Effects of Cage Enrichment on Heart Rate, Blood Pressure, and Activity of Female Sprague–Dawley and Spontaneously Hypertensive Rats at Rest and after Acute Challenges

Abstract

Here we sought to determine whether a nonsocial cage enrichment program, identical to one we previously used with male rats, was effective in reducing heart rate or systolic blood pressure (SBP) in female Sprague–Dawley rats and spontaneously hypertensive rats (SHR). Young adult rats, each instrumented with a radiotelemetry pressure transmitter, were housed individually under enriched or nonenriched conditions. Heart rate and SBP were monitored at 5- and 1-min intervals, respectively, when the rats were undisturbed or after several different types of experimental manipulations some of which are considered stressful. Cage enrichment did not significantly alter heart rate or SBP of undisturbed rats in either strain at any time during the day or night. However, activity of female SHR was increased in the afternoon and at night under enriched conditions compared with nonenriched conditions. The enrichment program did not significantly reduce heart rate or SBP responses to most acute manipulations in either strain. However, cage enrichment increased the responses to some procedures (Sprague–Dawley: handling, 1-h restraint; SHR: subcutaneous injection, tail-vein injection, handling). We conclude that a nonsocial cage enrichment program did not reduce physiologic indicators of stress in female Sprague–Dawley rats or SHR.

Cage enrichment for individually housed experimental rodents is recommended currently and soon may be required in many countries. These recommendations and requirements are supported by an extensive literature on the positive effects of environmental enrichment on brain development, cognitive function, and recovery from neural injury^{5,7,10,11,13,17,18,20-25,30,31} and by reports that environmental enrichment may reduce stress and improve wellbeing in rodents.^{1,6,9,16,26-28} However, the effects of environmental enrichment vary between strains, sexes, stressful conditions, types of enrichment, and parameters measured. For example, the cognitive responses of SHR—but not Wistar rats—were improved by environmental enrichment.²¹ In addition, that group also showed that enrichment had no effect on some noncognitive responses of SHR (for example, hypertension).²¹ In addition, environmental enrichment improved spacial memory after traumatic brain injury in Sprague–Dawley male but not female rats. Similarly, enriched housing had greater effects in male than female Sprague–Dawley rats.¹⁰ The authors also reported¹⁰ that social enrichment had greater effects on locomotor activity than did physical enrichment.¹⁰ However, the responses of female Sprague–Dawley rats to cat odors were more affected by environmental enrichment than were those of male rats.¹⁶ We have shown that a nonsocial enrichment program had less effect on in male Sprague–Dawley rats than on SHR.²⁶ Further, social enrichment (group housing) did not reduce heart rate and blood pressure responses to some experimental manipulations to the same degree in female rats as observed in male rats.^27,28 Others have observed that group housing reduced corticosterone secretion in female rats compared with levels in individually housed controls; however, corticosterone secretion in group-housed male rats was increased relative to that in those singly housed.⁸ Finally, we have observed that both social and nonsocial enrichment reduced heart rate and blood pressure responses to some—but not all—stressful procedures.^26-28

The objective of the current study was to determine whether a nonsocial enrichment program, identical to one we previously used with male rats,²⁶ was effective in reducing heart rate or systolic blood pressure (SBP) in female Sprague–Dawley rats and female SHR. The working hypothesis was that nonsocial cage enrichment would reduce heart rate and SBP under both basal conditions and after several types of acute experimental procedures, some of which were stressful.

Materials and Methods

Adult female Holtzman Sprague–Dawley rats and SHR (n = 12 of each strain) were purchased from Harlan Sprague–Dawley (Indianapolis, IN) at 175 to 200 g body weight. All rats were obtained from colonies reported by the vendor to be free from adventitious viruses, mycoplasma, respiratory and enteric bacteria (except several strains of Helicobacter), and ecto- and endoparasites (except a nonpathogenic commensal protozoa).

Routine husbandry.

The rats were allowed to acclimate to the animal room conditions and husbandry procedures for 2 wk prior to surgical implantation of a radiotelemetry transmitter. Environmental conditions in the animal room were: temperature, 22 to 26 °C; relative humidity, 30% to 60%; and lighting, 200 lx at cage level and lights on from 0700 to 1900 h. During this 2-wk acclimation period, rats were housed individually in conventional solid-bottom polycarbonate cages (nominal floor area, 930cm²) with standard stainless-steel lids and hardwood chip bedding (depth, 4 to 5 cm; Sanichip, PJ Murphy Forest Products, Montville, NJ). Cages were changed once each week (Mondays). Pelleted rat chow (Purina 5001, Purina Mills, Richmond, IN) was provided ad libitum, and tap water was provided in water bottles with sipper tube. Rats of both strains and, subsequently, both housing conditions (enriched and nonenriched) were housed in the same animal room at the same time.

Surgical procedures.

A radiotelemetry transmitter (model TA11PA-C40, Data Sciences International, St Paul, MN) was implanted aseptically into the abdominal cavity of each rat, with the pressure-sensing catheter inserted into the descending aorta via the left femoral artery as described previously.²⁶ The anesthetic was a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) given intraperitoneally. Postoperative analgesia (ketoprofen, 16 mg/kg SC) and postsurgical monitoring precisely followed procedures previously reported.²⁶

Enrichment program.

Beginning 10 to 14 d after telemetry transmitter implantation, half of the rats of each strain were assigned randomly to control (nonenriched conditions; individual housing with husbandry as described earlier) or enriched conditions. Cage enrichment consisted of 3 items added to the rat's home cage: a simulated burrow (2 red, telescoped, rectangular rat houses; Rodent Retreats, BioServ, Frenchtown, NJ) that was continuously present in the cage; a gnawing and food foraging object consisting of a hollow edible cylinder (Rodent Enrichment Habitat, Kong Veterinary Products, Irwindale, CA) into which a small filter paper bag (WF Fisher and Son, Watertown, TN) containing three 1-g chocolate-flavored rodent treats (SupremeTreats, BioServ) was placed on Wednesdays at 1300 h; and a shredding and nesting item (150-g Nestpak filled with a mixture of corn cob and wood chips; WF Fisher and Son) that was placed in the cage on Fridays at 1300. The foraging object and nesting item generally were consumed or completely shredded in 1 to 3 d. If destroyed, they were not replaced until the next scheduled addition. The simulated burrow was replaced with a clean one when the cages were changed at 1300 on Mondays. The selection of enrichment items was based on their probable stimulation of species-specific behaviors (for example, hiding, gnawing, food foraging, shredding, and nesting), commercial availability, and their known composition and suitability for Good Laboratory Practices studies. Rats were acclimated to the enrichment program for 2 wk prior to the onset of experiments outlined below.

Experimental procedures.

After the 2-wk acclimation to the enrichment program, heart rate, blood pressure, and activity data were collected at times when the rats were undisturbed (from 0800 to 0900 and from 1300 to 0700 when no humans were present in the animal room). In addition, heart rate and blood pressure data were collected during and for 3 h (1000 to 1300) after exposure to each of a battery of acute procedures. These procedures were selected to be representative of the following functional categories: husbandry procedures (routine cage change, 1 min of gentle handling, 1 h of exposure to an unfamiliar conspecific, removal of a familiar conspecific after 5 d of cohabitation); experimental procedures (hand restraint and subcutaneous injection, transport from animal room to lab and subcutaneous injection with hand restraint, 1 to 2 min of restraint in a rat restrainer with tail vein injection, hand restraint with intraperitoneal injection); and stressful procedures (15 min of exposure to the odors of urine and feces from stressed male and female rats, 15 min of exposure to the odor of dried rat blood, 60 min of restraint in a rat restrainer in the home cage).

Husbandry procedures.

Cage change.

The cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, and the rat was gently grasped at the base of its tail and transferred to a clean cage containing fresh wood-chip bedding. A clean cage lid and clean water bottle were placed on the cage, and the cage was replaced on the rack. The procedure required 20 to 30 s per cage.

Handling.

Each cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, and the rat was gently grasped at the base of its tail, removed from the cage, and placed on the technician's arm, where the rat was stroked on the back and head for 1 min, after which it was returned to its home cage and replaced on the cage rack.

Introduction of a conspecific.

The cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, an unfamiliar female rat of the same strain and age was placed into the telemetry cage, and the cage was returned to the cage rack for 60 min, after which the conspecific rat was removed from the cage.

Removal of a conspecific.

Five days before the experiment, a conspecific rat was introduced to the telemetry cage at 1300 as described and cohabitated there until 1000 of the experimental day, at which time each telemetry cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, and the conspecific was removed. The telemetry cage was then returned to the cage rack.

Experimental procedures.

Subcutaneous injection.

The cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, and the rat was gently grasped at the base of its tail and removed from the cage to the workbench surface. Using one hand, the technician gently held the rat and lifted the loose skin at the nape of its neck. An injection of 0.2 mL sterile saline was made in this loose skin pocket by using a 1-mL tuberculin syringe and a 26-gauge needle. The rat was returned to its cage; the cage lid and water bottle were replaced, and the cage returned to the cage rack. The procedure required 20 to 30 s per cage.

Transport to another room and subcutaneous injection.

The cages of all the rats to be injected were removed from the cage rack, placed on a cart, covered with a large cloth drape, and transported to another lab approximately 100 ft from the animal room. Restraint and subcutaneous injection were performed as described earlier. Once all rats had been injected, they were transported back to the animal room on the cart, and their cages placed on the cage rack. The entire process took approximately 10 min.

Tail vein injection.

The cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, and the rat grasped at the base of its tail and gently placed into a large standard rodent restrainer on the workbench. The rat's tail was placed in a beaker of warm water for 30 s to induce vasodilation and then was swabbed with 70% ethanol. A tail vein injection of 0.2 mL sterile saline was given by using a 25-gauge butterfly infusion catheter, and the rat removed from the restrainer, placed in its home cage, and returned to the cage rack. No more than 2 attempts to insert the needle in the vein were done. The procedure required 2 to 3 min per cage.

Intraperitoneal injection.

This procedure was done in a manner similar to the subcutaneous injection described earlier, except that the technician restrained the rat by using one hand and then turned it on its back to expose the abdomen; the injection of 0.2 mL sterile saline was made in the lower left or right quadrants of the abdomen by using a 1-mL tuberculin syringe and a 26-gauge needle. The procedure required 20 to 30 s per cage.

Stressful procedures.

Exposure to odors of urine and feces from stressed male rats.

A paper towel collected from the floor of a large acrylic glass chamber in which 6 male rats had been placed together for 5 min was air-dried and positioned on fine screen mesh previously secured to the lid of each telemetry cage. The towel remained on the cage lid for 15 min. That the rats placed in the acrylic glass chamber were stressed was supported by their marked exploratory behavior and frequent urination and defecation, as well as by previous results that showed marked elevations in heart rate (approximately 100 bpm above resting levels) and mean arterial blood pressure (approximately 20 mm Hg above resting levels) of rats bearing telemetry transmitters within 30 s of their placement in the same chamber.

Exposure to odor of urine and feces from stressed female rats.

This procedure was done as described for male rats, except that 6 adult female rats were placed in the acrylic glass chamber for collection of urine and feces.

Exposure to odor of dried rat blood.

A paper towel containing rat blood collected at decapitation of male rats was air-dried and placed on fine screen mesh on the cage lid as described for exposure to urine and feces. The towel remained on the cage lid for 15 min.

Prolonged restraint.

The cage was removed from the cage rack and placed on a workbench. The water bottle and cage lid were removed, and the rat was gently grasped at the base of its tail and placed into a restrainer that had been fabricated locally from a transparent acrylic cylinder (diameter, 7.6 cm; length, 17.8 cm) glued horizontally to a 15.2 × 15.2 × 5.1-cm acrylic riser (S and S Acrylics, Norcross, GA); the top and front of the acrylic cylinder contained multiple 6-mm holes for ventilation. The open end of the restrainer was closed by using a disposable plastic lid that had a 13-mm hole in the center for the rat's tail; the lid was secured to the acrylic cylinder with tape. The restrainer containing the rat then was placed in the animal's home cage, which was returned to the cage rack for 60 min, after which time the rat was removed from the restrainer and returned to its home cage.

Six rats of each strain from control and enriched groups individually underwent the acute procedures on Mondays, Wednesdays, and Fridays (Figure 1). Over the course of the study, each instrumented rat experienced every procedure once. The every-other-day schedule was established to reduce any potential carryover effects from one procedure to the next. There were no indications that the rats became conditioned to procedures being conducted on this schedule (that is, there were no changes in heart rate at 1000 on intervening nonexperimental days in the current study and no differences in heart rate responses to some of the same procedures applied on the thrice-weekly schedule for 2 consecutive weeks in a separate study). To reduce experimental error due to personnel, all routine animal care and experimental procedures were performed by the same 2 persons, with care taken to ensure that both used the same techniques.

Figure 1.

Schedule of experimental treatments.

All procedures were approved by the Wayne State University IACUC.

Collection of radiotelemetry data.

Output from the telemetry transmitters was collected by using hardware and software from Data Sciences International (St Paul, MN). Data were sampled for 10 s at 1- or 5-min intervals after the various acute procedures and during undisturbed periods, respectively. These data were saved to the hard drive of a desktop computer as spreadsheet files by using Excel (Microsoft Corporation, Redmond, WA), and these files subsequently were transferred to other computers for summarization and statistical analyses. To simplify the Results section, only heart rate, SBP, and (in some cases) activity (movement in the cage) are shown.

Statistical analysis.

The telemetry data collected at 5-min intervals during undisturbed periods were averaged across each respective time period (0800 to 0900, 1300 to 1900, and 1900 to 0700) for each of the 6 rats in the control and enriched groups for each strain; the mean ± SEM for that time period was calculated from these 6 values. Telemetry data collected during the 3 undisturbed periods were analyzed separately because time of day or lights-on or lights-off periods were thought to affect heart rate, SBP, and activity.

The heart rate and SBP responses to the various acute procedures are expressed as the mean ± SEM of the area under the response curve values obtained from the 6 rats in control and enriched groups within each strain. These values were computed for each rat as the sum of the changes in heart rate (or SBP) from the mean for the 0800 to 0900 period beginning at the onset of the procedure to the point when the response returned to the mean value for that period. Data from control and enriched groups were compared within each strain by using one-factor ANOVA followed by Tukey posthoc test by using SigmaStat statistical software (Systat Corporation, Point Richmond, CA). Differences between control and enriched groups were declared statistically significant at a P value of 0.05 or less.

Results

Cage enrichment did not significantly alter undisturbed heart rates or systolic blood pressures in either female Sprague–Dawley rats or SHR at any time during the day or night. However, activity of SHR females was significantly (P < 0.05) greater under enriched conditions in the afternoon and at night than was that of nonenriched controls (Figure 2).

Figure 2.

Heart rate, systolic blood pressure, and activity in the morning (0800 to 0900; panels A and B), afternoon (1300 to 1900; panels C, D, and E), or night (1900 to 0700; panels F, G, and H) in undisturbed female Sprague–Dawley rats (SD) and spontaneously hypertensive rats (SHR) housed under enriched or nonenriched conditions. Values are mean ± SEM. *, Significant (P < 0.05, one-way ANOVA and Tukey multiple-comparison test; n = 6) difference between enriched and nonenriched groups.

Heart rate responses to the acute manipulations were not significantly reduced by the enrichment program in either strain (Figure 3). In contrast, the responses to some procedures were significantly (P ≤ 0.05) increased by cage enrichment (Sprague–Dawley: 60-min restraint and handling; SHR: subcutaneous injection, tail vein injection and handling).

Figure 3.

Heart rate responses after experimental manipulation of female Sprague–Dawley rats (SD) and spontaneously hypertensive rats (SHR) housed under enriched or nonenriched conditions. Values are mean ± SEM of areas under the response curve. *, Significant (P < 0.05, one-way ANOVA and Tukey multiple-comparison test; n = 6) difference between enriched and nonenriched groups. U&F, urine and feces.

Similarly, SBP responses to the acute procedures were not significantly reduced by cage enrichment in either strain. In Sprague–Dawley female rats, enrichment significantly (P ≤ 0.05) increased SBP responses to subcutaneous injection and removal of a familiar cagemate, whereas SBP responses of female SHR to any of the acute manipulations did not differ after cage enrichment (Figure 4).

Figure 4.

Systolic blood pressure responses after experimental manipulation of female Sprague–Dawley rats (SD) and spontaneously hypertensive rats (SHR) housed under enriched or nonenriched conditions. Values are mean ± SEM of areas under the response curve. *, Significant (P < 0.05, one-way ANOVA and Tukey multiple-comparison test; n = 6) difference between enriched and nonenriched groups. U&F, urine and feces.

Discussion

The present results show that a nonsocial cage enrichment program had no significant effect on heart rate or SBP in undisturbed female Sprague–Dawley rats or SHR and that this program either did not change or significantly increased heart rate and SBP responses to acute procedures. These results do not support the working hypothesis and differ from the previously reported effects of this same enrichment program on male rats of the same 2 strains. In male rats, cage enrichment decreased the heart rates of undisturbed male SHR during the day and at night and decreased heart rate and SBP responses to several acute procedures in both strains.²⁶

The different response of female rats to cage enrichment in the current study as compared with that of male rats of the same strains in our previous study²⁶ agrees with our earlier observations that female Sprague–Dawley rats exposed to social enrichment (group housing) responded less to some experimental manipulations²⁷ than did male rats.²⁸ In this regard, other authors have reported that female rats showed a lower glucocorticoid response to crowded housing conditions than did male rats.⁸ In addition, enrichment had a greater effect on the locomotor activity of male rats than that of female rats.¹⁰ Furthermore, environmental enrichment enhanced spacial memory performance after traumatic brain injury in male but not female rats.³⁰ In our current results, females housed under enriched conditions showed increased responses to some of the experimental manipulations (for example, restraint and handling in Sprague–Dawley female rats and tail vein injection and handling in female SHR), indicating significant effects of cage enrichment but not in the hypothesized direction.

The mechanisms that underlie the greater responses of female rats are unknown, but we speculate that this sex-associated difference is due in part to gonadal hormones. Resolution of this particular issue depends on future studies using gonadectomized rats and the assessment of other physiologic parameters in male compared with female rats housed in enriched and nonenriched conditions. At present, these male–female differences complicate the discussion of recommending or requiring environmental enrichment for all rats.

We recognize that some studies support for the use of cage enrichment for singly housed rats (for example, studies of neural trauma and cognitive function).^{5,7,10,11,13,17,18,21-25,30,31} However, our current observations do not support nonsocial cage enrichment as a means to reduce stress in female rats. We did not evaluate whether this type of nonsocial enrichment affects parameters such as growth, immune competence, reproductive function, and normal species-specific behaviors. Although some recent studies show that environmental enrichment improves indices of wellbeing,^1,4 more studies are needed to resolve these questions. More importantly, the specifics of environmental enrichment must be considered carefully. As noted in a recent review,²⁹ enrichment can have many components, and multiple protocols have been used with variable effects. Cage enrichment is a subset of environmental enrichment. What happens outside of the cage (for example, lighting intensity and schedule,³ temperature, natural² and man-made sounds) may be as important as what happens inside the cage. Cage enrichment can incorporate social (group housing) or nonsocial (physical) schemes or both at the same time. Social enrichment can be provided by the addition of one or many cagemates, but doing so can add complications by the decreasing amount of cage space each rat occupies and by a possible crowding effect that can be stressful.⁸ Nonsocial enrichment can include exercise^12,14,15,19 (for example, running wheels), but voluntary exercise produces many physiologic changes that could confound studies in which it is used as an enrichment. Nonsocial enrichment can include objects that promote several different normal rat behaviors (for example, hiding, nesting, food gathering, gnawing, and chewing), but using these objects introduces questions about which ones should be used and which behaviors should be promoted. Therefore, at present, identifying a method of cage enrichment that is accepted as practical and valid from both scientific and animal welfare perspectives is difficult.

Finally, a potential unintended consequence of cage enrichment is that physiologic data from studies on animals housed under enriched conditions may not be comparable to information collected from animals housed under nonenriched conditions, due to confounding by housing method. This issue is especially important because many physiologic parameters have not yet been evaluated under enriched compared with nonenriched housing. Therefore, until these studies are done, care must be exercised to ensure that Methods sections of publications detail housing procedures, including whether any enrichment of the cage or housing area was included. Although a more detailed account of housing methods would be helpful regarding interpretation of data, a more direct approach would be to include appropriate control groups housed in enriched and nonenriched conditions. Perhaps the traditional rat models used as normal controls (that is, nonenriched, sedentary, and ad libitum fed) are inappropriate and need to be reevaluated.