Comparative Medicine Copyright 2016 by the American Association for Laboratory Animal Science
Vol 66, No 5 October 2016 Pages 384–391
Original Research
Effects of Colored Enrichment Devices on Circadian Metabolism and Physiology in Male Sprague–Dawley Rats Melissa A Wren-Dail,1,2,* Robert T Dauchy,2 Tara G Ooms,3 Kate C Baker,4 David E Blask,2 Steven M Hill,2 Lynell M Dupepe,1 and Rudolf P Bohm, Jr4 Environmental enrichment (EE) gives laboratory animals opportunities to engage in species-specific behaviors. However, the effects of EE devices on normal physiology and scientific outcomes must be evaluated. We hypothesized that the spectral transmittance (color) of light to which rats are exposed when inside colored enrichment devices (CED) affects the circadian rhythms of various plasma markers. Pair-housed male Crl:SD rats were maintained in ventilated racks under a 12:12-h light:dark environment (265.0 lx; lights on, 0600); room lighting intensity and schedule remained constant throughout the study. Treatment groups of 6 subjects were exposed for 25 d to a colored enrichment tunnel: amber, red, clear, or opaque. We measured the proportion of time rats spent inside their CED. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and analyzed for plasma melatonin, total fatty acids, and corticosterone. Rats spent more time in amber, red, and opaque CED than in clear tunnels. All tubes were used significantly less after blood draws had started, except for the clear tunnel, which showed no change in use from before blood sampling began. Normal peak nighttime melatonin concentrations showed significant disruption in the opaque CED group. Food and water intakes and body weight change in rats with red-tinted CED and total fatty acid concentrations in the opaque CED group differed from those in other groups. These results demonstrate that the color of CED altered normal circadian rhythms of plasma measures of metabolism and physiology in rats and therefore might influence the outcomes of scientific investigations. Abbreviations: CED, colored enrichment devices; EE, environmental enrichment; SCN, suprachiasmatic nucleus; TFA, total fatty acids
The visual system of mammals encompasses the anatomic structures of the eye and the primary optic tract. Because rats are nocturnal animals, their retinas are rod-dominated but also contain cones. This visual system contains 2 main classes of cones, which contain photopigment with spectral sensitivity in either the 359-nm (UV) or 509-nm (M cone) wavelengths. Rudimentary color discrimination is believed to be supported by these 2 cone classes, but this discrimination may be only dichromatic in nature.27 This dichromatic nature is a characteristic of the visual system and is reflected in rats’ preference for a dark environment: darkness likely is preferred due in part to the effects of light on retinal degeneration over time, which perhaps is painful, causing rats to shy away from light.33 Changes in lighting intensity, duration, and wavelength (visually perceived as color or tint) at various times of the day can disrupt circadian rhythms of metabolism and endocrine hormone concentrations.7,12,13,15,41 These responses are primarily elicited by means of a nonvisual system, also located in the retina, through Received: 11 Nov 2015. Revision requested: 05 Jan 2016. Accepted: 12 May 2016. 1 Departments of Comparative Medicine and 2Structural & Cellular Biology, Tulane University School of Medicine, New Orleans, Louisiana, 3Section of Laboratory Animal Medicine, IIT Research Institute, Chicago, Illinois and 4Division of Veterinary Medicine, Tulane National Primate Research Center, Covington, Louisiana *Corresponding author. Email: [email protected]
intrinsically photosensitive retinal ganglion cells5,8,11,17,21,24,25,32 and not through the visual primary optic tract. Circadian rhythms are entrained by the nonvisual light–dark cycle through signals from the retinohypothalamic tract to the suprachiasmatic nucleus (SCN). The SCN regulates circadian rhythms and is termed the ‘master biologic clock.’5,7 Wavelengths between 450 and 484 nm (that is, blue light) are the strongest inciters of neuroendocrine and circadian responses in mammals, however high-intensity, longer-wavelength (that is, red) light can have this effect also.7,14,15,23,35,41 There is now overwhelming evidence of the influence of the retinohypothalamic tract system on the regulation of behavior20 and metabolism.7,22,30,41 Prior research12-16,41 from our laboratory revealed significant disruptions of various circadian rhythms and increased tumor growth from light leaks around doorways at night. In addition, we investigated the effect of spectral transmittance (cage tint) through standard laboratory caging in female pigmented athymic nude rats and male Sprague–Dawley rats12,41 and showed that these animals developed chronobiologic disruptions in various plasma measures of physiology and metabolism. Although the circadian rhythm of total fatty acids (TFA) in plasma remained unchanged, all other metabolic and physiologic rhythms were significantly altered when exposed to amber-, red-, or blue-tinted caging as compared with clear caging. We also have shown that
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Sprague–Dawley rats have significant circadian disruptions in melatonin, corticosterone, leptin, insulin, and glucose concentrations when exposed to various color caging during the day.12,41 These disruptions in plasma levels included duration, phasing (timing), amplitude, or some combination of these elements. On inspection of items currently placed into the animal cage for environmental enrichment (EE), we noticed that many of these items are color-tinted. Most are made from transparent durable plastic for ease of cleaning and sanitation. The premise for using these color-tinted items is that they allow for monitoring and health checking yet afford rodents the opportunity to retreat from stressful stimuli or cage mates and to regulate body temperature.27 However, some objects added to the animals’ environment are known to induce stress.27 Although some studies1,4,18,26,37,38,40 have explored the effect of EE on behavior, few studies4,40 have examined the influence of EE, particularly colored enrichment devices (CED), on plasma measures of circadian metabolism and physiology in laboratory animals. Therefore, we sought to determine whether various CED affected animal measures of metabolism or neuroendocrine hormones. For the current study, we explored the effect of spectral transmittance exposure from commonly used CED (amber, clear, red, and opaque; Figure 1) during the light period in male albino Sprague–Dawley rats. We chose this rat stock because it is among the most widely used in laboratory research, comprising approximately 53% of EE studies.37 Melatonin, TFA, and corticosterone were chosen to examine common circadian rhythms through plasma measures of metabolism and physiology. We hypothesized that changing the tinting (spectral transmittance or quality) of EE by using various CED affects circadian rhythms of these measures in male Sprague–Dawley rats.
Materials and Methods
Reagents. HPLC-grade chloroform, ethyl ether, methanol, glacial acetic acid, heptane, and hexane were purchased from Fisher Scientific (Pittsburg, PA). Free fatty acids, rapeseed oil methyl ester standards, boron–trifluoride–methanol, potassium chloride, sodium chloride, and perchloric and trichloroacetic acids were purchased from Sigma Scientific (St Louis, MO). Ultrapure water (catalog no. 400000) was purchased from Cayman Chemical (Ann Arbor, MI). Animals, housing condition, and diet. Male Sprague–Dawley rats (age, 4 to 5 wk) were purchased from Charles River (Crl:CD[SD]; Kingston, NY) and certified by the vendor to be free of all known rodent bacterial, viral, and parasitic pathogens. According to the vendor, rats at this age were provided with only group social enrichment during rearing. Animals were maintained in Tulane University School of Medicine Vivarium, which has been an AAALAC-accredited facility since 1962. All animal use and accompanying procedures were in accordance with The Guide for the Care and Use of Laboratory Animals27 and were IACUCapproved. Rats were maintained in ventilated cages using hardwood maple bedding (no. 7090, Sanichips, Harlan Teklad, Madison, WI; 1 bedding change weekly). Serum samples from sentinel animals on combined soiled bedding from project animals were tested quarterly (Multiplex Fluorescent Immunoassay 2, IDEXX Research Animal Diagnostic Laboratory, Colombia, MO) for rat coronavirus, Sendai virus, pneumonia virus of mice, sialodacryoadenitis virus, Kilham rat virus, Toolan H1 virus, reovirus type 3,
Figure 1. Colored enrichment devices used in this experiment were (from left to right) translucent amber, clear, red (polycarbonate) and opaque (polyvinyl chloride) and had identical internal diameters and lengths.
Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus types 1 and 2, Hantaan virus, Encephalitozoon cuniculi, cilia-associated respiratory bacillus, parvovirus NS1, rat parvoviruses, rat murine virus, and rat theilovirus as well as external and internal parasites; all test results were negative. Rats had free access to a commercial diet (no. 5053 Irradiated Laboratory Rodent Diet, Purina, Richmond, IN) and acidified water. Quadruplicate determinations of this diet’s TFA composition were reported previously.41 Food and water intakes were measured 2 or 3 times weekly (every 2 to 3 d), and body weight was measured weekly throughout the 36-d experimental period. Food and water were measured (500 g and 500 mL, respectively) and placed in the stainless steel holder or water bottle. After 2 or 3 d, each was removed from each cage and measured. Any food on the cage floor was added to the remaining amount (counted as uneaten). Each of the 2 rats were assumed and recorded to have consumed half, but for statistical analysis, each counted as one measurement, such that each treatment group had 3 measurements per time point (n = 3). Caging, enrichment devices, and lighting regimen. Upon arrival, rats were housed in standard translucent ventilated laboratory rat cages (2 rats per cage; 10.5 in. × 19.0 in. × 8.0 in.; wall thickness, 0.1 in.) with identical stainless steel lids (for cradling food and the water bottle; catalog no. 10SS, Ancare, Bellmore, NY) that were covered with matching polysulfone translucent microfilter tops (catalog no. N10MBT, Ancare) for a 1-wk acclimation period. After this time, subjects were assigned at random to 1 of 4 treatment groups that contained either an amber, red (no. K3326, polycarbonate translucent amber, and no. K3325, polycarbonate translucent red; BioServe Flemington, NJ), clear (polycarbonate translucent clear, model no. R20, Pharmacia, Uppsala, Sweden), or opaque (PVC ASTM F891-10, Charlotte Pipe, Charlotte, NC) tunnel (length, 6 in.; inner diameter, 3 in.; wall thickness, 1/8 in.). Rats were housed in pairs so that each animal had ample opportunity to rest inside the tunnel; thus, each treatment group (n = 6 animals) incorporated 3 separate cages. The CED was continuously in the cage throughout the 36-d experiment and was replaced weekly during cage changes with a clean CED of the same tint. The rats were maintained in climate-controlled rooms (21 to 24 °C; 50% to 55% humidity) with diurnal lighting (12:12-h light:dark photoperiod; lights on, at 0600). As described previously,12 animal rooms were lighted with overhead white fluorescent lamps and were completely devoid of light contamination during the dark phase. All cages were rotated daily from left to right on
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the 2 ventilated rack rows to ensure that there were no significant differences in lighting intensity inside the cages (at rodent eye level). Weekly at 0800 during the course of this experiment, the animal room lighting intensity (spectral power distribution) was measured at 1 m above the floor in the right, left, and center of the individually ventilated cage rack, and at the front within the animal cages, as well as facing up inside the enrichment device, by using a radiometer–photometer and radiance detector with filter and diffuser which were calibrated regularly, as previously reported.12 Cage cleaning procedures12 did not cause any variations in light-intensity measurements throughout the course of the study. Data collection timeline. During days 1 through 7, rats were acclimated to the facility. On day 8, animals were housed with their respective CED; behavioral observations were recorded on days 10 and 11 (phase 1) and 24 and 25 (phase II). On days 14, 18, 22, 26, 30, and 34, blood samples were collected at a single circadian time point each day. The experimental period was 36 d in total. Behavioral observations. Instantaneous scan sampling2 with a 15-min intersample interval was used to quantify time spent inside each respective CED during 2 separate phases of the experiment. On experimental days 10 and 11, before any blood draws were started (phase 1), 1 of 3 coinvestigators observed the rats for 120 min; animals were coded as being inside the tunnel when the entire head and shoulders were within it. Every 15 min for 2-h segments, we recorded the animal’s position (inside or outside of CED) for all rats during the 12-h light phase. On day 10, recording occurred from 0800 to 1000, 1200 to 1400, and 1600 to 1800. On day 11, alternate times were recorded (0600 to 800, 1000 to 1200, 1400 to 1600). For phase 1 sampling, rats had their respective CED inside the cage for 3 and 4 d (experimental days 10 and 11, respectively) and had not undergone any other experimental manipulations at this point. Dark-phase measurements were not taken in complete darkness because there is no spectral transmittance of any light through the CED that might affect the animals. Phase 2 occurred on experimental days 24 and 25, when instantaneous scan sampling was repeated. A total of 720 min (12-h light phase) was recorded for each observation phase. Behavioral data were collected no sooner than 2 d after any blood draw. Blood collection. After a 2-wk exposure under the respective CED and lighting conditions, blood was collected from rats through cardiocentesis under gas anesthesia for a total of 6 lowvolume collections (0400, 0800, 1200, 1600, 2000, and 2400; 0.5 to 1.0 mL, less than 5% total blood volume per blood draw); draws were 3 to 5 d apart, as previously reported.12,16 Briefly, a 70% CO2–30% air mixture was passed into an acrylic gas anesthesia chamber.6,19,31 Upon loss of righting reflex, each rat was removed and allowed to breathe room air while blood was collected by using a 3-mL luer-lock syringe (Tyco Healthcare, Mansfield, MA) and 25-gauge, 3/8-in. needle (Tyco) moistened with sodium heparin (5000 USP U/mL, Sagent Pharmaceuticals, Schaumburg, IL). At our facility over the last decade of cardiocentesis in rats, mortality and morbidity have been less than 5%.16 Whole-blood samples were centrifuged at 12,000 × g for 10 min at 4 °C (model Micro17R, accuSpin centrifuge, Fisher Scientific, Fair Lawn, NJ) for plasma collection. Plasma samples were stored at −20 °C until assayed for melatonin, TFA, and corticosterone. Dark-phase sampling was performed in less than 1 min (from removal until recovery) in the home cage under 1 or 2 low-intensity safelight red lamps (120 V, 15 W; 1A model B, catalog no. 1521517;
Kodak, Rochester, NY) to preserve the normal nocturnal melatonin surge. Exposure at rodent eye level was 45.0 to 179.5 µW/ cm2, depending on the number and position of safelights used, but all measurements remained in the low, safe range that does not affect melatonin.39 Melatonin analysis. Plasma melatonin levels were measured by using the melatonin I125 radioimmunoassay kit (Bühlmann, Schönenbuch, Switzerland) and analyzed by using an automated gamma counter (Cobra 5005, Packard, Palo Alto, CA) as previously described.12 Briefly, C18 reverse-phase extraction columns included in the kit were used to extract the melatonin from the samples, by using 0.125 mL of plasma for the 2400 and 0400 time points from 3 animals chosen at random. For the 0800, 1200, 1600, and 2000 time points, 0.125 mL of plasma from each of 2 animals within the same treatment group were pooled to make a 0.25-mL sample, due to the low levels of melatonin at these time points, giving a total n number of 3. The functional sensitivity for the assay was 0.9 pg/mL. The intraassay precision of column extraction and radioimmunoassay combined is 8.2%. Fatty acid extraction and analysis. Plasma free fatty acids were extracted as previously described12 from 0.05-mL samples in duplicate by using an internal standard consisting of heptadecanoic acid (100 µg) dissolved in chloroform. A gas chromatograph fitted with a flame ionization detector, auto injector (both adjusted to 220 °C), and integrator (model 58990A, Hewlett Packard, Palo Alto, CA) was used to analyze the methyl esters of fatty acids according to their retention time compared with known standards. A 0.25 mm × 30 cm capillary column with helium as the carrier gas was used for separations. The minimum detection level for the assay was 0.05 µg/mL. ELISA of corticosterone. Plasma samples were prepared in duplicate for measurement of corticosterone levels by using chemiluminescent ELISA diagnostic kit (catalog no. 55-CORMS-E01, ALPCO, Salem, NH) and measured with a microplate reader (450 nM; VersaMax, Molecular Devices, Sunnyvale, CA) as previously described.12 Detection sensitivity for corticosterone was 4.5 ng/ mL, the lower limit of detection was 15 ng/mL and the coefficient of variation was less than 4.0%. Statistical analysis. All data are compared by using one-way ANOVA followed by the Bonferroni multiple comparison test and 2-tailed paired or unpaired t test by using Prism software (GraphPad, La Jolla, CA) and presented as mean ± 1 SD unless otherwise noted. Figures are double-plotted to facilitate visualization of circadian time. The sample size for TFA analysis is n = 6, except for the following time points due to low sample volume: 0800: amber, n = 5; red n = 4; 1200: amber, clear, and red, n = 5 each; 1600: amber, red, and clear, n = 5 each; 2000: amber n = 5; red and clear, n = 4 each. Statistical significance among the group means was set at a P value of less than 0.05.
Results
Animal room illumination and CED measurements. Mean daytime animal room illumination at the left, right, and center of the rack 1 m above the floor with the detector facing the ceiling had relatively small variance and measured 265.0 ± 43.2 lx (n = 21 [7 wk, 3 positions]). Measurements in the inside front of the cages were averaged for all cage positions and had little variance (57.6 ± 10.5 lx, n = 84 [7 wk, 12 cages]). Laboratory CED used in this experiment are shown in Figure 1. Measurements of radiometric irradiance (lx) from inside the CED positioned in the middle of
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each cage were made daily (amber, 2.4 ± 0.7 lx [n = 15, P = 0.0046]; red, 1.3 ± 0.5 lx [n = 12, P < 0.0001]; opaque, 1.8 ± 0.5 lx [n = 7, P < 0.0001); and clear, 3.9 ± 1.8 lx [n = 14]), and the measurements for amber, red, and opaque CED differed from that for the control clear CED. In addition, irradiance differed (P < 0.0001) between amber and red CED. Sample sizes (n) for statistical analysis differed due to the exclusion of measurements taken when only the running lighting (1/2 task lighting) was illuminated in the room, which was corrected for subsequent measurements. Behavioral observations. Figure 2 depicts the average amount of time that male rats (n = 6 per treatment group; 3 cages) chose to spend inside each CED, recorded after a 10-d exposure (before blood draws) and again after a 24-d exposure, which was 2 or 3 d after a scheduled blood draw. Before beginning scheduled blood sampling (phase 1, days 10 and 11), rats spent significantly more time in the red (mean ± SEM, 290 ± 74 min, P = 0.0059), amber (110 ± 28 min, P = 0.008), and opaque (68 ± 20 min, P = 0.0248) CED compared with the clear CED tunnel (8 ± 5 min). Rats used all tunnels significantly less during the blood-sampling phase (phase 2, days 24 and 25; red: 38 ± 22 min, P = 0.0312; amber: 10 ± 9 min, P = 0.0305; opaque: 8 ± 3 min, P = 0.0367) than before blood collection, except for the clear tunnel (8 ± 5 min), which showed similar use during both phases. Dietary and water intakes and body weight. Daily dietary intake (P < 0.0001) and water intake (P = 0.006) differed significantly between the red and clear CED groups (Figure 3), and dietary intake differed between the opaque and clear CED groups (P = 0.0225; food intake [n = 168; 14 measurements, 3 cages, 4 treatment groups]: clear, 27.0 ± 2.6 g; amber, 27.0 ± 2.5 g; red, 30.6 ± 2.9 g; and opaque, 28.6 ± 3.4 g; water intake [n = 168]: clear, 43.1 ± 3.7 mL; amber, 42.0 ± 4.1 mL; red, 47.3 ± 7.4 mL; and opaque, 43.4 ± 4.7 mL). In addition, body weight change differed significantly between amber (P = 0.0023) and red (P = 0.0022) CED groups compared with the clear control CED group (clear, 269.3 ± 40.8 g; amber, 260.5 ± 40.0 g; red, 280.2 ± 42.6 g; opaque, 274.5 ± 44.0 g; n = 36 [6 measurements, 6 rats per group]) over the 36-d experimental period (Figure 4). Plasma melatonin. Figure 5 depicts diurnal rhythms of plasma melatonin concentrations (mean ± SEM) for each CED treatment group (n = 3 for all groups). Melatonin concentrations were low for all treatment groups at 0800, 1200, and 1600 (23.2 ± 6.8 pg/ mL), as expected for rats during the light phase, although the hormone levels were significantly higher at the 0800 time point for the red CED group (32.5 ± 0.50 pg/mL; P = 0.035) as compared with the clear group (24.9 ± 4.13 pg/mL). All rats in all 4 CED groups demonstrated significant circadian rhythms in plasma melatonin concentrations. At 2000, melatonin levels in amber (26.9 ± 1.8 pg/mL; P = 0.012) and opaque (23.5 ± 1.6 pg/mL; P = 0.035) CED groups were significantly higher than in the clear CED group (15.3 ± 4.2 pg/mL). Peak levels of melatonin occurred at 2400 for all CED groups, but the concentration in the opaque CED group (83.5 ± 33.3 pg/mL; P = 0.20) was significantly lower than that for the clear CED group (247.0 ± 68.0 pg/mL). At 0400, the amber CED group melatonin level (252.0 ± 42.2 pg/mL) was significantly higher than the value for the clear CED group (78.2 ± 57.1 pg/mL). Plasma TFA. Significant differences for plasma TFA concentration between clear and opaque CED groups (P = 0.0231) occurred at 0400 (clear, 5387 ± 77 µg/mL; opaque: 5598 ± 160 µg/mL, n = 6 per treatment group). All plasma TFA values in
Figure 2. Instantaneous scan sampling for the time (mean ± SEM) that rats spent inside either the amber, clear, red, or opaque colored enrichment device (CED) before (phase I) and during (phase II) the experiment. During phase 1, rats spent significantly (*) more time in the red (290 ± 74 min, P = 0.0059), amber (110 ± 28 min, P = 0.008), and opaque (68 ± 20 min, P = 0.0248) than in the clear (8 ± 5 min) CED. During phase 2, rats spent less time in all tunnels (red: 38 ± 22 min, P = 0.0312; amber: 10 ± 9 min, P = 0.0305; opaque: 8 ± 3 min, P = 0.0367) except for the clear CED (8 ± 5 min), for which there was no difference compared with phase 1 data.
Figure 3. Dietary (bottom, g) and water (top, mL) intakes (mean ± 1 SD) for rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). The integrated mean dietary intake differed between the red and clear CED groups (P < 0.0001) and between the opaque and clear CED groups (P = 0.0178), and water intake differed (P = 0.006) between the red and clear CED groups. Food intake (n = 168, 14 measurements, 3 cages, 4 treatment groups): red, 30.5 ± 2.7 g; opaque, 28.6 ± 3.4 g; amber, 27.2 ± 2.8 g; clear, 27.2 ± 2.05 g. Water intake (n = 168): red, 47.3 ± 7.4 mL; amber, 42 ± 4.1 mL; opaque, 43.4 ± 4.7 mL; clear, 43.1 ± 3.7 mL.
male Sprague–Dawley rats with free access to food displayed a significant rhythm in all CED treatment groups (Figure 6), which reached the nadir at 1600 (1041 ± 43 µg/mL) and peaked at 0400 (5598 ± 161 µg/mL). Plasma corticosterone. Plasma corticosterone circadian rhythms are depicted in Figure 7. Values for plasma corticosterone in rats of all CED treatment groups followed diurnal rhythms, which increased continuously from the nadir at 0800 until their peak at 2000. At the 2400 and 0400 time points, the red CED group’s values were significantly lower (P = 0.0121) than those for the clear CED control group (2400: red, 12.1 ± 2.2 ng/mL; clear, 20.6 ± 1.4 ng/mL; 0400: red, 2.2 ± 1.2 ng/mL; clear, 12.3 ± 0.5 ng/mL; P = 0.025), n = 3 per treatment group).
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Figure 4. Integrated body weight change (mean ± SEM, n = 6) over the 36-d experimental period differed (2-tailed t test) between the clear control group and both the amber (P = 0.0023) and red (P = 0.0022) CED groups (red, 280.2 ± 42.6 g; amber, 260.5 ± 40.0 g; opaque, 274.5 ± 44.0 g; clear, 269.3 ± 40.8 g).
Figure 6. Circadian rhythm of plasma total fatty acids (mean ± 1 SD) concentrations for rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). Black bars represent the dark phase; concentrations with asterisks differ (P < 0.05) when using the clear CED as the control. Data are plotted twice to show rhythmicity. Significant differences for plasma total fatty acids between clear and opaque CED groups (P = 0.0231) occurred at 0400 (clear, 5387 ± 77 µg/mL; opaque, 5598 ± 161 µg/mL). All CED treatment groups displayed a nadir at 1600 (1041 ± 43 µg/mL) and a peak at 0400 (5598 ± 161 µg/mL).
Figure 5. Circadian rhythm of plasma melatonin concentration (mean ± SEM) in rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). Black bars represent the dark phase; concentrations with asterisks differ (P < 0.05) compared with the clear CED. Data are plotted twice to show rhythmicity. Melatonin concentrations were low for all treatment groups at 0800, 1200, and 1600 (23.2 ± 6.8 pg/mL) during the light phase as expected, although the hormone levels were significantly higher (P = 0.0348) at the 0800 time point in the red CED group (32.5 ± 0.50 pg/mL) as compared with the clear group (24.9 ± 4.13 pg/mL). At 2000, values for the amber (26.9 ± 1.76 pg/mL, P = 0.0116) and opaque (23.5 ± 1.62 pg/mL, P = 0.0351) CED groups were significantly higher than for the clear (15.3 ± 4.21 pg/mL) CED group. Peak levels of melatonin occurred at 2400 for all CED groups, but the opaque CED group (83.5 ± 33.3 pg/mL) was significantly lower than the clear CED group (247 ± 68 pg/mL, P = 0.0202). At 0400, the amber CED group melatonin level (252.0 ± 42.2 pg/mL) was significantly higher than the clear CED group (78.2 ± 57.1 pg/mL, P = 0.0163).
Figure 7. Circadian rhythm of plasma corticosterone (mean ± SEM) concentrations for rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). Black bars represent the dark phase; concentrations with asterisks differ (P < 0.05) when using the clear CED as the control. Data are plotted twice to show rhythmicity. All CED groups nadir at 0800, and rise continuously until their peak at 2000 and were not significantly different. However, at 2400 and 0400 time points, the red CED group’s values were significantly lower (P = 0.0121) when compared with those for the clear CED (2400: red, 12.1 ± 2.19 ng/mL; clear, 20.6 ± 1.38 ng/mL; 0400: red, 2.17 ± 1.15 ng/mL; clear, 12.3 ± 0.493 ng/mL; P = 0.025).
Discussion
Animal health and wellbeing are affected by the environment, housing, and management of the species, and these components should account for their physical, physiologic, and behavioral needs.27 Recent methods have been suggested to standardize rodent care, so that research models are better defined and more stable, thus requiring fewer animal subjects 40 and increasing
scientific validity.27 EE is an independent variable27 and refers to the provision of a more naturalistic environment and to the addition of objects or resources to the environment for increasing sensory and motor stimulation, the expression of species-typical behaviors, and the repression of stereotypies.27,37 The success of EE can be measured by using parameters of normal health, including growth, development, and reproduction.27,40 For rodents, EE can include bedding depth and type, which can promote species-typical foraging, burrowing, and nest-building behaviors in addition to opportunities for thermoregulation, as well as nestbuilding materials, novel food stuffs, chew toys, tunnels, igloos or houses, social housing, and cage modifications.4,27 However,
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little is known about the effects of these EE items on scientific outcomes.4 We chose to evaluate the commonly used tinted rat enrichment tunnels in this study. We used 4 types of CED: the 2 colors (amber and red) commonly available from manufacturers of rat enrichment tubes; opaque tubes, which were included because CED produced inhouse are often constructed of PVC; and clear tunnels, which eliminated the mere presence of an EE device as an independent variable; several studies have found that the presence of EE, when compared with isolation or impoverished conditions, has effects on cognition, development, and performance.18,37,38,40 In circadian light studies, 3 major variables include duration and timing of exposure, intensity, and tint (or wavelength). Lighting intensities inside each cage were measured at all cage locations and had little variance. However, radiometric irradiance measured inside each CED was significantly different between clear and colored (red, amber, and opaque) tunnels as well as between amber and red CED. Adherence to a strict 12:12-h light:dark environmental lighting cycle throughout the study, with complete darkness at night, ensured that any observed effects result only from variations in color tint. We found significant differences in daily food and water intake between animals with red compared with clear CED and in dietary intake between groups in opaque and clear CED. Food intake for rats with red CED was higher, and correspondingly, average body weight changes were significantly greater in the red CED group when compared with the clear CED group over the 36-d experimental period. The amber CED group had significantly lower average body weight changes. Other investigators reported significant increases in food and water consumption for rats under isolation or impoverished conditions compared with cages with EE and standard social conditions (4 rats per cage) but found significantly heavier body weights only after the first week of differential housing and no significant differences in body weight between EE and standard-condition treatment groups.38 The current study used social housing of rat pairs with EE, which resulted in the red CED group consuming more food and water with a corresponding increase in body weight. More studies should be conducted to reveal the mechanisms underlying this finding—perhaps by adding activity monitoring, recording food and water intake at shorter intervals, and measuring other neurohormones, such as plasma leptin and ghrelin, blood glucose, and insulin concentrations—to elucidate any possible central circadian disruption as a cause. Monitoring locomotor activity would be helpful in determining whether the significant decreases in use of the amber, red, and opaque CED are a result of these rats acclimating to the novel environment. In a behavioral context, locomotor activity is a reliable and consistent index of learning or information processing, and persistently high levels suggest a lack of information processing or the absence of acclimation to the novel environment.18 Results from the current study suggest that rats spend more time inside red CED as compared with all other CED tested. Because intensities were held constant, our results indicate that the tint of the CED causes this effect. However, the total time spent in each CED was markedly lower approximately 2 wk after presentation (phase 2) compared with 2 d after introduction (phase 1). There was no feasible way of controlling for this animal-dependent variable. However, this reduction in use and associated brief exposure times might have mitigated significant effects in some cases.
Several difficulties have been encountered in implementing EE for laboratory rodents. First, EE can have negative effects, including the induction of aggressive behaviors in male mice and rats,1 changes in fecundity or breeding pattern, and litter size;40 allergies and skin rashes caused by bedding type; and the ingestion of bedding or EE material.27,40 Second, difficulties have been encountered with the interpretation of behavioral and chemical effects as being positive, negative, or neutral in terms of their influence on health and wellbeing.40 For this reason, we believe that including physiologic and metabolic measures in behavioral studies is imperative so that all parameters of health and wellbeing can be taken into account. In addition, more studies are needed to determine whether any of these changes are biologically or clinically significant.40 Last, the literature demonstrates a lack of detail concerning the reporting all aspects of the environment and EE.40 Reviews show the great diversity in control housing conditions, the amount of space provided in cages, social group number, rat strain and sex, age at onset of EE,26 duration of exposure to EE, types of physical objects used for EE, and behavioral tests used.37,40 Social companionship has been found to act as a form of EE.4 Social enrichment has a greater effect when compared with EE or isolation or impoverished conditions.18 More importantly, these affects are lasting,18 unlike the acclimation seen with physical enrichment. Reports have demonstrated that toys used for EE have a very short (1 d) period of interest, whereas toys related to food retain their attraction longer, likely due to the primary reinforcement of food.4,42 Given that rats in all groups spent less time in the CED, regardless of color, during phase II compared with phase I, acclimation to EE could have occurred. In addition, animals’ social needs should be considered,27 given that studies report adverse behaviors in isolation or impoverished conditions.1,37 Single housing for social animals requires justification and review by the IACUC and veterinarian, and time alone should be minimized to the shortest duration possible.27 Providing extra EE for animals housed in isolation, in small spaces or providing visual, auditory, olfactory, or tactile contact with conspecifics have been recommended.27 EE can help animals escape aggression and avoid social conflicts.27 However, when reports yield evidence of objectinduced changes in normal physiology, alternatives should be sought. Melatonin is a neurohormone released primarily at night in response to rhythms generated in the SCN and is the body’s biologic clock.36 As expected, melatonin concentrations were low for all CED groups during the light phase, although the hormone levels were statistically (but perhaps not clinically) significantly higher at the 0800 time point in the red CED group. None of the CED affected the normal circadian rhythm of melatonin, as evidenced by the coinciding peaks and troughs. At 2000, melatonin concentrations of the amber and opaque CED groups rose more rapidly than did that of the clear CED group. Interestingly, the opaque CED group’s melatonin concentration did not peak as high as those of the other groups. These results suggest that the low level of exposure was sufficiently significant to affect the circadian rhythm. Alternatively, the light–dark cycle might be altered in these rats, due to the presence of the CED, perhaps providing a darker area in the cage, which would affect overall sensory perception of light. Finally, the instantaneous scan-sampling method used for behavioral observations may not accurately reflect the true behavior of the rats and might otherwise be measured by
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videorecording the activity of single-housed subjects. Further studies should address these possibilities. Peak plasma TFA levels (0400) differed between the opaque and clear CED groups, who had free access to food and water. The TFA rhythm, timing, and intensity were similar to previously reported results from male Sprague–Dawley rats,13,41 confirming that these circadian rhythms remain entrained6 and are independent from the SCN-generated rhythms. However, because significant difference was found at one time point, additional investigation is needed to correlate each blood draw result with food and water intakes and plasma TFA. Corticosterone is a widely used parameter for the measurement of stress in rats and displays true circadian rhythmicity that is regulated directly by the SCN.19,35 The corticosterone concentration peaks around the onset of darkness, and the nadir occurs after the onset of light, with gradual increases throughout the light phase.19,35 Even though significant differences were found between the red CED group compared with the clear CED group, all nighttime values of corticosterone were less than 51 ng/mL, which is comparable to results from other studies3,41 and much lower than those obtained in posttraumatic stress models29 (700 ng/mL) using this stock. An animal’s enclosure and its components should withstand cleaning procedures and not interfere with the animals’ health or research use.27 The opaque tube, which is made from PVC in the exact measurements of the other manufactured tubes, poses no concern regarding leaching because this material does not contain any plasticizers,10 such as the commonly used di(2-ethylhexyl) phthalate, that reportedly alter hormones.9 In addition, tunnels were replaced weekly, thus eliminating stress due to changing objects too frequently; rotation and replacement of EE items are recommended to maintain their novelty.27 Behavioral observations were performed at least 2 d after blood sampling to account for the potential confound of side effects of blood collection on behavioral measures. Limitations of the current study include the rat-dependent variable of the time spent inside each CED, which thus altered the ‘dose’ of colored light that each animal received. In fact, this limitation is present anytime a CED or other EE is added to a cage as standard practice. We gave the rats the opportunity to use their respective CED how they saw fit, and replicated how, in practice, we provide EE. Remember that the goal of EE is to provide animals the opportunity for species-specific behaviors that fulfill their innate behavioral and physiologic needs. Therefore, all studies in which the facility uses CED—whether they are colored tunnels, huts, or other objects—are potentially confounded due to the variable exposures to each individual animal. Changes in experimental design would give us a method to calculate individual color ‘doses,’ and comparisons can be made to the experimental circadian time points. Because of this limitation, possible effects may not be recognized for some groups, due to brief exposure times; in addition, we do not know the exact exposure time that resulted in the physiologic changes that were significant. Experimental design changes, including single housing, precise measurement of individual food and water intake and recording time of intake, and activity monitoring by videorecording, might help to yield this information in subsequent studies. These experimental design changes would give us a better indication of the effects of a novel object (shortly after object introduced) compared with acclimation to the object (here, after 2 wk).
An animal’s coping to environmental changes is a complex behavioral reaction revealed by their responses to changes in novelty (well-known territory) and can have 2 differently motivated reactions. That is, animals respond to changes in novelty by displaying fear-associated defensive reactions, such as freezing and escape, or by mounting exploratory associated reactions, such as curiosity (neotic preference) and active exploration. Typically, fear-evoking objects are avoided at first, and this behavior may reflect a need to seek immediate safety. If the fear level is low or when fear subsides with time or distance, subsequent active exploration of the object and information-gathering allows animals to adapt to the environmental change (novelty). When exploration brings no signs of danger and after information is consolidated into memory, animals habituate to the changed environment, resulting in reduction or removal of fear and curiosity.34 This typical behavioral process is hard to predict and varies between species, sexes, housing conditions, and even EE objects.4,37,38 Other limitations in terms of our modified experimental design likely would be recovery time and blood sample volumes and thus would require larger groups to collect all of the circadian time points for comparison. Other variables include strain, sex, and species, as discussed earlier. Our next steps will be to add individual behavioral measurements by using activity monitors, which would require individual housing, and to include time-recorded food and water measurements to elucidate individual dosages for each CED type. In studies now underway, we are evaluating the effects of other commonly used CED, such as the popular plastic hut for mice,26 which is manufactured in amber, red, and blue plastics and as disposable cardboard (opaque) items, to evaluate whether differences due to the color of these enrichment devices emerge. The effects of colored EE tunnels we used—and likely other CED such as huts—cannot be completely controlled, because we can only provide the animal the opportunity to use the device and cannot hold constant the dose of each CED received, given that this variable is behavior-dependent. Regardless of this limitation, circadian rhythms of metabolism and physiology were significantly disrupted depending on the CED in the cage. Therefore, CED used for enrichment in laboratory rat cages might significantly confound scientific outcomes, and their use should be thoroughly reported or pilot-studied. Further testing of these previously assumed harmless EE objects is warranted.
Acknowledgments
We thank Michael Webb, Patricia Beavers, and Katie Castillo for their outstanding care of the animals and Erin Dauchy for her technical training and statistical expertise. This work was supported by NIH grants 1R25RR032028 (MAWD and RPB) and 1R56CA193518-01 (DEB and SMH).
References
1. Abou-Ismail UA, Mahboub HD. 2011. The effects of enriching laboratory cages using various physical structures on multiple measures of welfare in singly housed rats. Lab Anim 45:145–153. 2. Altmann J. 1974. Observational study of behavior: sampling methods. Behaviour 49:227–267. 3. Arnold M, Langhans W. 2010. Effects of anesthesia and blood sampling techniques on plasma metabolites and corticosterone in the rat. Physiol Behav 99:592–598. 4. Baumans V. 2005. Environmental enrichment for laboratory rodents and rabbits: requirements of rodents, rabbits, and research. ILAR J 46:162–170.
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5. Berson DM, Dunn FA, Takao M. 2002. Phototransduction by retinal ganglion cells that set the circadian clock. Science 295:1070–1073. 6. Blask DE, Brainard GC, Dauchy RT, Hanifin JP, Davidson LK, Krause JA, Sauer LA, Rivera-Bermudez MA, Dubocovich ML, Jasser SA, Lynch DT, Rollag MD, Zalatan F. 2005. Melatonindepleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats. Cancer Res 65:11174–11184. 7. Brainard GC, Hanifin JP. 2005. Photons, clocks, and consciousness. J Biol Rhythms 20:314–325. 8. Brainard GC, Hanifin JP, Rollag MD, Greeson J, Byrne B, Glickman G, Gerner E, Sanford B. 2001. Human melatonin regulation is not mediated by the 3-cone photopic visual system. J Clin Endocrinol Metab 86:433–436. 9. Carbone S, Ponzo OJ, Gobetto N, Samaniego YA, Reynoso R, Scacchi P, Moguilevsky JA, Cutrera R. 2013. Antiandrogenic effect of perinatal exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate increases anxiety-like behavior in male rats during sexual maturation. Horm Behav 63:692–699. 10. Charlotte Pipe and Foundry Company. 2014. Safety Data Sheet: PVC pipe and fittings. Monroe (NC): Charlotte Pipe and Foundry Company. 11. Dacey DM, Liao HW, Peterson BB, Robinson FR, Smith VC, Pokorny J, Yau KW, Gamlin PD. 2005. Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature 433:749–754. 12. Dauchy RT, Dauchy EM, Hanifin JP, Gauthreaux SL, Mao L, Belancio VP, Ooms TG, Dupepe LM, Jablonski MR, Warfield B, Wren MA, Brainard GC, Hill SM, Blask DE. 2013. Effects of spectral transmittance through standard laboratory cages on circadian metabolism and physiology in nude rats. J Am Assoc Lab Anim Sci 52:146–156. 13. Dauchy RT, Dauchy EM, Tirrell RP, Hill CR, Davidson LK, Greene MW, Tirrell PC, Wu J, Sauer LA, Blask DE. 2010. Dark-phase light contamination disrupts circadian rhythms in plasma measures of endocrine physiology and metabolism in rats. Comp Med 60:348–356. 14. Dauchy RT, Hoffman A, Wren-Dail MA, Hanifin JP, Warfield B, Brainard GC, Xiang S, Yuan L, Hill SM, Belancio VP, Dauchy EM, Smith K, Blask DE. 2015. Daytime blue light enhances nighttime circadian melatonin inhibition of human prostate cancer growth. Comp Med 65:473–485. 15. Dauchy RT, Wren MA, Dauchy EM, Hanifin JP, Jablonski MR, Warfield B, Brainard GC, Hill SM, Mao L, Dupepe LM, Ooms TG, Blask DE. 2013. Effect of spectral transmittance through red-tinted rodent cages on circadian metabolism and physiology in nude rats. J Am Assoc Lab Anim Sci 52:745–755. 16. Dauchy RT, Wren MA, Dauchy EM, Hoffman AE, Hanifin JP, Warfield B, Jablonski MR, Brainard GC, Hill SM, Mao L, Dobek GL, Dupepe LM, Blask DE. 2015. The influence of red light exposure at night on circadian metabolism and physiology in Sprague–Dawley rats. J Am Assoc Lab Anim Sci 54:40–50. 17. Ecker JL, Dumitrescu ON, Wong KY, Alam NM, Chen SK, LeGates T, Renna JM, Prusky GT, Berson DM, Hattar S. 2010. Melanopsinexpressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision. Neuron 67:49–60. 18. Elliott BM, Grunberg NE. 2005. Effects of social and physical enrichment on open-field activity differ in male and female Sprague–Dawley rats. Behav Brain Res 165:187–196. 19. Forslid A, Ingvar M, Rosen I, Ingvar DH. 1986. Carbon dioxide narcosis: influence of short-term high concentration carbon dioxide inhalation on EEG and cortical evoked responses in the rat. Acta Physiol Scand 127:281–287. 20. Freedman MS, Lucas RJ, Soni B, von Schantz M, Munoz M, DavidGray Z, Foster R. 1999. Regulation of mammalian circadian behavior by nonrod, noncone, ocular photoreceptors. Science 284:502–504. 21. Gamlin PD, McDougal DH, Pokorny J, Smith VC, Yau KW, Dacey DM. 2007. Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells. Vision Res 47:946–954. 22. Hanifin JP, Brainard GC. 2007. Photoreception for circadian, neuroendocrine, and neurobehavioral regulation. J Physiol Anthropol 26:87–94.
23. Hanifin JP, Stewart KT, Smith P, Tanner R, Rollag M, Brainard GC. 2009. High-intensity red light suppresses melatonin. Chronobiol Int 23:251–268. 24. Hattar S, Liao HW, Takao M, Berson DM, Yau KW. 2002. Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295:1065–1070. 2 5. Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, Hankins MW, Lem J, Biel M, Hofmann F, Foster RG, Yau KW. 2003. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424:76–81. 26. Hutchinson EK, Avery AC, VandeWoude S. 2012. Environmental enrichment during rearing alters corticosterone levels, thymocyte numbers, and aggression in female BALB/c mice. J Am Assoc Lab Anim Sci 51:18–24. 27. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 28. Jacobs GH, Fenwick JA, Williams GA. 2001. Cone-based vision of rats for ultraviolet and visible lights. J Exp Biol 204:2439–2446. 29. Jia M, Meng F, Smerin SE, Xing G, Zhang L, Su DM, Benedek D, Ursano R, Su YA, Li H. 2012. Biomarkers in an animal model for revealing neural, hematologic, and behavioral correlates of PTSD. J Vis Exp. 68:e3361. 30. Kennaway DJ, Voultsios A, Varcoe TJ, Moyer RW. 2002. Melatonin in mice: rhythms, response to light, adrenergic stimulation, and metabolism. Am J Physiol Regul Integr Comp Physiol 282:R358– R365. 31. Kohler I, Meier R, Busato A, Neiger-Aeschbacher G, Schatzmann U. 1999. Is carbon dioxide (CO2) a useful short-acting anaesthetic for small laboratory animals? Lab Anim 33:155–161. 32. Lucas RJ, Peirson SN, Berson DM, Brown TM, Cooper HM, Czeisler CA, Figueiro MG, Gamlin PD, Lockley SW, O’Hagan JB, Price LL, Provencio I, Skene DJ, Brainard GC. 2014. Measuring and using light in the melanopsin age. Trends Neurosci 37:1–9. 33. Osteen WK, Sundberg DK. 1980. Prolactin binding in eyes of rats with retinal dystrophy and normal retinas - an autoradiographic study. Anat Rec 196:A141–A142. 34. Pisula W, Turlejski K, Stryjek R, Nalecz-Tolak A, Grabiec M, Djavadian RL. 2012. Response to novelty in the laboratory Wistar rat, wild-captive WWCPS rat, and the gray short-tailed opossum (Monodelphis domestica). Behav Processes 91:145–151. 35. Rahman SA, Kollara A, Brown TJ, Casper RF. 2008. Selectively filtering short wavelengths attenuates the disruptive effects of nocturnal light on endocrine and molecular circadian phase markers in rats. Endocrinology 149:6125–6135. 36. Reiter RJ, Robinson J. 1995. Melatonin: your body’s natural wonder drug. New York (NY): Bantam Books. 37. Simpson J, Kelly JP. 2011. The impact of environmental enrichment in laboratory rats—behavioural and neurochemical aspects. Behav Brain Res 222:246–264. 38. Simpson J, Kelly JP. 2012. The effects of isolated and enriched housing conditions on baseline and drug-induced behavioural responses in the male rat. Behav Brain Res 234:175–183. 39. Sun JH, Yaga K, Reiter RJ, Garza M, Manchester LC, Tan DX, Poeggeler B. 1993. Reduction in pineal N-acetyltransferase activity and pineal and serum melatonin levels in rats after their exposure to red light at night. Neurosci Lett 149:56–58. 40. Toth LA, Kregel K, Leon L, Musch TI. 2011. Environmental enrichment of laboratory rodents: the answer depends on the question. Comp Med 61:314–321. 41. Wren MA, Dauchy RT, Hanifin JP, Jablonski MR, Warfield B, Brainard GC, Blask DE, Hill SM, Ooms TG, Bohm RP Jr. 2014. Effect of different spectral transmittances through tinted animal cages on circadian metabolism and physiology in Sprague–Dawley rats. J Am Assoc Lab Anim Sci 53:44–51. 42. Young RJ, editor. 2003. Environmental enrichment for captive animals. Oxford (UK): Blackwell Science.
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Original Research
Effects of Colored Enrichment Devices on Circadian Metabolism and Physiology in Male Sprague–Dawley Rats Melissa A Wren-Dail,1,2,* Robert T Dauchy,2 Tara G Ooms,3 Kate C Baker,4 David E Blask,2 Steven M Hill,2 Lynell M Dupepe,1 and Rudolf P Bohm, Jr4 Environmental enrichment (EE) gives laboratory animals opportunities to engage in species-specific behaviors. However, the effects of EE devices on normal physiology and scientific outcomes must be evaluated. We hypothesized that the spectral transmittance (color) of light to which rats are exposed when inside colored enrichment devices (CED) affects the circadian rhythms of various plasma markers. Pair-housed male Crl:SD rats were maintained in ventilated racks under a 12:12-h light:dark environment (265.0 lx; lights on, 0600); room lighting intensity and schedule remained constant throughout the study. Treatment groups of 6 subjects were exposed for 25 d to a colored enrichment tunnel: amber, red, clear, or opaque. We measured the proportion of time rats spent inside their CED. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and analyzed for plasma melatonin, total fatty acids, and corticosterone. Rats spent more time in amber, red, and opaque CED than in clear tunnels. All tubes were used significantly less after blood draws had started, except for the clear tunnel, which showed no change in use from before blood sampling began. Normal peak nighttime melatonin concentrations showed significant disruption in the opaque CED group. Food and water intakes and body weight change in rats with red-tinted CED and total fatty acid concentrations in the opaque CED group differed from those in other groups. These results demonstrate that the color of CED altered normal circadian rhythms of plasma measures of metabolism and physiology in rats and therefore might influence the outcomes of scientific investigations. Abbreviations: CED, colored enrichment devices; EE, environmental enrichment; SCN, suprachiasmatic nucleus; TFA, total fatty acids
The visual system of mammals encompasses the anatomic structures of the eye and the primary optic tract. Because rats are nocturnal animals, their retinas are rod-dominated but also contain cones. This visual system contains 2 main classes of cones, which contain photopigment with spectral sensitivity in either the 359-nm (UV) or 509-nm (M cone) wavelengths. Rudimentary color discrimination is believed to be supported by these 2 cone classes, but this discrimination may be only dichromatic in nature.27 This dichromatic nature is a characteristic of the visual system and is reflected in rats’ preference for a dark environment: darkness likely is preferred due in part to the effects of light on retinal degeneration over time, which perhaps is painful, causing rats to shy away from light.33 Changes in lighting intensity, duration, and wavelength (visually perceived as color or tint) at various times of the day can disrupt circadian rhythms of metabolism and endocrine hormone concentrations.7,12,13,15,41 These responses are primarily elicited by means of a nonvisual system, also located in the retina, through Received: 11 Nov 2015. Revision requested: 05 Jan 2016. Accepted: 12 May 2016. 1 Departments of Comparative Medicine and 2Structural & Cellular Biology, Tulane University School of Medicine, New Orleans, Louisiana, 3Section of Laboratory Animal Medicine, IIT Research Institute, Chicago, Illinois and 4Division of Veterinary Medicine, Tulane National Primate Research Center, Covington, Louisiana *Corresponding author. Email: [email protected]
intrinsically photosensitive retinal ganglion cells5,8,11,17,21,24,25,32 and not through the visual primary optic tract. Circadian rhythms are entrained by the nonvisual light–dark cycle through signals from the retinohypothalamic tract to the suprachiasmatic nucleus (SCN). The SCN regulates circadian rhythms and is termed the ‘master biologic clock.’5,7 Wavelengths between 450 and 484 nm (that is, blue light) are the strongest inciters of neuroendocrine and circadian responses in mammals, however high-intensity, longer-wavelength (that is, red) light can have this effect also.7,14,15,23,35,41 There is now overwhelming evidence of the influence of the retinohypothalamic tract system on the regulation of behavior20 and metabolism.7,22,30,41 Prior research12-16,41 from our laboratory revealed significant disruptions of various circadian rhythms and increased tumor growth from light leaks around doorways at night. In addition, we investigated the effect of spectral transmittance (cage tint) through standard laboratory caging in female pigmented athymic nude rats and male Sprague–Dawley rats12,41 and showed that these animals developed chronobiologic disruptions in various plasma measures of physiology and metabolism. Although the circadian rhythm of total fatty acids (TFA) in plasma remained unchanged, all other metabolic and physiologic rhythms were significantly altered when exposed to amber-, red-, or blue-tinted caging as compared with clear caging. We also have shown that
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Sprague–Dawley rats have significant circadian disruptions in melatonin, corticosterone, leptin, insulin, and glucose concentrations when exposed to various color caging during the day.12,41 These disruptions in plasma levels included duration, phasing (timing), amplitude, or some combination of these elements. On inspection of items currently placed into the animal cage for environmental enrichment (EE), we noticed that many of these items are color-tinted. Most are made from transparent durable plastic for ease of cleaning and sanitation. The premise for using these color-tinted items is that they allow for monitoring and health checking yet afford rodents the opportunity to retreat from stressful stimuli or cage mates and to regulate body temperature.27 However, some objects added to the animals’ environment are known to induce stress.27 Although some studies1,4,18,26,37,38,40 have explored the effect of EE on behavior, few studies4,40 have examined the influence of EE, particularly colored enrichment devices (CED), on plasma measures of circadian metabolism and physiology in laboratory animals. Therefore, we sought to determine whether various CED affected animal measures of metabolism or neuroendocrine hormones. For the current study, we explored the effect of spectral transmittance exposure from commonly used CED (amber, clear, red, and opaque; Figure 1) during the light period in male albino Sprague–Dawley rats. We chose this rat stock because it is among the most widely used in laboratory research, comprising approximately 53% of EE studies.37 Melatonin, TFA, and corticosterone were chosen to examine common circadian rhythms through plasma measures of metabolism and physiology. We hypothesized that changing the tinting (spectral transmittance or quality) of EE by using various CED affects circadian rhythms of these measures in male Sprague–Dawley rats.
Materials and Methods
Reagents. HPLC-grade chloroform, ethyl ether, methanol, glacial acetic acid, heptane, and hexane were purchased from Fisher Scientific (Pittsburg, PA). Free fatty acids, rapeseed oil methyl ester standards, boron–trifluoride–methanol, potassium chloride, sodium chloride, and perchloric and trichloroacetic acids were purchased from Sigma Scientific (St Louis, MO). Ultrapure water (catalog no. 400000) was purchased from Cayman Chemical (Ann Arbor, MI). Animals, housing condition, and diet. Male Sprague–Dawley rats (age, 4 to 5 wk) were purchased from Charles River (Crl:CD[SD]; Kingston, NY) and certified by the vendor to be free of all known rodent bacterial, viral, and parasitic pathogens. According to the vendor, rats at this age were provided with only group social enrichment during rearing. Animals were maintained in Tulane University School of Medicine Vivarium, which has been an AAALAC-accredited facility since 1962. All animal use and accompanying procedures were in accordance with The Guide for the Care and Use of Laboratory Animals27 and were IACUCapproved. Rats were maintained in ventilated cages using hardwood maple bedding (no. 7090, Sanichips, Harlan Teklad, Madison, WI; 1 bedding change weekly). Serum samples from sentinel animals on combined soiled bedding from project animals were tested quarterly (Multiplex Fluorescent Immunoassay 2, IDEXX Research Animal Diagnostic Laboratory, Colombia, MO) for rat coronavirus, Sendai virus, pneumonia virus of mice, sialodacryoadenitis virus, Kilham rat virus, Toolan H1 virus, reovirus type 3,
Figure 1. Colored enrichment devices used in this experiment were (from left to right) translucent amber, clear, red (polycarbonate) and opaque (polyvinyl chloride) and had identical internal diameters and lengths.
Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus types 1 and 2, Hantaan virus, Encephalitozoon cuniculi, cilia-associated respiratory bacillus, parvovirus NS1, rat parvoviruses, rat murine virus, and rat theilovirus as well as external and internal parasites; all test results were negative. Rats had free access to a commercial diet (no. 5053 Irradiated Laboratory Rodent Diet, Purina, Richmond, IN) and acidified water. Quadruplicate determinations of this diet’s TFA composition were reported previously.41 Food and water intakes were measured 2 or 3 times weekly (every 2 to 3 d), and body weight was measured weekly throughout the 36-d experimental period. Food and water were measured (500 g and 500 mL, respectively) and placed in the stainless steel holder or water bottle. After 2 or 3 d, each was removed from each cage and measured. Any food on the cage floor was added to the remaining amount (counted as uneaten). Each of the 2 rats were assumed and recorded to have consumed half, but for statistical analysis, each counted as one measurement, such that each treatment group had 3 measurements per time point (n = 3). Caging, enrichment devices, and lighting regimen. Upon arrival, rats were housed in standard translucent ventilated laboratory rat cages (2 rats per cage; 10.5 in. × 19.0 in. × 8.0 in.; wall thickness, 0.1 in.) with identical stainless steel lids (for cradling food and the water bottle; catalog no. 10SS, Ancare, Bellmore, NY) that were covered with matching polysulfone translucent microfilter tops (catalog no. N10MBT, Ancare) for a 1-wk acclimation period. After this time, subjects were assigned at random to 1 of 4 treatment groups that contained either an amber, red (no. K3326, polycarbonate translucent amber, and no. K3325, polycarbonate translucent red; BioServe Flemington, NJ), clear (polycarbonate translucent clear, model no. R20, Pharmacia, Uppsala, Sweden), or opaque (PVC ASTM F891-10, Charlotte Pipe, Charlotte, NC) tunnel (length, 6 in.; inner diameter, 3 in.; wall thickness, 1/8 in.). Rats were housed in pairs so that each animal had ample opportunity to rest inside the tunnel; thus, each treatment group (n = 6 animals) incorporated 3 separate cages. The CED was continuously in the cage throughout the 36-d experiment and was replaced weekly during cage changes with a clean CED of the same tint. The rats were maintained in climate-controlled rooms (21 to 24 °C; 50% to 55% humidity) with diurnal lighting (12:12-h light:dark photoperiod; lights on, at 0600). As described previously,12 animal rooms were lighted with overhead white fluorescent lamps and were completely devoid of light contamination during the dark phase. All cages were rotated daily from left to right on
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the 2 ventilated rack rows to ensure that there were no significant differences in lighting intensity inside the cages (at rodent eye level). Weekly at 0800 during the course of this experiment, the animal room lighting intensity (spectral power distribution) was measured at 1 m above the floor in the right, left, and center of the individually ventilated cage rack, and at the front within the animal cages, as well as facing up inside the enrichment device, by using a radiometer–photometer and radiance detector with filter and diffuser which were calibrated regularly, as previously reported.12 Cage cleaning procedures12 did not cause any variations in light-intensity measurements throughout the course of the study. Data collection timeline. During days 1 through 7, rats were acclimated to the facility. On day 8, animals were housed with their respective CED; behavioral observations were recorded on days 10 and 11 (phase 1) and 24 and 25 (phase II). On days 14, 18, 22, 26, 30, and 34, blood samples were collected at a single circadian time point each day. The experimental period was 36 d in total. Behavioral observations. Instantaneous scan sampling2 with a 15-min intersample interval was used to quantify time spent inside each respective CED during 2 separate phases of the experiment. On experimental days 10 and 11, before any blood draws were started (phase 1), 1 of 3 coinvestigators observed the rats for 120 min; animals were coded as being inside the tunnel when the entire head and shoulders were within it. Every 15 min for 2-h segments, we recorded the animal’s position (inside or outside of CED) for all rats during the 12-h light phase. On day 10, recording occurred from 0800 to 1000, 1200 to 1400, and 1600 to 1800. On day 11, alternate times were recorded (0600 to 800, 1000 to 1200, 1400 to 1600). For phase 1 sampling, rats had their respective CED inside the cage for 3 and 4 d (experimental days 10 and 11, respectively) and had not undergone any other experimental manipulations at this point. Dark-phase measurements were not taken in complete darkness because there is no spectral transmittance of any light through the CED that might affect the animals. Phase 2 occurred on experimental days 24 and 25, when instantaneous scan sampling was repeated. A total of 720 min (12-h light phase) was recorded for each observation phase. Behavioral data were collected no sooner than 2 d after any blood draw. Blood collection. After a 2-wk exposure under the respective CED and lighting conditions, blood was collected from rats through cardiocentesis under gas anesthesia for a total of 6 lowvolume collections (0400, 0800, 1200, 1600, 2000, and 2400; 0.5 to 1.0 mL, less than 5% total blood volume per blood draw); draws were 3 to 5 d apart, as previously reported.12,16 Briefly, a 70% CO2–30% air mixture was passed into an acrylic gas anesthesia chamber.6,19,31 Upon loss of righting reflex, each rat was removed and allowed to breathe room air while blood was collected by using a 3-mL luer-lock syringe (Tyco Healthcare, Mansfield, MA) and 25-gauge, 3/8-in. needle (Tyco) moistened with sodium heparin (5000 USP U/mL, Sagent Pharmaceuticals, Schaumburg, IL). At our facility over the last decade of cardiocentesis in rats, mortality and morbidity have been less than 5%.16 Whole-blood samples were centrifuged at 12,000 × g for 10 min at 4 °C (model Micro17R, accuSpin centrifuge, Fisher Scientific, Fair Lawn, NJ) for plasma collection. Plasma samples were stored at −20 °C until assayed for melatonin, TFA, and corticosterone. Dark-phase sampling was performed in less than 1 min (from removal until recovery) in the home cage under 1 or 2 low-intensity safelight red lamps (120 V, 15 W; 1A model B, catalog no. 1521517;
Kodak, Rochester, NY) to preserve the normal nocturnal melatonin surge. Exposure at rodent eye level was 45.0 to 179.5 µW/ cm2, depending on the number and position of safelights used, but all measurements remained in the low, safe range that does not affect melatonin.39 Melatonin analysis. Plasma melatonin levels were measured by using the melatonin I125 radioimmunoassay kit (Bühlmann, Schönenbuch, Switzerland) and analyzed by using an automated gamma counter (Cobra 5005, Packard, Palo Alto, CA) as previously described.12 Briefly, C18 reverse-phase extraction columns included in the kit were used to extract the melatonin from the samples, by using 0.125 mL of plasma for the 2400 and 0400 time points from 3 animals chosen at random. For the 0800, 1200, 1600, and 2000 time points, 0.125 mL of plasma from each of 2 animals within the same treatment group were pooled to make a 0.25-mL sample, due to the low levels of melatonin at these time points, giving a total n number of 3. The functional sensitivity for the assay was 0.9 pg/mL. The intraassay precision of column extraction and radioimmunoassay combined is 8.2%. Fatty acid extraction and analysis. Plasma free fatty acids were extracted as previously described12 from 0.05-mL samples in duplicate by using an internal standard consisting of heptadecanoic acid (100 µg) dissolved in chloroform. A gas chromatograph fitted with a flame ionization detector, auto injector (both adjusted to 220 °C), and integrator (model 58990A, Hewlett Packard, Palo Alto, CA) was used to analyze the methyl esters of fatty acids according to their retention time compared with known standards. A 0.25 mm × 30 cm capillary column with helium as the carrier gas was used for separations. The minimum detection level for the assay was 0.05 µg/mL. ELISA of corticosterone. Plasma samples were prepared in duplicate for measurement of corticosterone levels by using chemiluminescent ELISA diagnostic kit (catalog no. 55-CORMS-E01, ALPCO, Salem, NH) and measured with a microplate reader (450 nM; VersaMax, Molecular Devices, Sunnyvale, CA) as previously described.12 Detection sensitivity for corticosterone was 4.5 ng/ mL, the lower limit of detection was 15 ng/mL and the coefficient of variation was less than 4.0%. Statistical analysis. All data are compared by using one-way ANOVA followed by the Bonferroni multiple comparison test and 2-tailed paired or unpaired t test by using Prism software (GraphPad, La Jolla, CA) and presented as mean ± 1 SD unless otherwise noted. Figures are double-plotted to facilitate visualization of circadian time. The sample size for TFA analysis is n = 6, except for the following time points due to low sample volume: 0800: amber, n = 5; red n = 4; 1200: amber, clear, and red, n = 5 each; 1600: amber, red, and clear, n = 5 each; 2000: amber n = 5; red and clear, n = 4 each. Statistical significance among the group means was set at a P value of less than 0.05.
Results
Animal room illumination and CED measurements. Mean daytime animal room illumination at the left, right, and center of the rack 1 m above the floor with the detector facing the ceiling had relatively small variance and measured 265.0 ± 43.2 lx (n = 21 [7 wk, 3 positions]). Measurements in the inside front of the cages were averaged for all cage positions and had little variance (57.6 ± 10.5 lx, n = 84 [7 wk, 12 cages]). Laboratory CED used in this experiment are shown in Figure 1. Measurements of radiometric irradiance (lx) from inside the CED positioned in the middle of
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each cage were made daily (amber, 2.4 ± 0.7 lx [n = 15, P = 0.0046]; red, 1.3 ± 0.5 lx [n = 12, P < 0.0001]; opaque, 1.8 ± 0.5 lx [n = 7, P < 0.0001); and clear, 3.9 ± 1.8 lx [n = 14]), and the measurements for amber, red, and opaque CED differed from that for the control clear CED. In addition, irradiance differed (P < 0.0001) between amber and red CED. Sample sizes (n) for statistical analysis differed due to the exclusion of measurements taken when only the running lighting (1/2 task lighting) was illuminated in the room, which was corrected for subsequent measurements. Behavioral observations. Figure 2 depicts the average amount of time that male rats (n = 6 per treatment group; 3 cages) chose to spend inside each CED, recorded after a 10-d exposure (before blood draws) and again after a 24-d exposure, which was 2 or 3 d after a scheduled blood draw. Before beginning scheduled blood sampling (phase 1, days 10 and 11), rats spent significantly more time in the red (mean ± SEM, 290 ± 74 min, P = 0.0059), amber (110 ± 28 min, P = 0.008), and opaque (68 ± 20 min, P = 0.0248) CED compared with the clear CED tunnel (8 ± 5 min). Rats used all tunnels significantly less during the blood-sampling phase (phase 2, days 24 and 25; red: 38 ± 22 min, P = 0.0312; amber: 10 ± 9 min, P = 0.0305; opaque: 8 ± 3 min, P = 0.0367) than before blood collection, except for the clear tunnel (8 ± 5 min), which showed similar use during both phases. Dietary and water intakes and body weight. Daily dietary intake (P < 0.0001) and water intake (P = 0.006) differed significantly between the red and clear CED groups (Figure 3), and dietary intake differed between the opaque and clear CED groups (P = 0.0225; food intake [n = 168; 14 measurements, 3 cages, 4 treatment groups]: clear, 27.0 ± 2.6 g; amber, 27.0 ± 2.5 g; red, 30.6 ± 2.9 g; and opaque, 28.6 ± 3.4 g; water intake [n = 168]: clear, 43.1 ± 3.7 mL; amber, 42.0 ± 4.1 mL; red, 47.3 ± 7.4 mL; and opaque, 43.4 ± 4.7 mL). In addition, body weight change differed significantly between amber (P = 0.0023) and red (P = 0.0022) CED groups compared with the clear control CED group (clear, 269.3 ± 40.8 g; amber, 260.5 ± 40.0 g; red, 280.2 ± 42.6 g; opaque, 274.5 ± 44.0 g; n = 36 [6 measurements, 6 rats per group]) over the 36-d experimental period (Figure 4). Plasma melatonin. Figure 5 depicts diurnal rhythms of plasma melatonin concentrations (mean ± SEM) for each CED treatment group (n = 3 for all groups). Melatonin concentrations were low for all treatment groups at 0800, 1200, and 1600 (23.2 ± 6.8 pg/ mL), as expected for rats during the light phase, although the hormone levels were significantly higher at the 0800 time point for the red CED group (32.5 ± 0.50 pg/mL; P = 0.035) as compared with the clear group (24.9 ± 4.13 pg/mL). All rats in all 4 CED groups demonstrated significant circadian rhythms in plasma melatonin concentrations. At 2000, melatonin levels in amber (26.9 ± 1.8 pg/mL; P = 0.012) and opaque (23.5 ± 1.6 pg/mL; P = 0.035) CED groups were significantly higher than in the clear CED group (15.3 ± 4.2 pg/mL). Peak levels of melatonin occurred at 2400 for all CED groups, but the concentration in the opaque CED group (83.5 ± 33.3 pg/mL; P = 0.20) was significantly lower than that for the clear CED group (247.0 ± 68.0 pg/mL). At 0400, the amber CED group melatonin level (252.0 ± 42.2 pg/mL) was significantly higher than the value for the clear CED group (78.2 ± 57.1 pg/mL). Plasma TFA. Significant differences for plasma TFA concentration between clear and opaque CED groups (P = 0.0231) occurred at 0400 (clear, 5387 ± 77 µg/mL; opaque: 5598 ± 160 µg/mL, n = 6 per treatment group). All plasma TFA values in
Figure 2. Instantaneous scan sampling for the time (mean ± SEM) that rats spent inside either the amber, clear, red, or opaque colored enrichment device (CED) before (phase I) and during (phase II) the experiment. During phase 1, rats spent significantly (*) more time in the red (290 ± 74 min, P = 0.0059), amber (110 ± 28 min, P = 0.008), and opaque (68 ± 20 min, P = 0.0248) than in the clear (8 ± 5 min) CED. During phase 2, rats spent less time in all tunnels (red: 38 ± 22 min, P = 0.0312; amber: 10 ± 9 min, P = 0.0305; opaque: 8 ± 3 min, P = 0.0367) except for the clear CED (8 ± 5 min), for which there was no difference compared with phase 1 data.
Figure 3. Dietary (bottom, g) and water (top, mL) intakes (mean ± 1 SD) for rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). The integrated mean dietary intake differed between the red and clear CED groups (P < 0.0001) and between the opaque and clear CED groups (P = 0.0178), and water intake differed (P = 0.006) between the red and clear CED groups. Food intake (n = 168, 14 measurements, 3 cages, 4 treatment groups): red, 30.5 ± 2.7 g; opaque, 28.6 ± 3.4 g; amber, 27.2 ± 2.8 g; clear, 27.2 ± 2.05 g. Water intake (n = 168): red, 47.3 ± 7.4 mL; amber, 42 ± 4.1 mL; opaque, 43.4 ± 4.7 mL; clear, 43.1 ± 3.7 mL.
male Sprague–Dawley rats with free access to food displayed a significant rhythm in all CED treatment groups (Figure 6), which reached the nadir at 1600 (1041 ± 43 µg/mL) and peaked at 0400 (5598 ± 161 µg/mL). Plasma corticosterone. Plasma corticosterone circadian rhythms are depicted in Figure 7. Values for plasma corticosterone in rats of all CED treatment groups followed diurnal rhythms, which increased continuously from the nadir at 0800 until their peak at 2000. At the 2400 and 0400 time points, the red CED group’s values were significantly lower (P = 0.0121) than those for the clear CED control group (2400: red, 12.1 ± 2.2 ng/mL; clear, 20.6 ± 1.4 ng/mL; 0400: red, 2.2 ± 1.2 ng/mL; clear, 12.3 ± 0.5 ng/mL; P = 0.025), n = 3 per treatment group).
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Figure 4. Integrated body weight change (mean ± SEM, n = 6) over the 36-d experimental period differed (2-tailed t test) between the clear control group and both the amber (P = 0.0023) and red (P = 0.0022) CED groups (red, 280.2 ± 42.6 g; amber, 260.5 ± 40.0 g; opaque, 274.5 ± 44.0 g; clear, 269.3 ± 40.8 g).
Figure 6. Circadian rhythm of plasma total fatty acids (mean ± 1 SD) concentrations for rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). Black bars represent the dark phase; concentrations with asterisks differ (P < 0.05) when using the clear CED as the control. Data are plotted twice to show rhythmicity. Significant differences for plasma total fatty acids between clear and opaque CED groups (P = 0.0231) occurred at 0400 (clear, 5387 ± 77 µg/mL; opaque, 5598 ± 161 µg/mL). All CED treatment groups displayed a nadir at 1600 (1041 ± 43 µg/mL) and a peak at 0400 (5598 ± 161 µg/mL).
Figure 5. Circadian rhythm of plasma melatonin concentration (mean ± SEM) in rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). Black bars represent the dark phase; concentrations with asterisks differ (P < 0.05) compared with the clear CED. Data are plotted twice to show rhythmicity. Melatonin concentrations were low for all treatment groups at 0800, 1200, and 1600 (23.2 ± 6.8 pg/mL) during the light phase as expected, although the hormone levels were significantly higher (P = 0.0348) at the 0800 time point in the red CED group (32.5 ± 0.50 pg/mL) as compared with the clear group (24.9 ± 4.13 pg/mL). At 2000, values for the amber (26.9 ± 1.76 pg/mL, P = 0.0116) and opaque (23.5 ± 1.62 pg/mL, P = 0.0351) CED groups were significantly higher than for the clear (15.3 ± 4.21 pg/mL) CED group. Peak levels of melatonin occurred at 2400 for all CED groups, but the opaque CED group (83.5 ± 33.3 pg/mL) was significantly lower than the clear CED group (247 ± 68 pg/mL, P = 0.0202). At 0400, the amber CED group melatonin level (252.0 ± 42.2 pg/mL) was significantly higher than the clear CED group (78.2 ± 57.1 pg/mL, P = 0.0163).
Figure 7. Circadian rhythm of plasma corticosterone (mean ± SEM) concentrations for rats housed with either amber, clear, red, or opaque colored enrichment devices (CED). Black bars represent the dark phase; concentrations with asterisks differ (P < 0.05) when using the clear CED as the control. Data are plotted twice to show rhythmicity. All CED groups nadir at 0800, and rise continuously until their peak at 2000 and were not significantly different. However, at 2400 and 0400 time points, the red CED group’s values were significantly lower (P = 0.0121) when compared with those for the clear CED (2400: red, 12.1 ± 2.19 ng/mL; clear, 20.6 ± 1.38 ng/mL; 0400: red, 2.17 ± 1.15 ng/mL; clear, 12.3 ± 0.493 ng/mL; P = 0.025).
Discussion
Animal health and wellbeing are affected by the environment, housing, and management of the species, and these components should account for their physical, physiologic, and behavioral needs.27 Recent methods have been suggested to standardize rodent care, so that research models are better defined and more stable, thus requiring fewer animal subjects 40 and increasing
scientific validity.27 EE is an independent variable27 and refers to the provision of a more naturalistic environment and to the addition of objects or resources to the environment for increasing sensory and motor stimulation, the expression of species-typical behaviors, and the repression of stereotypies.27,37 The success of EE can be measured by using parameters of normal health, including growth, development, and reproduction.27,40 For rodents, EE can include bedding depth and type, which can promote species-typical foraging, burrowing, and nest-building behaviors in addition to opportunities for thermoregulation, as well as nestbuilding materials, novel food stuffs, chew toys, tunnels, igloos or houses, social housing, and cage modifications.4,27 However,
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little is known about the effects of these EE items on scientific outcomes.4 We chose to evaluate the commonly used tinted rat enrichment tunnels in this study. We used 4 types of CED: the 2 colors (amber and red) commonly available from manufacturers of rat enrichment tubes; opaque tubes, which were included because CED produced inhouse are often constructed of PVC; and clear tunnels, which eliminated the mere presence of an EE device as an independent variable; several studies have found that the presence of EE, when compared with isolation or impoverished conditions, has effects on cognition, development, and performance.18,37,38,40 In circadian light studies, 3 major variables include duration and timing of exposure, intensity, and tint (or wavelength). Lighting intensities inside each cage were measured at all cage locations and had little variance. However, radiometric irradiance measured inside each CED was significantly different between clear and colored (red, amber, and opaque) tunnels as well as between amber and red CED. Adherence to a strict 12:12-h light:dark environmental lighting cycle throughout the study, with complete darkness at night, ensured that any observed effects result only from variations in color tint. We found significant differences in daily food and water intake between animals with red compared with clear CED and in dietary intake between groups in opaque and clear CED. Food intake for rats with red CED was higher, and correspondingly, average body weight changes were significantly greater in the red CED group when compared with the clear CED group over the 36-d experimental period. The amber CED group had significantly lower average body weight changes. Other investigators reported significant increases in food and water consumption for rats under isolation or impoverished conditions compared with cages with EE and standard social conditions (4 rats per cage) but found significantly heavier body weights only after the first week of differential housing and no significant differences in body weight between EE and standard-condition treatment groups.38 The current study used social housing of rat pairs with EE, which resulted in the red CED group consuming more food and water with a corresponding increase in body weight. More studies should be conducted to reveal the mechanisms underlying this finding—perhaps by adding activity monitoring, recording food and water intake at shorter intervals, and measuring other neurohormones, such as plasma leptin and ghrelin, blood glucose, and insulin concentrations—to elucidate any possible central circadian disruption as a cause. Monitoring locomotor activity would be helpful in determining whether the significant decreases in use of the amber, red, and opaque CED are a result of these rats acclimating to the novel environment. In a behavioral context, locomotor activity is a reliable and consistent index of learning or information processing, and persistently high levels suggest a lack of information processing or the absence of acclimation to the novel environment.18 Results from the current study suggest that rats spend more time inside red CED as compared with all other CED tested. Because intensities were held constant, our results indicate that the tint of the CED causes this effect. However, the total time spent in each CED was markedly lower approximately 2 wk after presentation (phase 2) compared with 2 d after introduction (phase 1). There was no feasible way of controlling for this animal-dependent variable. However, this reduction in use and associated brief exposure times might have mitigated significant effects in some cases.
Several difficulties have been encountered in implementing EE for laboratory rodents. First, EE can have negative effects, including the induction of aggressive behaviors in male mice and rats,1 changes in fecundity or breeding pattern, and litter size;40 allergies and skin rashes caused by bedding type; and the ingestion of bedding or EE material.27,40 Second, difficulties have been encountered with the interpretation of behavioral and chemical effects as being positive, negative, or neutral in terms of their influence on health and wellbeing.40 For this reason, we believe that including physiologic and metabolic measures in behavioral studies is imperative so that all parameters of health and wellbeing can be taken into account. In addition, more studies are needed to determine whether any of these changes are biologically or clinically significant.40 Last, the literature demonstrates a lack of detail concerning the reporting all aspects of the environment and EE.40 Reviews show the great diversity in control housing conditions, the amount of space provided in cages, social group number, rat strain and sex, age at onset of EE,26 duration of exposure to EE, types of physical objects used for EE, and behavioral tests used.37,40 Social companionship has been found to act as a form of EE.4 Social enrichment has a greater effect when compared with EE or isolation or impoverished conditions.18 More importantly, these affects are lasting,18 unlike the acclimation seen with physical enrichment. Reports have demonstrated that toys used for EE have a very short (1 d) period of interest, whereas toys related to food retain their attraction longer, likely due to the primary reinforcement of food.4,42 Given that rats in all groups spent less time in the CED, regardless of color, during phase II compared with phase I, acclimation to EE could have occurred. In addition, animals’ social needs should be considered,27 given that studies report adverse behaviors in isolation or impoverished conditions.1,37 Single housing for social animals requires justification and review by the IACUC and veterinarian, and time alone should be minimized to the shortest duration possible.27 Providing extra EE for animals housed in isolation, in small spaces or providing visual, auditory, olfactory, or tactile contact with conspecifics have been recommended.27 EE can help animals escape aggression and avoid social conflicts.27 However, when reports yield evidence of objectinduced changes in normal physiology, alternatives should be sought. Melatonin is a neurohormone released primarily at night in response to rhythms generated in the SCN and is the body’s biologic clock.36 As expected, melatonin concentrations were low for all CED groups during the light phase, although the hormone levels were statistically (but perhaps not clinically) significantly higher at the 0800 time point in the red CED group. None of the CED affected the normal circadian rhythm of melatonin, as evidenced by the coinciding peaks and troughs. At 2000, melatonin concentrations of the amber and opaque CED groups rose more rapidly than did that of the clear CED group. Interestingly, the opaque CED group’s melatonin concentration did not peak as high as those of the other groups. These results suggest that the low level of exposure was sufficiently significant to affect the circadian rhythm. Alternatively, the light–dark cycle might be altered in these rats, due to the presence of the CED, perhaps providing a darker area in the cage, which would affect overall sensory perception of light. Finally, the instantaneous scan-sampling method used for behavioral observations may not accurately reflect the true behavior of the rats and might otherwise be measured by
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videorecording the activity of single-housed subjects. Further studies should address these possibilities. Peak plasma TFA levels (0400) differed between the opaque and clear CED groups, who had free access to food and water. The TFA rhythm, timing, and intensity were similar to previously reported results from male Sprague–Dawley rats,13,41 confirming that these circadian rhythms remain entrained6 and are independent from the SCN-generated rhythms. However, because significant difference was found at one time point, additional investigation is needed to correlate each blood draw result with food and water intakes and plasma TFA. Corticosterone is a widely used parameter for the measurement of stress in rats and displays true circadian rhythmicity that is regulated directly by the SCN.19,35 The corticosterone concentration peaks around the onset of darkness, and the nadir occurs after the onset of light, with gradual increases throughout the light phase.19,35 Even though significant differences were found between the red CED group compared with the clear CED group, all nighttime values of corticosterone were less than 51 ng/mL, which is comparable to results from other studies3,41 and much lower than those obtained in posttraumatic stress models29 (700 ng/mL) using this stock. An animal’s enclosure and its components should withstand cleaning procedures and not interfere with the animals’ health or research use.27 The opaque tube, which is made from PVC in the exact measurements of the other manufactured tubes, poses no concern regarding leaching because this material does not contain any plasticizers,10 such as the commonly used di(2-ethylhexyl) phthalate, that reportedly alter hormones.9 In addition, tunnels were replaced weekly, thus eliminating stress due to changing objects too frequently; rotation and replacement of EE items are recommended to maintain their novelty.27 Behavioral observations were performed at least 2 d after blood sampling to account for the potential confound of side effects of blood collection on behavioral measures. Limitations of the current study include the rat-dependent variable of the time spent inside each CED, which thus altered the ‘dose’ of colored light that each animal received. In fact, this limitation is present anytime a CED or other EE is added to a cage as standard practice. We gave the rats the opportunity to use their respective CED how they saw fit, and replicated how, in practice, we provide EE. Remember that the goal of EE is to provide animals the opportunity for species-specific behaviors that fulfill their innate behavioral and physiologic needs. Therefore, all studies in which the facility uses CED—whether they are colored tunnels, huts, or other objects—are potentially confounded due to the variable exposures to each individual animal. Changes in experimental design would give us a method to calculate individual color ‘doses,’ and comparisons can be made to the experimental circadian time points. Because of this limitation, possible effects may not be recognized for some groups, due to brief exposure times; in addition, we do not know the exact exposure time that resulted in the physiologic changes that were significant. Experimental design changes, including single housing, precise measurement of individual food and water intake and recording time of intake, and activity monitoring by videorecording, might help to yield this information in subsequent studies. These experimental design changes would give us a better indication of the effects of a novel object (shortly after object introduced) compared with acclimation to the object (here, after 2 wk).
An animal’s coping to environmental changes is a complex behavioral reaction revealed by their responses to changes in novelty (well-known territory) and can have 2 differently motivated reactions. That is, animals respond to changes in novelty by displaying fear-associated defensive reactions, such as freezing and escape, or by mounting exploratory associated reactions, such as curiosity (neotic preference) and active exploration. Typically, fear-evoking objects are avoided at first, and this behavior may reflect a need to seek immediate safety. If the fear level is low or when fear subsides with time or distance, subsequent active exploration of the object and information-gathering allows animals to adapt to the environmental change (novelty). When exploration brings no signs of danger and after information is consolidated into memory, animals habituate to the changed environment, resulting in reduction or removal of fear and curiosity.34 This typical behavioral process is hard to predict and varies between species, sexes, housing conditions, and even EE objects.4,37,38 Other limitations in terms of our modified experimental design likely would be recovery time and blood sample volumes and thus would require larger groups to collect all of the circadian time points for comparison. Other variables include strain, sex, and species, as discussed earlier. Our next steps will be to add individual behavioral measurements by using activity monitors, which would require individual housing, and to include time-recorded food and water measurements to elucidate individual dosages for each CED type. In studies now underway, we are evaluating the effects of other commonly used CED, such as the popular plastic hut for mice,26 which is manufactured in amber, red, and blue plastics and as disposable cardboard (opaque) items, to evaluate whether differences due to the color of these enrichment devices emerge. The effects of colored EE tunnels we used—and likely other CED such as huts—cannot be completely controlled, because we can only provide the animal the opportunity to use the device and cannot hold constant the dose of each CED received, given that this variable is behavior-dependent. Regardless of this limitation, circadian rhythms of metabolism and physiology were significantly disrupted depending on the CED in the cage. Therefore, CED used for enrichment in laboratory rat cages might significantly confound scientific outcomes, and their use should be thoroughly reported or pilot-studied. Further testing of these previously assumed harmless EE objects is warranted.
Acknowledgments
We thank Michael Webb, Patricia Beavers, and Katie Castillo for their outstanding care of the animals and Erin Dauchy for her technical training and statistical expertise. This work was supported by NIH grants 1R25RR032028 (MAWD and RPB) and 1R56CA193518-01 (DEB and SMH).
References
1. Abou-Ismail UA, Mahboub HD. 2011. The effects of enriching laboratory cages using various physical structures on multiple measures of welfare in singly housed rats. Lab Anim 45:145–153. 2. Altmann J. 1974. Observational study of behavior: sampling methods. Behaviour 49:227–267. 3. Arnold M, Langhans W. 2010. Effects of anesthesia and blood sampling techniques on plasma metabolites and corticosterone in the rat. Physiol Behav 99:592–598. 4. Baumans V. 2005. Environmental enrichment for laboratory rodents and rabbits: requirements of rodents, rabbits, and research. ILAR J 46:162–170.
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5. Berson DM, Dunn FA, Takao M. 2002. Phototransduction by retinal ganglion cells that set the circadian clock. Science 295:1070–1073. 6. Blask DE, Brainard GC, Dauchy RT, Hanifin JP, Davidson LK, Krause JA, Sauer LA, Rivera-Bermudez MA, Dubocovich ML, Jasser SA, Lynch DT, Rollag MD, Zalatan F. 2005. Melatonindepleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats. Cancer Res 65:11174–11184. 7. Brainard GC, Hanifin JP. 2005. Photons, clocks, and consciousness. J Biol Rhythms 20:314–325. 8. Brainard GC, Hanifin JP, Rollag MD, Greeson J, Byrne B, Glickman G, Gerner E, Sanford B. 2001. Human melatonin regulation is not mediated by the 3-cone photopic visual system. J Clin Endocrinol Metab 86:433–436. 9. Carbone S, Ponzo OJ, Gobetto N, Samaniego YA, Reynoso R, Scacchi P, Moguilevsky JA, Cutrera R. 2013. Antiandrogenic effect of perinatal exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate increases anxiety-like behavior in male rats during sexual maturation. Horm Behav 63:692–699. 10. Charlotte Pipe and Foundry Company. 2014. Safety Data Sheet: PVC pipe and fittings. Monroe (NC): Charlotte Pipe and Foundry Company. 11. Dacey DM, Liao HW, Peterson BB, Robinson FR, Smith VC, Pokorny J, Yau KW, Gamlin PD. 2005. Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature 433:749–754. 12. Dauchy RT, Dauchy EM, Hanifin JP, Gauthreaux SL, Mao L, Belancio VP, Ooms TG, Dupepe LM, Jablonski MR, Warfield B, Wren MA, Brainard GC, Hill SM, Blask DE. 2013. Effects of spectral transmittance through standard laboratory cages on circadian metabolism and physiology in nude rats. J Am Assoc Lab Anim Sci 52:146–156. 13. Dauchy RT, Dauchy EM, Tirrell RP, Hill CR, Davidson LK, Greene MW, Tirrell PC, Wu J, Sauer LA, Blask DE. 2010. Dark-phase light contamination disrupts circadian rhythms in plasma measures of endocrine physiology and metabolism in rats. Comp Med 60:348–356. 14. Dauchy RT, Hoffman A, Wren-Dail MA, Hanifin JP, Warfield B, Brainard GC, Xiang S, Yuan L, Hill SM, Belancio VP, Dauchy EM, Smith K, Blask DE. 2015. Daytime blue light enhances nighttime circadian melatonin inhibition of human prostate cancer growth. Comp Med 65:473–485. 15. Dauchy RT, Wren MA, Dauchy EM, Hanifin JP, Jablonski MR, Warfield B, Brainard GC, Hill SM, Mao L, Dupepe LM, Ooms TG, Blask DE. 2013. Effect of spectral transmittance through red-tinted rodent cages on circadian metabolism and physiology in nude rats. J Am Assoc Lab Anim Sci 52:745–755. 16. Dauchy RT, Wren MA, Dauchy EM, Hoffman AE, Hanifin JP, Warfield B, Jablonski MR, Brainard GC, Hill SM, Mao L, Dobek GL, Dupepe LM, Blask DE. 2015. The influence of red light exposure at night on circadian metabolism and physiology in Sprague–Dawley rats. J Am Assoc Lab Anim Sci 54:40–50. 17. Ecker JL, Dumitrescu ON, Wong KY, Alam NM, Chen SK, LeGates T, Renna JM, Prusky GT, Berson DM, Hattar S. 2010. Melanopsinexpressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision. Neuron 67:49–60. 18. Elliott BM, Grunberg NE. 2005. Effects of social and physical enrichment on open-field activity differ in male and female Sprague–Dawley rats. Behav Brain Res 165:187–196. 19. Forslid A, Ingvar M, Rosen I, Ingvar DH. 1986. Carbon dioxide narcosis: influence of short-term high concentration carbon dioxide inhalation on EEG and cortical evoked responses in the rat. Acta Physiol Scand 127:281–287. 20. Freedman MS, Lucas RJ, Soni B, von Schantz M, Munoz M, DavidGray Z, Foster R. 1999. Regulation of mammalian circadian behavior by nonrod, noncone, ocular photoreceptors. Science 284:502–504. 21. Gamlin PD, McDougal DH, Pokorny J, Smith VC, Yau KW, Dacey DM. 2007. Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells. Vision Res 47:946–954. 22. Hanifin JP, Brainard GC. 2007. Photoreception for circadian, neuroendocrine, and neurobehavioral regulation. J Physiol Anthropol 26:87–94.
23. Hanifin JP, Stewart KT, Smith P, Tanner R, Rollag M, Brainard GC. 2009. High-intensity red light suppresses melatonin. Chronobiol Int 23:251–268. 24. Hattar S, Liao HW, Takao M, Berson DM, Yau KW. 2002. Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295:1065–1070. 2 5. Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, Hankins MW, Lem J, Biel M, Hofmann F, Foster RG, Yau KW. 2003. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424:76–81. 26. Hutchinson EK, Avery AC, VandeWoude S. 2012. Environmental enrichment during rearing alters corticosterone levels, thymocyte numbers, and aggression in female BALB/c mice. J Am Assoc Lab Anim Sci 51:18–24. 27. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 28. Jacobs GH, Fenwick JA, Williams GA. 2001. Cone-based vision of rats for ultraviolet and visible lights. J Exp Biol 204:2439–2446. 29. Jia M, Meng F, Smerin SE, Xing G, Zhang L, Su DM, Benedek D, Ursano R, Su YA, Li H. 2012. Biomarkers in an animal model for revealing neural, hematologic, and behavioral correlates of PTSD. J Vis Exp. 68:e3361. 30. Kennaway DJ, Voultsios A, Varcoe TJ, Moyer RW. 2002. Melatonin in mice: rhythms, response to light, adrenergic stimulation, and metabolism. Am J Physiol Regul Integr Comp Physiol 282:R358– R365. 31. Kohler I, Meier R, Busato A, Neiger-Aeschbacher G, Schatzmann U. 1999. Is carbon dioxide (CO2) a useful short-acting anaesthetic for small laboratory animals? Lab Anim 33:155–161. 32. Lucas RJ, Peirson SN, Berson DM, Brown TM, Cooper HM, Czeisler CA, Figueiro MG, Gamlin PD, Lockley SW, O’Hagan JB, Price LL, Provencio I, Skene DJ, Brainard GC. 2014. Measuring and using light in the melanopsin age. Trends Neurosci 37:1–9. 33. Osteen WK, Sundberg DK. 1980. Prolactin binding in eyes of rats with retinal dystrophy and normal retinas - an autoradiographic study. Anat Rec 196:A141–A142. 34. Pisula W, Turlejski K, Stryjek R, Nalecz-Tolak A, Grabiec M, Djavadian RL. 2012. Response to novelty in the laboratory Wistar rat, wild-captive WWCPS rat, and the gray short-tailed opossum (Monodelphis domestica). Behav Processes 91:145–151. 35. Rahman SA, Kollara A, Brown TJ, Casper RF. 2008. Selectively filtering short wavelengths attenuates the disruptive effects of nocturnal light on endocrine and molecular circadian phase markers in rats. Endocrinology 149:6125–6135. 36. Reiter RJ, Robinson J. 1995. Melatonin: your body’s natural wonder drug. New York (NY): Bantam Books. 37. Simpson J, Kelly JP. 2011. The impact of environmental enrichment in laboratory rats—behavioural and neurochemical aspects. Behav Brain Res 222:246–264. 38. Simpson J, Kelly JP. 2012. The effects of isolated and enriched housing conditions on baseline and drug-induced behavioural responses in the male rat. Behav Brain Res 234:175–183. 39. Sun JH, Yaga K, Reiter RJ, Garza M, Manchester LC, Tan DX, Poeggeler B. 1993. Reduction in pineal N-acetyltransferase activity and pineal and serum melatonin levels in rats after their exposure to red light at night. Neurosci Lett 149:56–58. 40. Toth LA, Kregel K, Leon L, Musch TI. 2011. Environmental enrichment of laboratory rodents: the answer depends on the question. Comp Med 61:314–321. 41. Wren MA, Dauchy RT, Hanifin JP, Jablonski MR, Warfield B, Brainard GC, Blask DE, Hill SM, Ooms TG, Bohm RP Jr. 2014. Effect of different spectral transmittances through tinted animal cages on circadian metabolism and physiology in Sprague–Dawley rats. J Am Assoc Lab Anim Sci 53:44–51. 42. Young RJ, editor. 2003. Environmental enrichment for captive animals. Oxford (UK): Blackwell Science.
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