and activated microglia in the brain. Nest complexity scoring, automated home-cage behaviour analysis (HCS) and Elevated Plus Maze testing (EPM) were used to detect any behavioural alterations. Recordings were made before and after a 15-minute period of R&L in groups of mice handled via TH, NAH-T or NAH-M.
RESULTS: BAT temperature significantly decreased in all handling groups following R&L regardless of handling method. There was a difference, at the limit of significance (p = 0.06), in CXCL12 BM content among groups. CXCL12 content in BM of NAH-T animals was similar to that found in Sentinels, the less stressed group of animals. After R&L, mice undergoing NAH-T and NAH-M showed improved body-weight maintenance compared to those exposed to TH. Mice handled via NAH-M spent a significantly longer time on the open arms of the EPM. The HCS results showed that in all mice, regardless of handling method, R&L resulted in a significant reduction in walking and rearing, but not in total distance travelled. All mice also groomed more. No difference among the groups was found in Nest Score, in CCL2 BM content or in brain activated microglia.
CONCLUSIONS: Stress induced by a common restraint procedure caused metabolic and behavioural changes that might increase the risk of unexpected bias. In particular, the significant decrease in BAT temperature could affect the important metabolic pathways controlled by this tissue. R&L lowered the normal frequency of walking and rearing, increased grooming and probably carried a risk of low-grade neuro-inflammation. Some of the observed alterations can be mitigated by Non-aversive handlings.

Introduction

Animal models used to investigate conditions like heart failure, stroke, hypertension or cancer, and to develop rehabilitation procedures, must be refined such that they deliver results with the maximum possible translational relevance. Only then can they be used to effectively develop therapeutic and rehabilitative strategies for humans suffering similar conditions. This means limiting undesirable effects that might generate bias. It has been shown that chronic psychological stress in laboratory animals can lead to CNS neuroinflammation [1-3], an effect that may contribute to experimental bias. Such biases or sources of experimental ‘noise’ are undoubtedly contributors to what has been described as a current reproducibility crisis in pre-clinical research [4]. The extent to which routine but nevertheless potentially highly stressful procedures might negatively impact on the quality of the results of studies has not been extensively investigated. Studies concerning the impact of restraint are typically chronic in nature and the period of capture can last at least one hour [5-7], and are meant to model methods which may induce behaviours that have relevance to the clinical conditions of post-traumatic stress and major depressive disorders [8, 9]. However, mice are more commonly restrained for a period lasting only several minutes for procedures such as blood sampling or to receive injections, and during this time they are usually also exposed to abnormally high ambient lighting. Although it is likely that such shorter more acute restraint exposures are also highly stressful, only a few attempts have been made to address whether this represents a major problem [10-12]. Following the discovery that chronic psychological stress leads to neuroinflammation at a central level [1] our group wondered whether the same potentially confounding effect might arise following putatively less impactful procedures, such as acute restraint under bright light (R&L); a scenario mice often encounter in pharmacological studies, for example for repeated injections or blood withdrawals. If this were the case, then neuroinflammation, even low-grade, could lower the reliability of results, and ultimately lead to greater numbers of animals being needed [13]. This problem, addressed in the case of chronic psychological stress [1, 3, 14, 15], has not been sufficiently studied in the case of minor operative procedures. Therefore, in this study we investigated whether 15 minutes R&L affected CXCL12 and CCL2 content in the BM and activated microglia in the brain, and whether it also affected physiological homeostasis, like changes in BAT temperature, as detected with IRT, and body weight alteration. We also investigated possible effects on behavioural parameters.

A period of 15 minutes, incorporating bright lighting, was considered appropriate to model the above mentioned scenario (repeated injections or blood withdrawals); moreover, 15 minutes of restraint caused an increase in plasma corticosterone and ACTH levels, together with a significant rise of the anxiety levels, in rats [12], thus suggesting that also in mice 15 minutes of containment might affect physiological homeostasis.

CXCL12 and CCL2 were measured for their key role in the interplay between BM, microglia and immune mediators seen in chronic psychological stress neuroinflammation, where they are involved in the mobilization of inflammatory cells from the BM, followed by subsequent brain infiltration through the blood-brain barrier [1, 14–19] and undesirable central inflammation.

Regarding IRT, its benefit consists in the fact that it can be used unobtrusively in freely moving mice, thus representing a refinement compared to methods typically requiring placement of a transponder either onto or inside the body [20-24]. In our recent study using a model of spinal cord injury [21] we showed IRT to be effective for detecting links between post-surgical pain and increases temperature in the mice BAT, suggesting that surgical pain might influence BAT homeostasis and possibly systemic metabolism. We therefore wanted to assess whether also acute R&L stress might affect BAT temperature.

Also, the way mice are handled can make an important contribution to stress susceptibility, and handling them by the tail, which is still the most commonly used method [25], is now known to be aversive and unnecessarily stressful [26]. Therefore, a further aim was to verify whether, compared to standard TH, the NAH-T and NAH-M techniques might prevent or standardise acute R&L-induced stress.

NAH-T involves guiding mice into a Plexiglas tube, and once inside, transferring them between cages or other apparatus as necessary [26], while NAH-M consists in low-force, low-velocity stroking of the fur with brush resulting in MRGPRB4 G-protein-coupled receptor activation which has demonstrated anxiolytic effects [27, 28] and is found in social interactions involving grooming behaviors in mice [29].

Thereby, the hypothesis of the present study was that 15 minutes of R&L stress would produce neuroimmune and physiological alterations consistent with stress, and that the effects of this could be mitigated, and therefore welfare refined, via the use of two forms of non-aversive handlings. In particular, the primary endpoint was the CXCL12 chemokine content in the bone-marrow, [the decrease of this chemokine in BM indicates egression of potentially inflammatory cells from BM [1, 15]], and one of the main secondary endpoints were eventual changes in brown adipose tissue temperature detected by Infrared thermography. The behavioural data were collected to have a complete overview within this exploratory study, despite being not the primary endpoints.

Materials and methods

Ethical statement

All procedures were approved by the Italian Institute of Health (Permit Number 55/2019-PR) according to 26/2014 Italian Law on the protection of animals used for scientific purposes. The manuscript was prepared according to the ARRIVE guidelines [30, 31].

Husbandry

Forty-two male 8-week-old CD1 mice were supplied by Envigo srl (Milan, Italy). Only males were used to avoid the biological variable of sex. Animals were individually housed in polycarbonate cages (268x215x141 mm) with wood-shaving bedding (ENVIGO RMS) with free access to tap water and rodent feed (Teklad Global Diet 18%—ENVIGO RMS). Upon arrival they were allowed six days to acclimate to their new surroundings. Cages were open to the room environment and, when necessary for normal husbandry activities like cage cleaning, animals were handled two times a week by the staff involved in the experimental protocol. Strict adherence was made to the method of handling each mouse by its pre-assigned method, which was lifting by the tail in the TH and NAH-M groups and lifting by tunnel in NAH-T group. Room temperature was maintained at 20°±2°C with 55% ± 10% relative humidity and ventilated at 15–18 filtered air changes per hour. Animals were kept on a 12:12 light: dark cycle (lights on at 6am). Shredded paper strips were use as environmental enrichment [32]. Blood screening was performed on sentinel mice (not those involved in the experiment) every six months during the year, to certify the absence of endoparasites and ectoparasites in the animal facility, which is classified as conventional.

NAH-M, n = 10. A cosmetics brush was used to apply NAH-M stimulation [miniature paintbrush Cadrim n° 21 [27]], whose softness was tested on the base of our clinical experience to match the force parameters indicated by Delfini and colleagues [33]. Stimulation was performed by gently stroking the back of the animal with the brush for 5 minutes, in a cephalocaudal direction, without removing the mouse from the cage. The application force and brush velocity fell within the range described by Vrontou et al [27]; approximately 3 cm/s and a force of 2.5mN. When necessary (e.g. for cage cleaning), animals were moved by picking them up by holding the tail.

TH, n = 10. As described by Gouveia and Hurst [26], animals were captured by the base of the tail and lifted and then supported on the experimenter’s sleeve for 30 seconds (secs). The mouse was then released back into its home cage. The handler then stood back from the cage for 60 sec, and then repeated handling for a further 30 secs.

NAH-T, n = 10. Animals were lifted using a handling tunnel as demonstrated in an online video tutorial (https://www.nc3rs.org.uk/how-to-pick-up-a-mouse). They received a session of NAH-T handling as also described by Gouveia and Hurst [26]; i.e. once lifted using the handling tunnel (50 mm diameter, 100 mm long), mice were held for 30 secs, then placed back into their cage. The experimenter’s hands were loosely cupped around the end of the tunnel to prevent the mouse exiting. The experimenter then moved away from the cage for 60 secs and then handled the mice a second time for 30 secs.

Handling treatments method and experimental groups

A group of twelve sentinel mice were provided with shredded paper strips for nesting but were not handled and did not undergo any procedures during the entire duration of the study. When necessary for cage cleaning, they were transferred by tail handling. These animals were used as controls for biochemical parameters and represent the less stressful condition of housing.

Experimental design

The study design and order of data collection is depicted in Fig 1. The procedures were conducted during the light-on cycle (approximately between 7am and 3pm) in order to assess the impact of the R&L procedure as it is normally applied–i.e. during the day. In particular, in the vivarium light was on at 6 am and off at 6pm and the time when the behavioral tests and samples were collected was consistently during the light phase, at least one hour away from the transition phase. When necessary, caretakers entered the room at the same hour which was around 9 am. On arrival mice were randomly assigned using a fair coin toss to one of three handling groups, TH, NAH-T or NAH-M (n = 10 per group), or to Sentinel group (n = 12). Sentinels were provided with shredded paper strips for nesting, and apart from cage cleaning they were not handled and did not undergo any other procedures before sacrifice. They were exposed to the same environment the others mice were exposed to and were individually housed specifically on the same rack as the experimental animals. During cage cleaning they were transferred to their new cage via tail handling. They provided controls for the biochemical/neuroinflammatory parameters but no behaviour data were collected.

Fig 1

Study design.

One complete test lasted 5 consecutive days. In the first two days, the different handling methods (TH, NAH-M and NAH-T) and basal behavioural tests were performed, followed by R&L stress stimuli during the 3rd day. The hour before stress stimuli, animals received a session of handling. The 4th day, 24 hours after stress, post-stress behavioural tests were performed and the last day, after another handling session, mice were sacrificed and organ collected. The entire study lasted 3 weeks, repeating the complete test 3 times.

Once the acclimation period was complete, 3 to 4 mice from each handling group were tested each week, for a total of three study weeks. Mice in the TH and NAH-T groups were always handled according to their assigned method during transfers between the various types of test apparatus (details of this below). Mice in the NAH-M group were lifted by the tail for this purpose. The three experimental groups underwent behavioural testing and application of R&L according to the following timeline:

Day 1: One exposure to handling using TH, NAH-T or NAH-M

Day 2: Baseline assessment of behaviour and anxiety status (between 7am to 3pm, according to the following test sequence;

Nesting scoring (without disturbing the animals or touching the nest)

Recording of BAT temperature via IRT

EPM test (10 minutes)

Body weight recording

HCS Behaviour recording (in a separate room)

Day 3: Handling repeated before placing each mouse into the R&L device for 15 minutes (described below, R&L procedure, performed in a separate room).

Day 4: Behavioral testing repeated as on Day 2 (with the timing of events as far as possible matching Baseline timing);

Day 5: Sacrifice day, with the following sequence of actions between 7am and 3pm:

Handling

Sacrifice and organ removal (in a separate dedicated surgical room)

Tissues were harvested for later processing as described below. Fourteen animals were examined per week, for a total of three weeks of experiments.

Experimental procedures

Stress test

R&L Stress consisted of one 15-minute period of restraint in a small Plexiglas tube (Mouse Tailveiner Standard, diam. 25 mm, 2 Biological Instruments–Varese, Italy). During this time they had a 400–500 lux light source directed at them from their left at a distance of 20 cm [Bouwknecht [34]; Gameiro [12]].

Nesting building

Nest quality was recorded by taking ~30 secs of video footage and then 10 photographs using a digital camera (Sony Cybershot W830). After opening each cage and without disturbing the mouse, approximately 30 seconds of video footage of the completed nest was taken (without continuously following the construction process). The statistical analysis was therefore conducted on nest score at 24 h. Ten photographs were also taken, according to the method of Hess [32]. Mice were considered to have built a nest only if the nesting material contained a hole in the middle and there was a clear sign of a depression in the centre. Mice that had not done so received a score of 0. If there was an interaction with the nesting material, consisting in shredded paper strips, the score was 1 (Fig 2A). When the nest was flat, with a cavity in the middle but with no, or incomplete, walls, nest score was 2 (Fig 2B). Score 3 corresponded to a nest with walls forming a “cup” shape (Fig 2C) and a score of 4 was awarded if the walls reached the widest point of an imaginary half of a sphere (Fig 2D). Finally, a nest forming a complete dome with walls completely enclosing the nest was given a score of 5 (Fig 2E).

Fig 2

Nesting.

Example of nest building. Nest score from 1 (A) to 2, 3, 4 and 5 (B to-E, see text for detailed description). There was no difference in nest score between the groups, either before stress, or after stress test.

IRT

A microbolometric high resolution infrared camera (FLIR A65 model; 640 X 512 pixel sensor) was placed on a tripod 60 cm above the open cage. Mice were continuously recorded while freely moving for 3 minutes. Three still images were chosen from the IRT video footage for each mouse; selected when the image was in focus and the BAT tissue area, chosen on the base of previous studies [21, 35, 36], was clearly visible. The median value of the maximum temperature in this area was calculated using two software: FLIR Tools (FLIR Systems, https://www.flir.it/) and IRT Analyzer (GRAYESS software, https://www.grayess.com/).

EPM

The EPM is a well-established method for anxiety assessment [37]. The EPM platform was 50 cm from the ground and consisted 2 two open and 2 close arms facing one another. Each arm was 50 cm length and 10 cm wide. The closed arm walls were 30 cm high. After lifting each mouse by the appropriate handling method, they were placed into the centre of the maze facing an open arm. They were then recorded using a Sony Digital Handycam video camera placed on a tripod 50 cm above the apparatus for 5 minutes.

HCS

Automated behaviour analysis software (www.cleversysinc.com) was used to record the spontaneous unconstrained behaviour of the mice. The setup for this has previously been described [38]. Briefly, mice were filmed for 10 minutes using a Sony Digital Handycam video camera placed on a tripod approximately 30 cm from the cage front. The cages were lit from behind using a neon light covered with a thin sheet of paper, to evenly distribute the light, thus creating the contrast necessary for subsequent analysis with the HCS software [38]. Recordings lasted ten minutes. The total time needed to undertake each of the 3 types of recording (IRT, EPM and HCS) was approximately 23 minutes; including roughly 10 minutes for handling and moving mice from one apparatus to the next.

Anaesthesia and sacrifice

Each mouse was anaesthetized by intraperitoneal injection of 40 mg/kg Tiletamine/zolazepam (Zoletil 100) and 8 mg/kg xylazine (Xilor), as recommended by 26/2014 Italian Law on the protection of animals used for scientific purposes. Once fully unconscious (verified by toe-pinch method) they were killed by surgical cervical dislocation (surgical cutting of the cervical vertebrae), to avoid brain damage. Femur bones and brain were collected.

Cytokine quantification in BM

Isolation of femur bone from mice was adapted from the method of Madaan et al [39]. In brief, isolated femur was rinsed in cold PBS, both ends were cut and BM was flushed by 29G × ½ needle. BM was flushed from the other end by inverting the femur, directly in the Eppendorf tube containing RIPA extraction buffer and immediately stored in dry ice. ELISA assays for CXCL12 and CCL2 cytokine quantification were performed by LABOSPACE Srl (Via Ranzato, 12–20128 –Milan, Italy), by using R&D MCX120 Mouse CXCL12/SDF-1 alpha Quantikine ELISA kit and R&D MJE00B Kit Mouse CCL2/JE/MCP-1 Quantikine ELISA Kit, following kit instructions. In particular, for Mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit, the Assay Range was defined from 0.2 to 10 ng/mL in tissue lysed and relative sensitivity 0.069 ng/mL. Vendor evaluated Intra–and Inter assay precision at 3.7–5.1% and 7.2–7.5%. Vendor indicated a recovery of above 100% in the dilution’s ranges indicated for assay.

For Mouse CCL2/JE/MCP-1 Quantikine ELISA Kit, the assay range was defined from 7.8 to 500 pg/mL in tissue lysed with sensitivity 0.666 pg/mL. Vendor evaluated Intra–and Inter assay precision at 2.5–4% and 5.1–7.5% which indicated a recovery of above 100% in the dilution’s ranges indicated for assay.

Both ELISA assays are specific for mouse species with < 0.5% cross-reactivity observed with available related molecules. Each ELISA assay included one blank and 7 points for the standard curve. Each plate was loaded with 40 samples. All samples were run in duplicate. Data were validated by testing laboratory and QC was performed. A Four Parameter Curve was applied for standard samples fitting. Values of the standard curve were compared with the values provided by the ELISA vendor not exceeding a R2 of 10% and all of the above parameters were applied on at least 90% of the standard curve values. All CVs of unknown samples met specifications of both vendor’s ELISA.

Activated brain microglia assessment

Activated microglia in the brain were analysed using immunohistochemistry according to manufacturer instructions. After buffered formalin fixation, coronal cutting of the whole brain was made and paraffin embedding was performed. Three micrometer (3 μm) thin sections were cut and stained with Haematoxylin-Eosin and Iba-1 antibody (Abcam Microglia marker panel Ab226482). The section was pre-treated using heat mediated antigen retrieval with Ethylenediaminetetraacetic acid (EDTA) for 3 cycles of 5 minutes each in microwave oven. The section was then incubated with recombinant Iba-1 antibody, 1/2000 dilution, for 2 hours at room temperature. Abcam Iba-1 is a recombinant, non-pre-absorbed antibody. In order to obtain a better result, we used the MACH4 HRP polymer, a very effective amplification system, as a secondary antibody. DAB was used as the chromogen. Negative control was performed by removing the recombinant Iba-1 antibody. The section was then counterstained with haematoxylin and mounted.

Two areas were evaluated for assessment of the activated brain microglia: Paraventricular nucleus (PVN) [1] and the entire first section posterior to the midpoint of the brain. In order to define the brain areas, we used the atlas of Paxinos and Franklin [40]: the entire brain section posterior to the midpoint was taken 2 mm posterior to the bregma. According to the Atlas, we identify PVN just beneath the 3rd ventricle, at the following coordinates: -1.5 rostro-caudal; +/- 0.1 lateral (i.e. millimeters from bregma and from the midline, respectively). The histological slides were digitalized by Hamamatsu Nanozoomer scanner; then Iba-1 positive cells were counted using the ImageJ software as follows: image was transformed into black and white and was subjected to threshold filtering shape and size of the staining in order to identify cell nuclei; in order to measure the Iba-1 positive cells in a non-biased manner, areas and signals were automatically counted by the software and researchers performing histology were blinded to the treatment.

The two areas of interest, the PVN or the whole brain section, were measured in mm2, then Iba-1+ cells density was calculated for each sample. Due to the small size of the PVN, Iba-1 positive cells in this region was expressed differently from those in the entire brain sections. For the PVN, the calculation was the number of Iba-1+ cells/0,08 mm2, whereas in the entire brain section it was the number of Iba-1+ cells/mm2. The entire brain section results were then multiplied for the same factor (x100), to avoid decimals numbers lower than 1.

Statistical analysis

The numerosity within the groups derived from a power calculation for the primary endpoint which was CXCL12 chemokine concentration in the BM (software available at http://www.biomath.info/). On the base of the literature [1, 41], a standard deviation of 50 and a difference of 70 pg/ml was expected between groups to have an alpha value less than 0,05 and a power of 0,80.

The description of the groups of interest with respect to clinical characteristics has been performed by means of median and interquartile range for continuous covariates and by means of absolute and relative frequencies for categorical ones.

Due to the exploratory aim of this study and the small sample size a non-parametric approach was used to investigate the differences within and between groups and we did not perform significance testing to avoid inflation of type 1 error, so generating an excess of false positive signals. To evaluate differences between two groups and intra group (before vs after stress) Wilcoxon-Mann-Whitney test and Wilcoxon signed rank sum test have been performed respectively. Moreover, Kruskal-Wallis (KW) test was used to study the differences among groups. Furthermore, eta-squared based on the H-statistic was calculated to evaluate the percentage of variance in the dependent variable explained by handling groups [42]. This index ranges from 0% to 100% and values between 0% to 6% indicate small effect, from 6% to 14% moderate effect and more than 14% large effect [43]. Statistical analysis was generated using SAS/STAT software, version 9.4 of the SAS system for Windows. Range plots with capped spikes were generated using Stata Software, version 15.1 (StataCorp. 2017. College Station, TX: StataCorp LLC). All statistical tests are interpreted at the 5% significance level considering two-sided test.

Results

IRT

There was no significant difference in temperatures among the handling treatment groups, but the temperature significantly decreased in all groups after stress test in BAT area. In particular, temperature of NAH-M treated mice before stress was 30,5°C (range IQ: 30–30,7) and after stress was 29,5°C (range IQ: 29–30), p = 0,037. In the NAH-T handled animals temperature decreased from 30,4°C before stress test (range IQ: 30,26–30,66) to 29,2 after test (range IQ: 28,5–29,7), p = 0,0097. Control animals, TH, decreased their temperature from 30,2°C to 29,4°C (range IQ: 29,8–30,7 before stress and range IQ: 29,2–30 after stress), p = 0,019 (Fig 3).

Fig 3

IRT image and measurements.

A-Example of IRT image of a mouse: the part in yellow on the dorsal region is the BAT area. B-IRT measurements: after R&L stress test, the temperatures recorded in the BAT area in all experimental groups were lower than those recorded at baseline, suggesting that this stress stimuli might affect BAT homeostasis, with possible consequences on metabolism (*p< 0,03 vs baseline).

BM CCL2 and CXCL12 content

There was no significant difference in CCL2 content in BM among groups [TH 8,014 pg/ml (range IQ:4,352–9,568), Sentinel mice 9,858 pg/ml (range IQ:6,055–10,663), NAH-T 6,411 pg/ml (range IQ:3,875–11,056) and NAH-M 7,597 pg/ml (range IQ:3,935–11,689) H of KW = 0.71, p-value KW = 0,87, Eta2 = 0%] (Fig 4A).

Fig 4

Cytokines content in the bone marrow.

A) CCL2 content: no significant difference was found among groups. B) R&L stress did not cause massive neuroinflammatory activation, despite a trend of CXCL12 decrease in TH and NAH-M groups with respect to Sentinels (Kruskal-Wallis test: p = 0,06), which might indicate an inflammatory process at its final stage.

There was a difference, at the limit of significance (p = 0,06), in CXCL12 BM content among groups. Interestingly, CXCL12 content in BM of NAH-T animals was similar to that found in Sentinels, the less stressed group of animals [TH 1,528 ng/ml (range IQ:1,344–2,025), Sentinel mice 2,531 ng/ml (range IQ:1,631–3,122), NAH-T 2,346 ng/ml (range IQ:1,762–2,741), and NAH-M 1,114 ng/ml, (range IQ:1,036–1,546), H = 7.31, p- value KW = 0,06, Eta2 = 13%] (Fig 4B).

Immunohistochemistry

Considering observation by Ataka et al [1], who found stress-related activated microglia in PVN, this was analysed first, then the entire brain slice was analysed. Histological analysis revealed normal brain architecture and activated microglia were present, but there were no significant group differences.

[PVN: TH 5,29 Iba1+cells/0,08mm2 (median area) (range IQ:2,84–7,09), Sentinel 3,47 Iba1+cells/0,08mm2 (range IQ:2,17–6,39), NAH-T 5,39 Iba1+cells/0,08mm2 (range IQ:3,65–5,71) and NAH-M 4,15 Iba1+cells/0,08mm2 (range IQ:2,88–5,08) H of KW = 2.51, p-value KW = 0.473, Eta2 = 0%].

[Entire Brain Slice: TH 1,936x10-2 Iba1+cells/mm2 (range IQ:1,145–3,051) Sentinel 1,4x10-2 Iba1+cells/mm2 (range IQ:0,933–2,584) NAH-T 1,420x10-2 Iba1+cells/mm2 (range IQ:0,953–2,891) NAH-M 1,373x10-2 Iba1+cells/mm2 (range IQ:0,771–2,583) H of KW = 1.69, p-value KW = 0.638, Eta2 = 0%] (Fig 5 and Table 1).

Fig 5

Activated microglia in the brain.

Histological analysis of the brain. Presence of activated microglia was found (Iba1+ cells in the red circle), but there was not significant difference between R&L stressed mice, regardless of handling, and Sentinels, suggesting that if an inflammatory process occurred in BM, it did not reach the central nervous system. Left: above a x2 magnification of negative control without Iba1 antibody, in the middle a x2 magnification of positive control with Iba1 antibody, below a 4x magnification of PVN (area inside the circle, see methods for stereotaxic coordinates). Center: a representative photomicrograph for each handling group (x5 magnification). Right: a series of images with different degrees of magnification of Iba1+ cells (x20 and x40).

Table 1

Immunohistochemistry staining.

Median values of Iba1+ cells number, with interquartile ranges (25° ptcl-75° ptcl), present in the PVN (H of KW = 2.51, p-value KW = 0.473, Eta2 0%) and in the entire brain slice (H of KW = 1.69, p-value KW = 0.638, Eta2 = 0%) after immunohistochemistry staining.

PVN
Treatment	n	Median Iba1+cells/0,08mm 2	25° pctl	75° pctl
Sentinel	9	3,47	2,17	6,39
TH	10	5,29	2,84	7,09
NAH-T	10	5,39	3,65	5,71
NAH-M	10	4,15	2,88	5,08
Entire Brain
Treatment	n	Median Iba1+cells/mm 2	25° pctl	75° pctl
Sentinel	9	1,4x10-2	0,933	2,584
TH	10	1,936x10-2	1,145	3,051
NAH-T	10	1,420x10-2	0,953	2,891
NAH-M	10	1,373x10-2	0,771	2,583

Body weight

After stress, there was a difference in percentage of body weight gain among groups (H of KW = 6.886 p = 0.032, Eta2 18%). In particular, NAH-T and NAH-M were associated with an increase in body weight with respect to TH, suggesting that Non-aversive handlings might protect mice from the lack of weight gain seen in TH group. [NAH-M was 37 gr before stress (range IQ:36–40) and 38 gr after stress (range IQ: 36–40), TH was 36 gr (range IQ:35–37,5) before stress and 36 gr (range IQ: 35,5–37,5) after stress, NAH-T was 38 gr (range IQ: 37,25–40) before stress and 39 gr (range IQ: 38–40,75, after stress] (Fig 6 and Table 2).

Fig 6

Body weight.

Difference in percentage of body weight gain among groups. NAH-T and NAH-M were associated with an increase in body weight with respect to TH, suggesting that Non-aversive handlings might protect mice from the lack of weight gain seen in TH group (H of KW = 6.886 p = 0.032, Eta2 18%).

Table 2

Percentage of body weight increase after R&L stress, (H value of Kruskal Wallis = 6,88; p = 0,032, Eta2 18%).

NAH-T and NAH-M were associated with a significant increase in BW after stress with respect to TH. TH handled animals showed no body weight increase after R&L stress, therefore the BW percentage value was 0.

BW percentage ((Post -pre)/pre)*100	H of KW = 6.88 p-value = 0.03, Eta2 = 18%
TREATMENT	Median	25° pctl	75° pctl
NAH-M	1.21951	0	2.7027
TH	0	0	0
NAH-T	1.21951	0	2.63158

Behavioural tests

After R&L stress, there was a significant increase in the ratio post/pre of time spent on open arm in mice treated with NAH-M with respect to NAH-T and TH handled groups (H of KW = 6.4585, p-value KW = 0.0396, Eta2 = 18%) (Fig 7 and Table 3). No difference was found before and after stress in TH and NAH-T groups [TH = 40 secs (range IQ:31–75) before stress and 54 secs (range IQ:40–70) after stress, p = 0,89; NAH-T = 64 secs (range IQ:50–79) before stress and 62 secs (range IQ:33–85) after stress; p = 0,49]. No difference was found before and after stress neither in the number of open arm entries nor in the number of protected stretch attends in any of the experimental groups (Table 3).

Fig 7

EPM test.

Time spent on open arm after R&L stress expressed as ratio of post/pre-stress. Mice treated with NAH-M spent a significantly longer time on open arm after stress, (H value of Kruskal Wallis = 6,458, p = 0,0396, Eta2 = 18%.) suggesting a beneficial effect on anxiety in this experimental group.

Table 3

EPM test: A) time spent on open arm, number of open arm entries and protected stretch attends before and after R&L stress.

Median values with interquartile ranges (25° ptcl-75° ptcl.) are reported. Wilcoxon signed rank sum test: *p = 0.025 vs pre-stress value-. B) ratio post/pre increase of time spent on open arm, H value of Kruskal Wallis = 6,458, p = 0,0396, Eta2 = 18%.

A
EPM results		Handling	Pre-stress		p25	p75	Post-stress		p25	p75
Time spent on open arms (sec)		NAH-M	47,5		36	67	90*		58	131
Time spent on open arms (sec)		TH	40		31	75	54		40	70
Time spent on open arms (sec)		NAH-T	64		50	79	62		33	85
Number of open arm entries		NAH-M	4		3	6	4		3	6
Number of open arm entries		TH	3,5		3	5	3		3	4
Number of open arm entries		NAH-T	5		4	6	4		2	6
Number of protected stretch attend		NAH-M	2,5		1	4	3		2	4
Number of protected stretch attend		TH	2,5		0	4	0		0	2
Number of protected stretch attend		NAH-T	1		1	2	1,5		0	4
B
Ratio post/pre of time spent on open arm
H of KW = 6.46, p-value KW = 0.04, Eta2 = 18%
Handling	Median			25° pctl				75° pctl
NAH-M	1.56137			0.8773				3.23529
TH	0.85281			0.58246				1.44921
NAH-T	0.86207			0.57971				1.09231

R&L stress caused a significant reduction in walking and rearing frequency (p = 0.04, p = 0.027, respectively), an increase in grooming duration (in average bout duration seconds; p = 0.0004) and frequency (p<0.0001) in all animals (Fig 8 and Table 4). Handling method had no significant effects on these changes. No difference was seen in total distance travelled.

Fig 8

HCS.

The results of automated behaviour analysis (with HCS) showing behaviour from before and after exposure to restraint stress in mice undergoing each method of handling (NAH-T, TH or NAH-M). P-values refer to comparison between pre-post R&L stress on all animals. Walking and Rearing frequencies significantly declined following stress, although the Total Distance mice moved (metres) tended to decline, not significantly. The average duration of bouts (in seconds) of Grooming increased. Handling method had no significant effects.

Table 4

HCS median values with interquartile ranges (25° ptcl-75° ptcl).

Irrespectively to handling method, R&L stress caused a significant reduction in walking and rearing frequency, and an increase in grooming duration and frequency in all animals.

HCS VALUES		PRE-STRESS				POST-STRESS
Variable	n	Median	25° pctl	75° pctl	n	Median	25° pctl	75° pctl	P-value
Allwalk	30	71	50	87.5	30	47.75	31	58	0.0445
Groom_frequency	30	2	0	4	30	5.5	2	8	<0.0001
RearUp_frequency	30	10	3	15	30	5	2	11	0.027
TravDist_frequency	30	19.62	13.2	24.79	30	14.52	11.29	17.13	0.057
Groom_duration	30	22.3	0	56.68	30	59.98	19.2	101.96	0.0004

Nest score

There was no difference in nest score between the groups, either before stress, or after stress test (Table 5). Before stress test, 24 hours after nesting material availability TH mice displayed a score of 4,75 (range IQ 4,75–5), NAH-T a score of 4,5 (range IQ 0–5), and NAH-M a score of 4,75 (range IQ 4,5–5). H of KW = 3.00, p-value KW = 0.223, Eta2 = 4%. After stress test, 24 hours after nesting material availability TH mice displayed a score of 5 (range IQ 4,75–5), NAH-T a score of 4,75 (range IQ 4,75–5) and NAH-M a score of 5 (range IQ 4,75–5). H of KW = 1.93 p-value KW = 0.381, Eta2 = 0%.

Table 5

Nest score increased in 24 hours in all experimental groups, both before and after R&L stress test, suggesting that 15 minutes of acute R&L did not affect nest building ability.

In particular, before stress test, 24 hours after nesting material availability, TH mice displayed a score of 4,75, NAH-T a score of 4,5 and NAH-M a score of 4,75 (H of KW = 3.00, p-value KW = 0.223, Eta2 = 4%). After stress test, 24 hours after nesting material availability TH mice displayed a score of 5, NAH-T a score of 4,75 and NAH-M a score of 5 (H of KW = 1.93 p-value KW = 0.381, Eta2 = 0%).

NEST SCORE
PRE-STRESS: H of KW = 3.00, p-value KW = 0.223, Eta2 = 4%
POST-STRESS: H of KW = 1.93 p-value KW = 0.381, Eta2 = 0%
Handling	PRE-STRESS	POST-STRESS
NAH-M	4.75	5
TH	4.75	5
NAH-T	4.5	4.75

Discussion

Restraining mice is a common requirement in biomedical research. This is likely to be highly stressful and to influence research findings, and the number of studies assessing if this is a problem is steadily rising [26, 44–46]. However, the focus of these has been on stress-indicative behavioural alterations following restraint by manual handling (e.g., scruffing), and whether these can be minimised using non-aversive (tunnel) handling. There have been no studies to determine the effects of another restraint method that is common for intravenous substance injection, i.e., restraint in an injection tube. The aim was to determine whether this not only has harmful behavioural, but also physiological and neurological consequences. As a secondary aim we also wished to determine if the effects of this type of restraint can be minimised using tunnel handling, but we also included a less well known potentially non-aversive option: mechanoceptive handling.

The choice to work only with male mice was based on evidence that sex can influence responses to handling [45, 47]. In their original article Hurst and West [44] found that although both sexes had lowered anxiety following tunnel handling, females were more responsive. It was later confirmed that male mice are more sensitive to handling-related stress [45, 47]. As our study was exploratory in nature we wished to avoid this potential sex-related influence.

A significant and unexpected finding was that R&L stress caused a significant decrease in BAT temperature in all animals. Several studies have shown that decreased peripheral body temperature (usually caused by adrenergic mediated vasoconstriction) is a fear and stress-related phenomenon, especially in the case of long-lasting inescapable restraint [48-51]. In our case R&L was applied for a moderately long period so it is reasonable to assume that the temperature reductions we recorded were directly attributable to the stress test. Several studies have shown that BAT has an important role in bone health and in glucose homeostasis [35, 36], consequently the exposure to animals of stimuli affecting this tissue might create bias in other types of physiological data. Thus, those studies using procedures similar to ours would be advised to consider the impact that this type of restraint may have when interpreting findings. That said, contrary to what has been observed in models eliciting chronic psychological stress [1], the present procedure had relatively little impact in terms of neuroinflammatory activation. However, there was a trend of inflammation in the BM which might reach significance by anticipating the measurement at a time closer to R&L stress. In fact, 48 hours after R&L two groups of animals (TH and NAH-M) presented decreased CXCL12 content in the BM with respect to Sentinels, a sign of possible progenitor cells egression and differentiation in inflammatory cells [15, 18]. The difference among groups was almost significant (p = 0,06), but suggested an inflammatory process at its final stage, or at least at a later stage. Only NAH-T handled mice showed a CXCL12 content in the BM like that seen in Sentinels; potentially underlying the beneficial effect of tunnel handling on welfare. Further investigations are needed to determine the timescale of this inflammation more precisely.

R&L caused anxiety-like behaviour and reduced body weight that were to a limited extent modulated by handling. According to our EPM results NAH-M seemed to reduce anxiety, and compared to tail-handled mice, both NAH groups were protected from significant post-procedural weight loss. Mechanoceptive handling has a strong clinical background both in animal and human models [27, 52–55]. It relies on activation of the unmyelinated C-fibers polymodal afferent system. C-fibers differentiate in free tactile arborizations on the skin creating a secondary touch system that is specifically processed by the interoceptive system [56]. Due to the strong ties between human pain perception and anxiety, intriguingly, and perhaps unsurprisingly, both human and animal studies have shown that interoceptive touch (involved in NAH-M) can modulate (usually reduce) chronic stress and anxiety [27], pain severity [33, 57], and can improve heart rate variability [58]. It also has a prominent role in social and emotional bonding [59] and in modulating the endogenous u-opioid system [60]. Although our data did not provide such firm evidence of an improvement in the response of mice undergoing NAH-M, they were still consistent with these previous findings. As such, it seems that in some circumstances it could have potential to be an alternative to tunnel handling to at least partially protect animals from unnecessary stress.

Despite substantial evidence that NAH-T can be an effective means of anxiety control [45, 46], it is still used far less frequently than tail handling [25], probably because it is perceived as being more time consuming. While this aspect may be particularly important in the routine of cage cleaning, NAH-T use during experimental procedures could have considerable advantages. Handling mice using a tunnel or using cupped hands rapidly reduces stress susceptibility [26, 45], can mitigate against stress due to injections or anaesthesia and can minimise stress following identification procedures [61]. As far as reproducibility of findings is concerned, non-aversive handling may be an important means of improving the precision of research findings [46, 62, 63].

Being similar to the restraint methods often needed for substance administration, an important study objective was to try to determine the severity of stress or anxiety caused by R&L. We found physiological/metabolic and behavioural alterations consistent with stress, including reduced BAT temperature, reduced spontaneous movements (rearing, walking) and increased grooming. However, despite these changes it had virtually no impact on the ability to mice to nest-build; an outcome that is more commonly associated with more severely impactful disease states or procedures such as surgery [64]. The neuroinflammatory response was also less severe than in conditions of chronic psychological stress [1]. These preliminary data therefore indicate that R&L probably induced stress of moderate severity. Nevertheless, they also indicate that what may be perceived to be relatively ordinary procedures still carry a risk of experimental bias, in which case the use of non-aversive handling methods, especially NAH-T seems a sensible strategy to attempt to mitigate against that risk.

Study limitations

In the present work it was not possible to measure peripheral corticosterone and plasma cytokines levels since the most common blood collection method for this, cardiac puncture, would likely have influenced our assessment of neuroinflammation; which was one of our principal measurements. Blood collection via the facial vein was an alternative possibility and has less impact of on cytokine up-regulation [65], but does not allow taking of the necessary blood volume for both corticosterone and plasma cytokines measurements.

Our assessment of behavioural differences may have been aided had we testing during the night when mice are more active. For example, it has been shown that superior EPM data may be obtained from animals kept on a reverse light cycle [37]. However, our study was meant as a ‘real world’ investigation of the impact of stress as it is normally applied (during the day) and to investigate the risk of increased bias potentially resulting from this, and if this risk can be ameliorated by lowering stress using non-aversive handling methods. Finally, previous EPM findings have indicated that there should be an approximate correlation between the number of open arm entries and time spent on the open arms. We cannot explain such low numbers of open arm entries but note that it is not unusual. For example, Henderson et al. [46] reported an average of less than 1 open arm entry following tail-handling, increasing to only 3.5 in mice handled non-aversively (using a tunnel). In their study the corresponding percentage of time mice in each handling group spent on the open arms was only 7 and 18%, respectively. In our study, ignoring the relatively minor effect that handling method had, the equivalent values were 5.6% before stress vs. 7.6 afterwards. We also checked to see if in our data there were parallel changes in open arm entries and open arm time. The proportion of entries vs. time was between 3–4 before handling and between 5 and 7 afterwards. We admit that these values are still low, but they do parallel one another. Nevertheless, the behavioural outcomes were subsidiary to our main objective of exploring a possible neuroinflammatory effect of acute restraint under bright light. Handling method, and whether it might ameliorate the response to this stressor was secondary.

12 Apr 2021

PONE-D-21-03073

Neuroinflammation, body temperature and behavioural changes in CD1 Male Mice Undergoing Acute Restraint Stress: an Exploratory Study.

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

**********

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Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This is a very interesting study to determine the impact of several factors common to animal handling that might impact study outcome. In particular, the authors focused on: 1. impact of bright light (500 lux) and 15 min restraint, procedures common to injections and type of handling (tail hold, tunnel or mechanoreceptive handling). While the overall intent of the study is laudatory, there are some concerns.

Unfortunately, the manuscript is underdeveloped. It is difficult to evaluate the study as it stands and require significant revisions before being properly evaluated. The experimental design is confusing - there are many factors that come into play and are not well controlled. The data presented does not show all the behavioral outcomes measured (1st after handling and 2nd after stress stimuli/handling). There is also no discussion of how each of the treatments (handling or stress) might have confounding effects.

Additional concerns are listed below:

1. The authors conducted the study using various non-parametric tests. More significantly, the authors used a power analysis centering on chemokine data to determine the number of replicates per treatment group. For this study, the authors should be using the behavioral outcomes to determine the number of replicates. It would appear from the figures and the raw data that the study lacks power. Figures 3, 4 and 8 all have very large error bars (not mentioned if this is standard deviation or error). In addition, the error bars in these figures are unusual - the upper and the lower error bars are different sizes. Its unclear how that can happen.

2. There is a lot of gaps in the methods. CXCL12 and CCL2 were quantified using an ELISA. What is the intra- and inter-assay CV? What is the sensitivity of the assay for each of the markers?

3. Immunohistochemistry - no controls were included in the figure. Was the antibody pre-absorbed? How was the antibody characterized? The low magnification image makes it difficult to evaluate whether the positive signals are real and not background noise - a 40X magnification is a minimum for this Iba1.

4. Elevated plus maze study. It is important to be sure that the baseline is at least 20-30% in the open arms compared to the overall (ie 60-90 s of 300 s). As it stands, the EPM data is not so valid.

5. More details are needed to the sequence of when the studies were conducted relative to each other.

6. There is no mention of when the studies are conducted during the light cycle and relative to zeitgeber (such as lights on or lights off). This is important as it affects the EPM data, body weight (is it right after main feeding?), nest building - affected by diurnal time etc.

7. The overall paper can be better develop and edited.

Reviewer #2: Redaelli and colleagues describe numerous physiological and behavioral effects of acute (15 min) restraint stress on mice that were handled using three different techniques, conventional tail handling, mechanoceptive handling and the non-invasive tunnel handling method. The authors provide a clear rationale for these studies, namely that the physiological and behavioral effects induced by routine procedural use of restraint under bright light conditions may induce variability in results of subsequent endpoints of interest and increase the number required per group due to this variability. Specifically, acute restraint stress reduced body temperature, produce a cessation in body weight gain, increased grooming behavior and decreased naturalistic behaviors in the home cage. No significant restraint induced changes in bone marrow chemokines, activated microglial number in the PVN and entire brain, or on nesting behavior. Furthermore, the authors demonstrate that tunnel handling mitigates the effects of the acute restraint stress/bright light conditions.

Overall the methods are clear and the interpretation of data is appropriate. The manuscript would be of interest to the general readership of PLOS One. There are some concerns/comments that the authors may wish to address.

Concerns/Comments

Regarding the statistical analysis, it is somewhat difficult to draw direct comparisons between the handling groups using non-parametric Mann Whitney within subjects’ evaluation of stress for each given handling technique. If you normalize the data and use Kruskal Wallis, you may wish to compute the effect size for Kruskal-Wallis test as the eta squared based on the H-statistic. That effect size will be a more objective method to compare between handling groups.

In the introduction, the authors suggest that chronic restraint is a well-established model to produce post-traumatic stress disorder or model depression. It is not really appropriate to say it produces PTSD, rather that it may induce behaviors that have relevance to the clinical conditions of PTSD and major depressive disorder.

Only male animals are used in this study. Is there evidence in the literature that show that females respond similarly to males in terms of stress responsivity following tunnel handling? It would be appropriate to add to the discussion to address the issue of sex as a biological variable. Useful reference include Sensini et al2020, DOI: 10.1038/s41598-020-74279-3 and Gouveia and Hurst 2019, DOI: 10.1038/s41598-019-56860-7, which is already referenced in this manuscript.

It may be useful to measure peripheral corticosterone levels in these animals to use as another physiological measure. I would expect that your tunnel handled animals may not have as high levels. Another control would be tail handled animals that are intermittently but repeatedly exposed to the restraint device. Would there be habituation of the behavioral effects observed in such animals.

Methods

• It would be helpful to explicitly state how the animals were transferred to the experimental apparatus. I assume sentinels were transferred by tail handling.

• With regards to the use of sentinels as control animals, were these animals exposed to other pathogens etc. in the room? Are they housed specifically on the same rack as the experimental animals?

• To improve the flow of the manuscript, handling methods should come directly after husbandry.

• Move the following information from the experimental design to the statistical section – “The numerosity within the groups derived from a power calculation for CXCL12 chemokine concentration in the BM (software available at http://www.biomath.info/). On the base of the literature, a standard deviation of 50 and a difference of 70 pg/ml was expected between groups to have an alpha value less than 0,05 and a power of 0,80.”

• For the nesting procedure, 30 seconds is a very short duration in which to observe construction of a nest. Were the additional scores observed at later time points? There may have been a delay in the onset of nesting building, and this would be more informative than a snap shot just after nesting material presentation or 24 h totals of nesting. I would draw your attention to newer publications that utilize nesting as a behavioral endpoint, for example Jacobson 2020 DOI: 10.1016/j.neuropharm.2020.108254. In that study, nesting was evaluated over a 5 hour period. Also, please clarify in the methods whether the statistical analysis was conducted on nest score at 24 h.

• For body weight, data is represented in grams. It perhaps may be most useful to have a normalized value of weight gain/weight loss. Using the percentage of body weight is a useful transformation for statistical analysis between groups.

• It is always difficult to interpret repeated assays of anxiety measures. It is most common that upon repeated testing animals tend to explore more as the aversive quality of the neophobia is diminished. This might explain some of the variance in the post stress time spent in the open arms. Have you considered transforming the data as ratio of post/pre stress?

Results/Figures

• For the data which was analyzed with Kruskal Wallis, please provide the H values.

• Provide the actual values for nesting in the results section and also in Figure 2. The photographs of the nests are appropriate, but the data should be included in that figure.

• Figure 5 - please place the appropriate symbols that indicate the significant effects of stress/handling on grooming and other parameters.

• Figure 10 - only one photomicrograph of Iba1+ immunoreactivity is provided. Please provide the data obtained from all groups and a representative photomicrograph for each handling group.

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Reviewer #1: No

Reviewer #2: No

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7 Jun 2021

Response to Reviewers

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

5. Review Comments to the Author

Reviewer #1: This is a very interesting study to determine the impact of several factors common to animal handling that might impact study outcome. In particular, the authors focused on: 1. impact of bright light (500 lux) and 15 min restraint, procedures common to injections and type of handling (tail hold, tunnel or mechanoreceptive handling). While the overall intent of the study is laudatory, there are some concerns.

Unfortunately, the manuscript is underdeveloped. It is difficult to evaluate the study as it stands and require significant revisions before being properly evaluated. The experimental design is confusing - there are many factors that come into play and are not well controlled.

We thank Reviewer for this comment which has allowed us to improve the experimental design description. A revised figure 1 and a more detailed explanation of our study design is now included in the manuscript (Experimental Design paragraph).

The data presented does not show all the behavioral outcomes measured (1st after handling and 2nd after stress stimuli/handling).

We thank the Reviewer for this observation. Following the reviewer’s suggestion we have now included a new table describing the behavioural outcomes measured and not displayed in the original manuscript (1st after handling and 2nd after stress stimuli/handling) (Tab. 3). Moreover, another table with immunohistochemistry values has been added (Tab. 1).

There is also no discussion of how each of the treatments (handling or stress) might have confounding effects.

We thank the Reviewer for this comment which was probably derived from an insufficiently clear description of the experimental design in our original manuscript. While handling was the study treatment factor, which was thus different among the three experimental groups, R&L stress was not a treatment, but rather the common condition which all treated mice were exposed to. Therefore, stress cannot have generated confounding effects, as it was administered in the same way to all experimental groups. Our study was aimed at assessing the occurrence of systematic differences among groups (all exposed to the same R&L stress), in response to a given specific treatment (handling). We have to thank the reviewer for this comment, which has allowed us to better describe the flow chart of the experimental design of our work, now included in the revised manuscript.

Additional concerns are listed below:

1. The authors conducted the study using various non-parametric tests. More significantly, the authors used a power analysis centering on chemokine data to determine the number of replicates per treatment group. For this study, the authors should be using the behavioral outcomes to determine the number of replicates. It would appear from the figures and the raw data that the study lacks power.

This reviewer’s comment is theoretically important, as it addresses a basic and fundamental aspect of any experimental study design, but, also in this case, it was probably originated by an insufficiently clear description of our primary endpoint in the original manuscript. The primary endpoint of this study was to explore a possible neuroinflammatory effect of acute restraint under bright light. This was the endpoint for which we received the grant NC/S000887/1 provided to by the UK NC3R’s. Assessing response to behavioural tests was not our main goal, but rather a secondary aim of our study, collected to obtain a more comprehensive assessment of animals’ response to stress. Thus, study power calculation was performed on the primary endpoint, i.e. on the CXCL12 chemokine content in the bone-marrow. The sample size determined through this power calculation came out to be adequate also to observe a significant change in the responses to two behavioural tests, EPM and HCS. We have now better clarified these issues in the revised paper.

In fact, exploring possible stress-related neuroinflammation was a fundamental point in our investigation, since Ataka and colleagues [1] revealed an important increase in bone marrow-derived microglia infiltration in the brain and a reduced stromal cell-derived factor-1 (SDF-1 or CXCL12) content in the bone marrow after chronic psychological stress. Being bone marrow-derived microglia infiltration in the brain related with neurogenic hypertension [2], it was a fundamental point to explore whether also common R&L stress might induce neuroinflammation, thus becoming a possible source of bias.

We applied non-parametric tests since in our intention this was to be an exploratory experiment useful to provide information for subsequent research in our laboratory, and we wanted to be conservative. Another intention we had in submitting this preliminary observation, was to disseminate the information of a possible risk of bias when a common procedure like restraint has to be used.

We wish to thank the reviewer for these comments which have allowed us to clarify in our revised paper our approach to study power analysis and the choice of the primary and secondary endpoints (see Statistical analysis paragraph and Introduction).

Figures 3, 4 and 8 all have very large error bars (not mentioned if this is standard deviation or error). In addition, the error bars in these figures are unusual - the upper and the lower error bars are different sizes. It’s unclear how that can happen.

Thank you for this comment. We have now deleted the error bars which represented the interquartile ranges and not the standard deviations. We have now also specified that the data showed in the figures were median values.

2. There is a lot of gaps in the methods. CXCL12 and CCL2 were quantified using an ELISA. What is the intra- and inter-assay CV? What is the sensitivity of the assay for each of the markers?

We thank the Reviewer for this comment which allowed to improve the Methods section. The required details are now in the revised manuscript.

3. Immunohistochemistry - no controls were included in the figure. Was the antibody pre-absorbed? How was the antibody characterized? The low magnification image makes it difficult to evaluate whether the positive signals are real and not background noise - a 40X magnification is a minimum for this Iba1.

Immunohistochemistry was performed according to manufacturer instructions. The section was pre-treated using heat mediated antigen retrieval with Ethylenediaminetetraacetic acid (EDTA) for 3 cycles of 5 minutes each in microwave oven. The section was then incubated with recombinant Iba-1 antibody, 1/2000 dilution, for 2 hours at room temperature. Abcam Iba-1 is a recombinant, non-pre-absorbed antibody. In order to obtain a better result, we used the MACH4 HRP polymer, a very effective amplification system, as a secondary antibody. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted. These details are now in the manuscript.

In our hands, immunohistochemistry worked very well, with only optimization of the retrieval conditions. We agree with the reviewer that higher magnification (40x) can better show the clean background and specificity of the signal. As stated by many authors, the positive controls are microglial cells in the rat brain: an image at high magnification (40x) will allow to make evident the well delineated signal in the cell body and in the dendrites. Thanks to the Reviewer’s suggestion, the methods section has been corrected accordingly and an updated immunohistochemistry image (now Fig. 6) is now present in the manuscript.

4. Elevated plus maze study. It is important to be sure that the baseline is at least 20-30% in the open arms compared to the overall (ie 60-90 s of 300 s). As it stands, the EPM data is not so valid.

We thank the Reviewer for this comment which allows to clarify an important point. According to Walf et al [7], we think that it is not possible to be ‘sure’ of anything with regard to the proportion of time mice will spend on the open arms at baseline versus after any stress/anxiety-causing procedure. In fact, this important paper describes some of the limitations of the test procedure, suggesting ideal conditions for its performance and giving example data. These authors describe how baseline open arm time can be confounded by mice ‘freezing’ on the open arms, i.e. if this is around 30% of the overall test time. Therefore, the time the reviewer suggests might actually represent a confounding effect rather than be an important value to be sure of. This was not the case with any of our baseline results and we consider this an advantage. In one study on anxiolytic treatment that Walf et al cite, they describe how, at baseline (control condition), 2% of time was spent on the open arms by mice in a 300s trial, whereas anxiolytic treated mice spent 7% time on the open arms, and this was deemed a significant difference. Therefore, we respectfully do not consider our EPM data to be not so valid.

5. More details are needed to the sequence of when the studies were conducted relative to each other.

We thank the Reviewer for this suggestion. A more detailed explanation on the sequence of the studies conducted relative to each other is now in the manuscript in the “experimental design” chapter as well as in the new Fig. 1

6. There is no mention of when the studies are conducted during the light cycle and relative to zeitgeber (such as lights on or lights off). This is important as it affects the EPM data, body weight (is it right after main feeding?), nest building - affected by diurnal time etc.

We thank the Reviewer for the comment. We agree that these aspects are all affected by the time during the day when the testing is done. However, we wished to perform a comparative investigation of the impact of the stress procedure as it is normally applied – ie during the day. We have acknowledged this potential limitation in the new Study Limitation paragraph and cited Walf and Frye [7]. The clarification about light cycle is also in the revised manuscript in the “experimental design” chapter.

7. The overall paper can be better developed and edited.

We have tried to improve our manuscript as suggested by the Reviewer.

Reviewer #2: Redaelli and colleagues describe numerous physiological and behavioral effects of acute (15 min) restraint stress on mice that were handled using three different techniques, conventional tail handling, mechanoceptive handling and the non-invasive tunnel handling method. The authors provide a clear rationale for these studies, namely that the physiological and behavioral effects induced by routine procedural use of restraint under bright light conditions may induce variability in results of subsequent endpoints of interest and increase the number required per group due to this variability. Specifically, acute restraint stress reduced body temperature, produce a cessation in body weight gain, increased grooming behavior and decreased naturalistic behaviors in the home cage. No significant restraint induced changes in bone marrow chemokines, activated microglial number in the PVN and entire brain, or on nesting behavior. Furthermore, the authors demonstrate that tunnel handling mitigates the effects of the acute restraint stress/bright light conditions. Overall the methods are clear and the interpretation of data is appropriate. The manuscript would be of interest to the general readership of PLOS One.

We thank Reviewer for this comment.

There are some concerns/comments that the authors may wish to address.

Concerns/Comments

Regarding the statistical analysis, it is somewhat difficult to draw direct comparisons between the handling groups using non-parametric Mann Whitney within subjects’ evaluation of stress for each given handling technique. If you normalize the data and use Kruskal Wallis, you may wish to compute the effect size for Kruskal-Wallis test as the eta squared based on the H-statistic. That effect size will be a more objective method to compare between handling groups.

We thank the Reviewer for this suggestion, now we included in the results section the eta-square based on H statistic and we added an explanation of this index in statistical analysis section.

In the introduction, the authors suggest that chronic restraint is a well-established model to produce post-traumatic stress disorder or model depression. It is not really appropriate to say it produces PTSD, rather that it may induce behaviors that have relevance to the clinical conditions of PTSD and major depressive disorder.

We thank the Reviewer for this clarification. The text is now corrected accordingly.

Only male animals are used in this study. Is there evidence in the literature that show that females respond similarly to males in terms of stress responsivity following tunnel handling? It would be appropriate to add to the discussion to address the issue of sex as a biological variable. Useful reference includes Sensini et al2020, DOI: 10.1038/s41598-020-74279-3 and Gouveia and Hurst 2019, DOI: 10.1038/s41598-019-56860-7, which is already referenced in this manuscript.

We thank the Reviewer for this suggestion. The original article by Hurst and West [8] showed that both males and females of three different strains responded well (i.e., had lowered anxiety in response) to tunnel handling, and there was a slight tendency for females to have even lower anxiety than males. A more recent study has replicated this finding but found that male mice may be more negatively impacted by tail handling than females. This did not drive our selection of a male-only design, which was based on the fact that, since gender can influence response to handling [9-10] and considering the exploratory nature of the study, we had to reduce possible confounding effects, avoiding to introduce this biological variable. Nevertheless, our choice to use males was a fortunate one as these may be more susceptible to the aversive tail handling than females [10]. A clarification about the issue of sex as a biological variable is now added in the Experimental Design and in the Discussion chapters.

It may be useful to measure peripheral corticosterone levels in these animals to use as another physiological measure. I would expect that your tunnel handled animals may not have as high levels.

These are all going point worth remembering in the design of future studies. Thank you for the suggestion. Nevertheless, while corticosterone assays can provide useful information with regard to the stressful consequences of procedures, the success of both of the commonest methods of sampling (plasma or faecal) are tightly bound to the timing of collection. In summary, there is some evidence for both positive and negative impacts on either faecal or plasma corticosterone (please see the summary table NC3Rs has produced at: https://www.nc3rs.org.uk/mouse-handling-research-papers). Moreover, in the present work it was not possible to safely withdraw plasma because the blood collection procedure might have influenced neuroinflammatory response, that was our primary endpoint. In fact, in order to have sufficient quantity of sample we should have performed a cardiac puncture, which has been demonstrated to cause a cytokine elevation with respect to collection of blood from the facial vein, which has a less impact on cytokines level, but does not allow to take the necessary amount of blood [11]. This clarification is now added in the new section “Study Limitations”

Another control would be tail handled animals that are intermittently but repeatedly exposed to the restraint device. Would there be habituation of the behavioral effects observed in such animals.

Again, we thank for this suggestion. Our primary goals were to verify possible stress-related alteration in neuroinflammation and in BAT temperature, caused by the common setting of restraint animals for injections or similar procedures. The approach suggested by the reviewer is certainly of interest. However, in our study we decided to consider as controls animals not exposed to any restraint procedure. In our future studies we will consider adding this additional control, and we are grateful for the advice.

Methods

• It would be helpful to explicitly state how the animals were transferred to the experimental apparatus. I assume sentinels were transferred by tail handling.

The reviewer’s comment is very appropriate: sentinels did not undergo any behavioural test, as specified in the first lines of experimental design chapter, nevertheless when necessary for cage cleaning, they were transferred by tail handling. This clarification is now added in the text.

• With regards to the use of sentinels as control animals, were these animals exposed to other pathogens etc. in the room? Are they housed specifically on the same rack as the experimental animals?

We thank the Reviewer for this question, sentinels were exposed to the same environment the others mice were exposed to and were housed specifically on the same rack as the experimental animals. This detail is now added in the experimental design chapter.

• To improve the flow of the manuscript, handling methods should come directly after husbandry.

We thank the Reviewer for this suggestion, the chapter has been moved accordingly.

• Move the following information from the experimental design to the statistical section – “The numerosity within the groups derived from a power calculation for CXCL12 chemokine concentration in the BM (software available at http://www.biomath.info/). On the base of the literature, a standard deviation of 50 and a difference of 70 pg/ml was expected between groups to have an alpha value less than 0,05 and a power of 0,80.”

We thank the Reviewer for this suggestion, this part is now in the statistical section.

• For the nesting procedure, 30 seconds is a very short duration in which to observe construction of a nest. Were the additional scores observed at later time points? There may have been a delay in the onset of nesting building, and this would be more informative than a snap shot just after nesting material presentation or 24 h totals of nesting. I would draw your attention to newer publications that utilize nesting as a behavioral endpoint, for example Jacobson 2020 DOI: 10.1016/j.neuropharm.2020.108254. In that study, nesting was evaluated over a 5 hour period. Also, please clarify in the methods whether the statistical analysis was conducted on nest score at 24 h.

Thirty seconds of footage was used to allow us to score the completed nest not its construction. Our description of the scoring procedure was obviously misleading and has been corrected, the revised manuscript now clarifies this.

We are also grateful for bringing this article to our attention. Unfortunately, this interesting work was published after completion of our experiments. Nevertheless, we took care to repeat the observations at the same time, around 7 am each day, and to follow the method described by Hess [12] who first published the nest score procedure. In our future studies, the suggested procedure of Jacobson will certainly be taken into consideration. The required clarification on the statistical analysis conducted on nest score at 24 h is now present in the revised manuscript.

• For body weight, data is represented in grams. It perhaps may be most useful to have a normalized value of weight gain/weight loss. Using the percentage of body weight is a useful transformation for statistical analysis between groups.

We are grateful for this suggestion. A new table and a new figure displaying the percentage of body weight increased is now in the revised manuscript (Tab. 2, Fig. 7).

• It is always difficult to interpret repeated assays of anxiety measures. It is most common that upon repeated testing animals tend to explore more as the aversive quality of the neophobia is diminished. This might explain some of the variance in the post stress time spent in the open arms. Have you considered transforming the data as ratio of post/pre stress?

We are grateful for this suggestion which we have followed by adding a new table (Tab.3) that shows all the EPM outcome measured as well as a ratio of post/pre-stress time spent in the open arms. Regarding the difficulty to interpret repeated assays of anxiety measures, it is true that the EPM is susceptible to variation if the test is applied successively, unless there are several weeks passing between the before and after conditions. If EPM had been our main endpoint, the design would have obviously to be different, measuring open-field responding first, then test using the EPM in one half of the mice, then the reverse in the other. Following this, we could have looked at proportional changes in anxiety-like responding in a more balanced manner while equalising the exposure of the mice in each treatment group to the novel environment each method poses. Again, this suggestion is extremely useful for our future studies.

The reason of adding EPM as further behavioural test, was to have a complete overview within this exploratory study, despite behavioural tests were not the primary endpoints of the project. We have now added this latter point in the text (Introduction).

Results/Figures

• For the data which was analyzed with Kruskal Wallis, please provide the H values.

Thanks for the suggestion, H values are now provided.

• Provide the actual values for nesting in the results section and also in Figure 2. The photographs of the nests are appropriate, but the data should be included in that figure.

Reviewer’s comment is very useful, results section and figure 2 are now corrected accordingly.

• Figure 5 - please place the appropriate symbols that indicate the significant effects of stress/handling on grooming and other parameters.

Thanks for the suggestion, an appropriate description of the significant effects of stress is now in the figure and in the legend.

• Figure 10 - only one photomicrograph of Iba1+ immunoreactivity is provided. Please provide the data obtained from all groups and a representative photomicrograph for each handling group.

We thank the Reviewer for this suggestion. A corrected figure representative for each handling group is now present, as well as a new table with the data described in immunohistochemistry results chapter and a series of images with different degrees of magnification (2x, 20x and 40x).

Reference

1. Ataka K, Asakawa A, Nagaishi K, Kaimoto K, Sawada A, Hayakawa Y, et al. Bone marrow-derived microglia infiltrate into the paraventricular nucleus of chronic psychological stress-loaded mice. PLoS One. 2013;8(11):1–14.

2. Calvillo, L. Gironacci, M.M. Crotti, L. Meroni, P.L. Parati G. Neuroimmune crosstalk in the pathophysiology of hypertension. Nat Rev Cardiol. 2019;16:476-490.

3. Redaelli, V., Papa, S. Marsella, G. Grignaschi, G., Bosi, A. Ludwig, N. Luzi, F., Vismara, I., Rimondo, S. Veglianese, P. Tepteva, S. Mazzola, S. Zerbi, P. Porcu, L. Roughan, J.V. Parati, G. and Calvillo L. A Refinement approach in a mouse model of rehabilitation research. Analgesia strategy, Reduction approach and infrared thermography in spinal cord injury. PLoS One. 2019;14:e0224337.

4. Devlin MJ. The “skinny” on brown fat, obesity, and bone. Am J Phys Anthropol. 2015;156(S59):98–115.

5. Stanford KI, Middelbeek RJW, Townsend KL, An D, Nygaard EB, Hitchcox KM, et al. Brown adipose tissue regulates glucose homeostasis and insulin sensitivity. J Clin Invest. 2013;123(1):215–23.

6. Fornasier M., Redaelli V., Tarantino A., Luzi F. VM. Infrared thermography (IRT) in nude mice: an alternative method for body temperature measurement. In: Atti Scand FELASA 2010, Helsinki, June 14-17, 2010. 2010.

7. Walf A., Frye C. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc. 2007;2(2):322–8.

8. Hurst JL, West RS. Taming anxiety in laboratory mice. Nat Methods [Internet]. 2010;7(10):825–6. Available from: http://dx.doi.org/10.1038/nmeth.1500

9. Gouveia K, Hurst JL. Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice. Sci Rep. 2019;9(1):1–19.

10. Sensini F, Inta D, Palme R, Brandwein C, Pfeiffer N, Riva MA, et al. The impact of handling technique and handling frequency on laboratory mouse welfare is sex-specific. Sci Rep [Internet]. 2020;10(1):1–9. Available from: https://doi.org/10.1038/s41598-020-74279-3

11. Mella JR, Chiswick EL, King E, Remick DG. Location, location, location: Cytokine concentrations are dependent on blood sampling site. Shock. 2014;42(4):337–42.

12. Hess SE, Rohr S, Dufour BD, Gaskill BN, Pajor EA, Garner JP. Home improvement: C57BL/6J mice given more naturalistic nesting materials build better nests. J Am Assoc Lab Anim Sci [Internet]. 2008;47(6):25–31. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2687128&tool=pmcentrez&rendertype=abstract

25 Jun 2021

PONE-D-21-03073R1

Neuroinflammation, body temperature and behavioural changes in CD1 Male Mice Undergoing Acute Restraint Stress: an Exploratory Study.

PLOS ONE

Dear Dr. Calvillo,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: (No Response)

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Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: I thank the authors for the improved manuscript. Figure 1 is helpful in interpreting the experiment conducted.

There are still a few points of concern:

1. NEW. It is unclear when the behavioral tests and samples were collected during the light phase of the day (0600 - 1800 h). Is it consistent? It makes a difference if it is close to the transitions (ie 0600 - ~ 0800 h and 1500 - ~1800 h) for the mice. Since this is a NC3R study, the authors should also state what time of the day the vivarium caretakers enter the room as this can also have an impact on the study.

2. The authors made a very good counter argument about the very low baseline time in open arms (significantly less than 20-30%). However, the data does not reflect their argument. There should be parallel changes in open arm time and open arm entry. I am okay to waive the 20-30% baseline but the authors should show that the mice were actively exploring. In addition to the measures in Table 3A, it would be good to include activity in the open arm and activity in the closed arm. Other measures of exploration are head dip, sniffing etc.

3. Immunohistochemistry.

The 40X is an improvement. Thank you.

Regarding controls for the Iba1+ cells, the authors still did not address what controls they used. Controls for immunohistochemistry typically includes: 1. removal of primary antibody, 2. preabsorption of the antibody using a peptide (this should be easy to purchase or synthesize) or 3. use a Iba1 knockout mouse. The third option is idea but not always possible. Option #2 is the more commonly used option. The reason it is necessary to conduct a negative control is that immunohistochemistry is sensitive to many subtle changes in protocol - fixative, antigen retrieval etc etc etc. These subtle changes can cause changes to the epitope which can lead to false positives or partial positives/negatives.

4. NEW. More details are needed for where Iba1+ cells were counted. More details are needed to define the PVN and the entire section posterior to the midpoint of the brain. More typical details include: 1. using a stereotaxic atlas to provide the coordinates (rostro-caudal and lateral) for what you are looking at and 2. a photomicrograph of the region of interest that is outlined. In addition, more information is also needed to how the authors measured the Iba+ cells in a non-biased manner.

5. NEW. In the response to reviewers, the authors state that "an image at high magnification (40X) will allow to make evident the well delineated signal in the cell body and in the dendrites." Microglia do not have dendrites.

Reviewer #2: Please ensure that the term sex is used throughout the manuscript not sex. Your mice have a sex, they do not have a gender.

Tables & Figures – with regard to presentation of the data. It would be best to present the data for the sentinels (true controls) in the first line.

If the data are presented as a figure it is not necessary to present a table also, for example Table 3b contains the same information as presented in figure 8.

Table 2 – data from TH animals were not included here, so perhaps you may wish to omit this line from the table to avoid confusion.

Figures 3 and 4 should be combined into one Figure.

The data for the nesting behavior has not been added in table or figure format.

For the EPM it is not conventional to use the term fold increase. Rather choose a better descriptor, such as ratio of post/pre, or normalized to prestress baseline.

There remains a number of grammatical and typographical errors. The manuscript would benefit from editing by a native English speaker.

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9 Oct 2021

Please pay particular attention to the request of reviewer 1 to provide additional controls for data analysis and interpretation.

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank the authors for the improved manuscript. Figure 1 is helpful in interpreting the experiment conducted.

There are still a few points of concern:

1. NEW. It is unclear when the behavioral tests and samples were collected during the light phase of the day (0600 - 1800 h). Is it consistent? It makes a difference if it is close to the transitions (ie 0600 - ~ 0800 h and 1500 - ~1800 h) for the mice. Since this is a NC3R study, the authors should also state what time of the day the vivarium caretakers enter the room as this can also have an impact on the study.

We thank the reviewer for highlighting this important detail. The time when the behavioral tests and samples were collected was consistently during the light phase, at least one hour away from the transition phase. The specific time of the day is reported in the “Experimental Design” paragraph, where the procedures performed in Day 2, 4 and 5 were described. In the vivarium, light was on at 6 am and off at 6pm. When necessary, caretakers entered the room always at the same hour which was around 9 am. This information is now in the manuscript in the “Experimental Design” paragraph (from line 214 of the Revised Manuscript with Track Changes).

2. The authors made a very good counter argument about the very low baseline time in open arms (significantly less than 20-30%). However, the data does not reflect their argument. There should be parallel changes in open arm time and open arm entry. I am okay to waive the 20-30% baseline but the authors should show that the mice were actively exploring. In addition to the measures in Table 3A, it would be good to include activity in the open arm and activity in the closed arm. Other measures of exploration are head dip, sniffing etc.

Thank you for acknowledging the validity of the counter argument in our initial reply. We agree that there should be an approximate correlation between the number of open arm entries and time spent on the open arms of the EPM. We cannot explain such low numbers of open arm entries but note that it is not unusual. For example, Henderson et al. reported an average of less than 1 open arm entry following tail-handling, increasing to only 3.5 in mice handled non-aversively (using a tunnel: https://doi.org/10.1038/s41598-020-71476-y). In their study the corresponding percentage of time mice in each handling group spent on the open arms was only 7 and 18%, respectively. In our study, ignoring the relatively minor effect that handling method had, the equivalent values were 5.6% before stress vs. 7.6 afterwards. We also checked to see if our data met the reviewers correct assertion that there should be parallel changes in open arm entries and open arm time. The proportion of entries vs. time was between 3-4 before handling and between 5 and 7 afterwards. We admit that these values are still low, but actually do parallel one another. Furthermore, and as we previously said, the behavioural outcomes were subsidiary to our main objective of exploring a possible neuroinflammatory effect of acute restraint under bright light. Handling method, and whether it might ameliorate the response to this stressor was secondary. Since there were not significant effects of this on the other parameter we evaluated (protected-stretched attend), we do not think it would be a sensible use of time to re-evaluate the activity of the mice on the open and closed arms, as we would likely arrive at the same conclusion – that handling had minimal effect. That’s said we do agree with the reviewer that these values are low, and have acknowledged this in the “Study Limitation” paragraph (from line 609 of the Revised Manuscript with Track Changes).

3. Immunohistochemistry.

The 40X is an improvement. Thank you.

Regarding controls for the Iba1+ cells, the authors still did not address what controls they used. Controls for immunohistochemistry typically includes: 1. removal of primary antibody, 2. preabsorption of the antibody using a peptide (this should be easy to purchase or synthesize) or 3. use an Iba1 knockout mouse. The third option is idea but not always possible. Option #2 is the more commonly used option. The reason it is necessary to conduct a negative control is that immunohistochemistry is sensitive to many subtle changes in protocol - fixative, antigen retrieval etc etc etc. These subtle changes can cause changes to the epitope which can lead to false positives or partial positives/negatives.

Thank you for acknowledging the improvement of the 40X figure. In order to have controls for immunohistochemistry we repeated reactions on a double set of slides of all brain samples, simultaneously, with Iba1 antibody and without it. The reactions on slides performed by removal of primary antibody did not show any signal. We have now specified the negative control used in the text (line 368) and modified figure 5 accordingly.

4. NEW. More details are needed for where Iba1+ cells were counted. More details are needed to define the PVN and the entire section posterior to the midpoint of the brain. More typical details include: 1. using a stereotaxic atlas to provide the coordinates (rostro-caudal and lateral) for what you are looking at and 2. a photomicrograph of the region of interest that is outlined. In addition, more information is also needed to how the authors measured the Iba+ cells in a non-biased manner.

Thanks for the comment. We used the following atlas: Paxinos G. and Franklin, K.B.J., The mouse brain in stereotaxic coordinates, Academic Press, New York, 2001, ISBN 0-12-547637-X, in order to define the brain areas. The entire brain section posterior to the midpoint was taken 2 mm posterior to the bregma. According to Paxinos Atlas, we identify PVN just beneath the 3rd ventricle, at the following coordinates: -1.5 rostro-caudal; +/- 0.1 lateral (i.e. millimeters from bregma and from the midline, respectively). Atlas information and coordinates are now provided in the text (from line 372) and the photomicrograph of the region of interest that is outlined (PVN) is now in the modified fig. 5. Regarding information to how we measured the Iba+ cells in a non-biased manner, we provided a detailed description of our use of ImageJ software in the method paragraph of our original submission (from line 372); in particular, we specified that signals were automatically counted. Nevertheless, Reviewer’s comment is important and we have now added a sentence accordingly, also specifying that researchers performing histology were blinded to the treatment (from line 379).

5. NEW. In the response to reviewers, the authors state that "an image at high magnification (40X) will allow to make evident the well delineated signal in the cell body and in the dendrites." Microglia do not have dendrites.

The Reviewer is right, thanks for highlighting this error in our previous response.

Reviewer #2: Please ensure that the term sex is used throughout the manuscript not sex. Your mice have a sex, they do not have a gender.

Thanks for highlighting this error, which we have corrected accordingly.

Tables & Figures – with regard to presentation of the data. It would be best to present the data for the sentinels (true controls) in the first line.

Thank you for the suggestion. We have now modified all tables and figures accordingly.

If the data are presented as a figure it is not necessary to present a table also, for example Table 3b contains the same information as presented in figure 8.

We thank for this comment; nevertheless, a complete presentation with both table and figure for the EPM parameters was necessary to address the Reviewer 1’s comments.

Table 2 – data from TH animals were not included here, so perhaps you may wish to omit this line from the table to avoid confusion.

The meaning of these values was that there were no differences. Omitting these values would be in our opinion more and not less confusing. To address the reviewer’s concern, however, we have tried to clarify this issue in the legend. Thank you for the suggestion.

Figures 3 and 4 should be combined into one Figure.

Thank you for this suggestion. We have now combined Figure 3 and figure 4, accordingly.

The data for the nesting behavior has not been added in table or figure format.

Thank you for the suggestion. A new table 5 with the data for the nesting behaviour is now present in the manuscript

For the EPM it is not conventional to use the term fold increase. Rather choose a better descriptor, such as ratio of post/pre, or normalized to prestress baseline.

We thank the Reviewer for this comment. The table 3b and the EPM figure actually showed a ratio of post/pre, and we have double checked this by repeating all calculations. We have now edited figures, tables and manuscript text accordingly.

There remains a number of grammatical and typographical errors. The manuscript would benefit from editing by a native English speaker.

Thank you for the comment, the native English speaker co-author Dr. Roughan has edited the manuscript.

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Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Nov 2021

Neuroinflammation, body temperature and behavioural changes in CD1 Male Mice Undergoing Acute Restraint Stress: an Exploratory Study.

PONE-D-21-03073R2

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Table 2 has been updated adequately. The nesting scores are very helpful to have in the table form and the data from the EPM are explained very well.

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4 Nov 2021

PONE-D-21-03073R2

Neuroinflammation, body temperature and behavioural changes in CD1 Male Mice Undergoing Acute Restraint Stress: an Exploratory Study.

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