Timecourse of recovery after surgical intraperitoneal implantation of radiotelemetry transmitters in rats

Journal of Pharmacological and Toxicological Methods 56 (2007) 218 – 222 www.elsevier.com/locate/jpharmtox

Original article

Timecourse of recovery after surgical intraperitoneal implantation of radiotelemetry transmitters in rats Adrianne N. Greene ⁎, Shannon L. Clapp, Richard H. Alper Pfizer Inc., Drug Safety Research and Development, Safety Pharmacology, Eastern Point Rd, MS 8274-1303, Groton, CT 06340, United States Received 1 December 2006; accepted 16 April 2007

Abstract Introduction: Rats are used in drug safety because they satisfy regulatory guidelines, there is a large historical database, and their cardiovascular systems respond similarly to humans. The use of radiotelemetry offers a variety of advantages over non-invasive techniques. Implantation of telemetry devices for cardiovascular assessments in rats is not novel, but the time to recover from the surgery has not been reported. In order to assess this, mean arterial blood pressure (MAP), heart rate (HR), body temperature (BT), body weight (BW), food consumption (FC), activity, and general health were determined in male Sprague–Dawley rats for 2 weeks following surgery. Methods: Radiotelemetry transmitters (TL11M2-C50-PXT, Data Sciences® International, St. Paul, MN) were implanted into the peritoneal cavity of 15 rats under isoflurane anesthesia; 11 rats were used in the study. Data were collected (500 Hz for 24 h) using PONEMAH® Physiology Platform software (Gould Instruments, Inc, Valley View, OH, version 4.0) on days 3, 8 and 15 following surgery. Body weight and 24-h food consumption were recorded 1 day prior to surgery and on days 3, 8 and 15 post-surgery; general health was recorded daily. Results: MAP tended to be greater on day 3 (104 ± 2 mmHg) than on days 8 or 15 (99 ± 2 mmHg and 99 ± 2 mmHg, respectively). By contrast, activity (arbitrary units) tended to be lower on day 3 (1.9 ± 0.2) than on days 8 or 15 (2.9± 1.2 and 3.0 ± 1.0, respectively). HR (∼460 bpm) and BT (∼37.5 °C) remained constant throughout the study. FC on day 4 postsurgery was less than pre-surgery (16.7 ± 6.6 g vs. 23.6 ± 1.2 g) and returned by day 9 (23.6 ± 5.4 g). BW on day 4 (295 ± 20 g) was not different from presurgery (297 ± 11 g) and the rats gained weight on subsequent days. There were no effects on overall appearance and behavior due to surgery. Discussion: It is concluded that male Sprague–Dawley rats recover from surgery and can be used ∼1 week following intraperitoneal implantation of radiotelemetry transmitters with monitoring of body weight and food consumption. © 2007 Elsevier Inc. All rights reserved. Keywords: Body weight; Body temperature; Food consumption; Heart rate; Hemodynamics; Mean arterial pressure; Methods; Rats; Surgical recovery; Radiotelemetry

1. Introduction Rats are used in drug safety because they satisfy regulatory guidelines for both general toxicology and safety pharmacology, there is a large historical data base for comparison, they are relatively small thus do not require large amounts of drug, and their cardiovascular systems respond to vasoactive substances similar to humans. The use of radiotelemetry offers advantages over non-invasive techniques including reduction of stress, elimination of restraint and anesthesia, and reduction of animal use (Kramer & Kinter, 2003).

Although the surgical techniques employed to implant telemetry devices in rats and other species are not novel, (Kramer & Kinter, 2003; Brockway et al., 1991; Deveney et al., 1998) a description of recovery following abdominal implantation of telemetry devices in rats has not been reported. To assess this, mean arterial blood pressure, heart rate, body temperature, body weight, food consumption, activity, and general health was monitored in male Sprague–Dawley rats during a 2-week postsurgical period. 2. Materials and methods 2.1. Animals

⁎ Corresponding author. Tel.: +1 860 686 1810; fax: +1 860 686 0433. E-mail address: [email protected] (A.N. Greene). 1056-8719/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2007.04.006

This study was conducted in accordance with the current guidelines for animal welfare (Guide for the Care and Use of

A.N. Greene et al. / Journal of Pharmacological and Toxicological Methods 56 (2007) 218–222

Laboratory Animals, 1996; Animal Welfare Act, 1966, as amended in 1970, 1976, and 1985, 9 CFR Parts 1, 2, 3). All procedures were reviewed and approved by the Pfizer IACUC. Male Sprague–Dawley rats [Crl:CD (SD)] used in this study were purchased from Charles River Breeding Laboratories (Kingston, NY). Animals were housed individually in plexiglass cages on standard bedding with food (Certified Rodent Diet 5002: PMI®) and water available at all times. The rats were acclimated in the vivarium for at least 7 days prior to surgery. A total of 15 rats underwent surgery and 11 were used in the study. An additional 5 rats that did not undergo anesthesia or surgery served as control. The rats weighed 288 ± 20 g (n = 20) prior to study initiation.

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Table 1 Body weight and food consumption (grams) prior to surgery (day ­1) and on days 3, 8, and 15 postsurgery Day of study

Body weight (surgery) a Body weight (control) b Food consumption (surgery) a Food consumption (control) b

−1

3

8

15

297 ± 11 267 ± 22 24 ± 1 23 ± 1

295 ± 20 293 ± 12 17 ± 7 ⁎ 28 ± 2

326 ± 46 ⁎ 343 ± 16 ⁎ 24 ± 5 28 ± 4

387 ± 44 ⁎ 395 ± 28 ⁎ 28 ± 4 29 ± 4

Body weight and food consumption were recorded presurgery and on days 3, 8 and 15. Values represent the mean ± SD. a n = 11. b n = 5. ⁎ Significantly different from presurgery (p ≤ 0.05).

2.2. Radiotelemetry transmitters The catheter tips of previously used transmitters (TL11M2C50-PXT Physiotel™ multiplus; Data Sciences International®, St. Paul, MN) were re-gelled and the pressure and temperature offsets were verified following instructions provided by the vendor. The telemetry implants were used to determine mean arterial blood pressure, body temperature and activity (as arbitrary units, dependent on the distance of the rat from the receiver as well as the speed of its movement as described by the vendor). Since ECG and EMG were not being assessed in these rats, the biopotential leads were removed. The transmitters were sterilized in a 2% glutaraldehyde solution for approximately 24 hours then washed and soaked in sterile 0.9% saline immediately prior to implantation. 2.3. Surgery Surgery was performed under isoflurane anesthesia using minor modifications of procedures described previously (Kramer & Kinter, 2003, Brockway et al., 1991; Deveney et al., 1998). The non-steroidal anti-inflammatory drug, carprofen (5 mg/kg, SC; Pfizer Inc, NY, NY) was administered prior to surgery to the anesthetized rats (Hawk et al., 1999). An abdominal incision (4–5 cm) was made through the skin and underlying muscle wall along the ventral midline using a scalpel and scissors. The descending aorta was carefully isolated using sterile cotton applicators and blunt dissection. An occlusion ligature (2-0 braided silk; Ethicon, Inc., Somerville, NJ) was inserted between the aorta and the vena cava to prevent backflow. Using the ligature, the aorta was elevated and punctured just cranial to the bifurcation using an appropriate size needle (21 or 23 g) bent 90° at the beveled end. The tip of the catheter was inserted under the needle as a guide and passed cranially until the entire thin-walled section was within the vessel. Placement of the catheter was verified by a rapid check of the blood pressure signal using the PONEMAH® Data Acquisition software. Once verified, the puncture site was dried, surgical glue (∼ 1–2 drops of 3 M™ Vetbond™ Tissue Adhesive, St. Paul, MN) was applied to the puncture site to secure the catheter and the tension on the occlusion suture was released. The transmitter was secured in the abdomen by

suturing the muscle wall to the suture rib on the implant using silk, a non-absorbable suture (4-0; braided silk; Ethicon, Inc., Somerville, NJ) with simple interrupted surgeon's knots. The skin was closed with 9 mm stainless steel wound clips (Reflex Skin Closure System, Gaithersburg, MD). The anesthesia was stopped and the rat was allowed to recover on a heating pad where it was monitored until it regained its toe-pinch reflex, approximately 15–20 min when it was returned to its home cage. Additional carprofen (5 mg/kg, SC) was administered the morning following surgery. 2.4. Data collection 2.4.1. Physiologic assessments Rats were weighed pre-surgery and on days 3, 8 and 15 post surgery immediately prior to data collection. At the same time, the food container was weighed and re-weighed 24 h later at the end of the data acquisition period as an estimate of food consumption. The rats were checked for morbidity, mortality, general health and appearance at least once daily prior to surgery and on the days following surgery. All animals were observed at least twice on the day of surgery. 2.4.2. Telemetric assessments The rats remained in their home cages with access to food and water throughout the data collection periods. Signals from the implanted radiotelemetry transmitters were detected by DSI PhysioTel® receivers (RPC-1; Data Sciences® International, St. Paul, MN) placed ∼ 6–8 cm directly beneath the suspended cage. To ensure that the signal from one transmitter could not be detected by adjacent receivers, the cages were positioned at least 45 cm apart as recommended by the vender. Systolic blood pressure, diastolic blood pressure, activity and body temperature were acquired for 24 h on days 3, 8 and 15 at a sampling rate of 500 Hz, logged every 20 s, stored as 60-minute averages and reduced into 24-hour intervals for each rat using PONEMAH® Physiology Platform (version 4.0, Gould Instruments, Inc, Valley View, OH) installed on a standard personal computer. The same software was used to calculate mean arterial pressure (the area under the pressure curve for a valid cardiac cycle) and heart

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Table 2 Absolute values for mean arterial pressure, heart rate, body temperature and activity on days 3, 8 and 15 following surgery Days following surgery

Mean arterial pressure (mmHg) Heart rate (bpm) Body temperature (°C) Activity (arbitrary units)

comparison of all pairs (Jmp release 5.1.1, SAS Institute, Inc., Cary, NC, USA). 3. Results

3

8

15

104 ± 2 463 ± 28 37.5 ± 0.6 1.9 ± 0.2

99 ± 2 459 ± 20 37.4 ± 0.1 2.9 ± 1.2

99 ± 2 448 ± 18 37.5 ± 0.2 3.0 ± 1.0

Mean arterial pressure, heart rate, body temperature and activity were recorded presurgery and on days 3, 8 and 15 following the abdominal implantation of radiotelemetry transmitters. Values represent the 24-hour mean ± SD as determined from 11 rats. There were no significant differences among the groups for any of the measured values ( p N 0.05).

rate (the reciprocal of the time interval for the cardiac cycle multiplied by 60). 2.4.3. Statistical analysis All data are presented as the mean ± SD (n =11) and were analyzed by 1-way ANOVA followed by Tukey–Kramer HSD for

3.1. Physiologic assessments Of the 15 rats that underwent surgery, 3 were euthanized during surgery due to excessive blood loss or transmitter failure discovered subsequent to the catheter being secured in the vessel. One additional rat died 7 days following surgery (cause of death unknown) the remaining 11 rats were used throughout the 2-week study. There appeared to be no effects on general health due to surgery. The body weight of rats that underwent surgery was not different on day 3 than from day ­1 (Table 1). All rats gained weight on subsequent days. Control rats, those that did not undergo surgery, gained weight throughout the 2-week period with statistical increases from prestudy (day ­1) noted on days 8 and 15.

Fig. 1. Mean arterial pressure and heart rate were monitored continuously for 24 h on days 3, 8 and 15 postsurgery. Data were averaged for 60-minute intervals over the entire 24-hour period. The symbols represent the average for 11 rats.

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Fig. 2. Body temperature and activity were monitored continuously for 24 hours on days 3, 8 and 15 postsurgery. Data were averaged for 60-minute intervals over the entire 24-hour period. The symbols represent the average for 11 rats.

Food consumption was less in rats on day 3 following surgery than day ­1. Food consumption returned to day ­1 presurgery values by day 8 and was similar to that in rats that did not undergo surgery. Control rats maintained steady food consumption throughout the 2-week period. 3.2. Telemetric assessments Although there was no statistical effect, 24-hour mean arterial pressure tended to be greater on day 3 than on days 8 or 15 (Table 2). By contrast, activity tended to be lower on day 3 than on day 8 or 15. Heart rate and body temperature remained constant and did not change significantly over the 2-week observation period. Presented in Figs. 1 and 2 are the same data plotted as hourly intervals. From this representation, it can be seen that there are circadian rhythms in all 4 measured parameters which appear as early as day 3. 4. Discussion Prior to establishing a long-standing colony of rats for cardiovascular assessments in safety pharmacology studies, a

question arose regarding the appropriate recovery period to allow the rats prior to their use in experiments. Thus, a study was designed to evaluate the post-surgical recovery of male Sprague–Dawley rats following intraperitoneal implantation of radiotelemetry transmitters and insertion of a chronic indwelling catheter in the abdominal aorta. The weight of rats subjected to surgery decreased slightly from day ­1 to day 3 whereas control rats tended to gain weight. In addition, food consumption was reduced approximately 30% on day 3. As food consumption increased, returning to control values by day 8, the rats were able to restore normal weight gain similar to the control rats which were not subjected to surgery. The stress induced by anesthesia and subsequent abdominal surgery could likely be the cause of the acute reduction in body weight, food consumption and/or activity as demonstrated previously (Kuntz et al., 1998; O'Neil & Kaufman, 1990). It was found that parameters such as heart rate and body temperature do not vary from first time of observation (3 days post surgery) throughout the duration of this study. Mean arterial pressure tended to be higher and activity lower on day 3 as compared to days 8 and 15 yet the 24-hour value (104 ± 2 mmHg) is well within the range reported for rats in the literature (Bohus, 1974;

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Brockway et al., 1991: Sharp & LaRegina, 1998). The lower activity is particularly noticeable during the dark phase of their light/dark period when the rats are typically eating, drinking and display greater activity. It was thought that circadian rhythms in blood pressure, heart rate, activity and body temperature might be a sensitive indicator to surgical stress. Specifically, activity, heart rate and mean arterial pressure naturally tend to be higher during the 12 h of dark as compared to the 12 h of light (Zhang et al., 2000). In this study the differences between light and dark were small in magnitude, yet as early as 3 days following surgery the normal circadian rhythms were apparent. There are advantages and disadvantages to all animal models in drug discovery and development. Some of the advantages for using rats with radiotelemetry implants in drug safety cardiovascular assessment include the ease of husbandry and handling, availability of historical data in publications for comparison and rapid recovery from surgery. Other critical issues have been reviewed in detail by Kramer & Kinter (2003). Additionally, it has been found that rats can be maintained for at least 4 months (up to the weight of 650 g) without any substantial drift in the radiotelemetry signal or change in baseline parameters and general health. Furthermore, rats are appropriate for use in safety pharmacology because they are an acceptable small animal according to regulatory guidelines (Anonymous, 2001), they respond to vasoactive substances similar to humans and they often are used to support discovery, drug safety and toxicology studies. There are some disadvantages to the use of rats in telemetry studies in safety pharmacology. As reported by De Clerck et al. (2002), the rat does not express the type and ratio of cardiac ion channels to appropriately assess QT interval as a predictor of Torsade de Pointes. When this is the primary focus of studies, the most appropriate small animal is the guinea pig for which surgical procedures have been well documented (Provan et al., 2005; Kayar et al., 1998). Similar to the report of O'Neil & Kaufman (1990), the current data suggest that if the primary end-point of a study is food intake and/or body weight, surgery should be avoided if possible. It is concluded that approximately 1 week is required for rats to recover following the surgical implantation of radiotelemetry transmitters into the abdomen. Body weight, food consumption and activity appear to be the most sensitive markers and should be monitored to assess postsurgical recovery. 5. Conflict of interest statement This work was conducted at and supported by Pfizer Inc. The authors are employed by Pfizer Inc. and there are no conflicts of interest.

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