Evidence that the RFID transponders do not elicit an inflammatory tissue reaction.

<p>Subcutaneous empty pocket (arrowed) displays site of former localisation of RFID microchip. No or slight cellular reaction (focal chronic reactive granulation tissue) was observed histologically after H&E staining of skin collected from the area of implantation.</p

Schematic overview of the Rodent Big Brother (Home Cage Analyzer; ActualHCA™) system.

<p>Rats are housed in social groups in standard IVC cages with the Home Cage Analyzer equipment slotted inside an adjacent cage void. The sealed baseplate RFID reader derives positional and temperature information for each animal individually from their subcutaneous RFID chip. The infrared HD video camera captures 25 fps, infrared gray scale video, continuously. An array of infrared LEDs above the cage provides even illumination day and night. The IVC home cage sits immediately above a baseplate RFID reader. The mini-computer captures the video and baseplate data. The system enables manual behavioral analysis at any time of day or night, overall motion detection (whole-cage activity) for the group of rats, and automated detection of ambulatory and vertical activity, and subcutaneous temperature. Representative 7-day readouts are shown for ambulatory activity and subcutaneous temperature; the 12 h light-dark cycle is indicated by white-gray shading.</p

Validation of baseplate-derived ambulatory activity by comparison to manual tracking.

<p>Data shown are from the ventral midline RFID placement following ‘shielding upgrade’. (a) Ambulatory movement (distance travelled) of the rats derived from the baseplate RFID reader (red line) is overlaid with distance travelled measured by manual tracking (blue line) over a 60 min period (plotted in 1-minute bins; mean of 6 rats ± SEM), for each RFID implantation site. (b) Correlation plot of baseplate and manual activity data (distance travelled) from the 6 rats plotted in 15-minute bins from the 60-minute monitoring period. The dotted line is the equivalence line. (c) Bland-Altman plot showing the average difference between the baseplate and manual measurements as a function of the average reading.</p

Validation of vertical activity and rearing measurements by temporal correlation.

<p>(a) Broken column charts showing the automated detection of vertical activity and manual analyses of vertical activity and rearing, in 1 h samples of light and dark phase video footage, arranged in sequential 15-min bins (videos 1–4). Gray columns indicate the time spent in vertical activity by automated detection, red columns indicate the time spent in vertical activity by manual annotation, blue columns indicate the time spent rearing by manual annotation. The frame-by-frame accuracy against the gray column of each bin was determined. (b) Correlation between time spent in vertical activity detected automatically versus manual annotation of vertical activity; R² = 0.9236; ICC = 0.97. (c) A Bland-Altman plot, a visual tool to compare two techniques by plotting the difference against the signal to assess for bias in any one technique over the dynamic range assessed, was used to compare a manual measure of vertical activity and an automated measure of vertical activity. (d) Correlation between time spent in vertical activity detected automatically versus manual annotation of rearing; R² = 0.4774; ICC = 0.91. Two notable outliers are highlighted; red-encircled point: probable cause is rat on top of play tunnel but not actually rearing; blue-encircled point: rat rearing at rear of cage but nose just beneath horizontal line cut-off (illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181068#pone.0181068.g005" target="_blank">Fig 5</a>). (e) Bland-Altman plot comparing vertical measure between manual assessment of rearing and an automated assess of vertical activity.</p

Relationship between light and dark phase overall activity for 12 cages of 3 rats.

<p>Data are 7-day mean values for the light phase overall activity (average of each of the 24 30-minute bins of side-view video pixel movement) and dark phase overall activity (ditto) for 12 cages of 3 rats. (a) Box-and-whisker plot illustrating the difference between dark and light phase activity. (b) Correlation plot between light and dark phase activity for each cage. Note that for 3 of the groups of 3 rats, their light phase activity exceeded that of the dark phase activity for at least one of the other cages. (c) Box-and-whisker plot of the ratio of dark: light phase activity. Note that although there is a range of activities between cages of 3 rats (panel b), the ratio of dark: light phase activity remains within the range 1.2 to 1.7-fold, with a mean of approximately 1.4-fold (panel c).</p

Illustration of the effects of routine perturbations on ambulatory activity and temperature.

<p>(a) Effects of handling/dosing, cage changing and entry of staff into the holding room, on ambulatory activity in the home cage. Plot of baseplate-derived ambulatory activity (mean of 6 rats ± SEM). Data plotted in 10-minute bins. The baseplate detected a spike in activity lasting around an hour in response to the change in home cage. An earlier, smaller peak was in response to dosing (oral gavage) with vehicle. (b) Subcutaneous (flank) temperature of rats when single-housed (filled circles) vs. group-housed (open circles) throughout the dark phase (gray-shaded). Data plotted in 30-minute bins (mean of 6 rats ± SEM). Rats were single-housed at 16:00 hrs and re-grouped at 08:00 hrs the following day. There was an apparent reduction in subcutaneous temperature from 1 h after separation from cage mates and throughout the 16 h separation period. These data were not evaluated by statistical analysis, as they were casual observations rather than pre-designed experiments.</p

Concordance between baseplate-derived ambulatory activity and overall movement within the cage by video analysis.

<p>Overlay plot from one cage of 3 rats (a) and correlation plot for 2 cages of 3 rats (b) of ambulatory movement of the rats derived from the baseplate RFID reader versus side-view pixel movement detection, for the ventral midline site (after ‘shielding upgrade’), over 7 consecutive days of recording. Data are plotted as the mean of 3 rats per cage (2 cages), in 30-minute bins. For the overlay plot (a), the red line is mean transitions derived from the baseplate, the blue line is the total pixel movement derived from the side-view HD video; light and dark phases are indicated by the shading. Note that the video motion analysis reflects all movement (not just ambulatory activity), and may at times be exaggerated by a rat in the foreground grooming (for example).</p

Anticipated advantages and drawbacks of Actual Home Cage Analyzer™ over existing technologies in rats

<p>Anticipated advantages and drawbacks of Actual Home Cage Analyzer™ over existing technologies in rats</p

Comparison of the 4 subcutaneous RFID transponder implantation sites/ orientations.

<p>Guide to rating: +++++ (green): excellent; ++++ (yellow): optimal; +++ (light brown): near-optimal; ++ (amber): sub-optimal; + (red): poor (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181068#pone.0181068.t001" target="_blank">Table 1</a> for rationale). *Data for flank: vertical and ventral midline are post-‘shielding upgrade’. **Baseplate-derived ambulatory activity data for the 3 individual rats within a cage averaged to compare to whole-cage activity. On balance, the ventral midline location was deemed preferable.</p

Criteria used to select optimal subcutaneous RFID implantation site/orientation.

<p>Criteria used to select optimal subcutaneous RFID implantation site/orientation.</p