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  Vol 96, No 3May 2009Pages 292–295 Journal of the American Association for Laboratory Animal ScienceCopyright 2009 by the American Association for Laboratory Animal Science 292 For the purposes of risk assessment and species extrapolation, it is preferable to treat laboratory animals with a compound by the route of administration that would be used in humans. For many studies, drug delivery necessitates oral administration. A drug can be administered orally by dissolving it in the drink-ing water or mixing it into the feed. Other options include oral gavage and pipetted amounts that are directly consumed by the animal. In preparation for a study of oral methylphenidate treatment in rats, we found that each of these techniques had disadvantages for the focus and endpoints of our study, prompt-ing us to search for an alternative method.The most detrimental confounder for our developmental toxicology laboratory is the potential stress associated with oral gavage. Gavage is an extremely common method for oral administration, and daily gavage was used successfully in our previous studies. 7,8  However, questions have been raised regarding the potential stress induced by this procedure 3  (but see reference 4 for quantification of corticosterone levels after gavage). Significant stress during rodent pregnancy can alter offspring behavior, endocrinology, neuroanatomy, and neurochemistry. 1,5,15,19  Finally, where multiple daily adminis-trations are necessary due to a short half-life of the drug, oral gavage becomes less practical.Dietary or water delivery of test substances may avoid the confounding variable of stress, and these routes are particularly appropriate for studies in which the substance under investi-gation is found in food (for example, as used in our studies of genistein found in soy products 9,10 ). However, those routes of administration are not feasible with drugs or substances that are unpalatable. Further, the normal fluctuations in daily intake do not result in administration of an exact dose to each study subject. In addition, the effects of the drug itself may alter those normal variations, potentially further decreasing daily doses. For many drugs, human exposure occurs in bolus, rather than prolonged or ‘timed-release’ doses. Because rodents typically eat and drink over several hours during the dark period, 17,18  dietary drug exposure then occurs over an extended period, peaking during the dark period.To minimize stress, some studies implemented pipette ad-ministration of the drug directly to the animal. 11,12,14  In that procedure, the volume is collected into a pipette and the rodent voluntarily consumes the liquid from the pipette tip as it is dispensed. Like gavage, this method allows administration of an exact dose. This method works well with individual hous-ing; however, in social housing conditions, cagemates may interfere with the administration procedure. The method can  be time-consuming and labor-intensive because it requires that the technician remain at each cage for the necessary duration for the animal to fully consume the substance.For a study of oral methylphenidate treatment in rats, our laboratory needed an administration method that was time-efficient, used few personnel, and could be easily accomplished 3 times daily. A literature search revealed studies in which the drug was placed onto a small cracker or wafer that the rat then consumed, 2,20,21  but there was little description of the technical details for this procedure. Here, we describe our system for administration of methylphenidate 3 times daily to pair-housed adolescent rats; this system led to little or no observable change in behavior. Our method is highly accurate with regard to dose administration and efficient with regard to time and personnel. Materials and Methods We used a semiautomatic precision liquid processor capable of accurate and precise transfer pipetting (Microlab 500, Ham-ilton Company, Reno, NV). Two of these systems were in place, with 1 designated for use with control solutions, and the other for use with methylphenidate solutions, thereby decreasing the potential for cross-contamination. Each system was equipped with a push-button hand-pipettor (Concorde Push-button Hand Probe, Hamilton Company, Reno, NV) and a 250-µL Hamilton syringe. Each system was interfaced with animal data collection software developed at the National Center for Toxicological Research (NCTR) and allowed us to use an automated algo-rithm to calculate the volumes of solution needed based on the daily body weight of each rat. A balance (model PM11-K, Mettler–Toledo, Columbus, OH) that was interfaced with the data-collection software was used to weigh the animals.Wafers (Mini Nilla Wafers, Nabisco, Kraft Foods, Northfield, IL) were quartered into approximately equal pieces, which were placed individually in 30-mL polypropylene cups (Ted Pella, Use of Food Wafers for Multiple Daily Oral Treatments in Young Rats Sherry A Ferguson *  and Sherin Y BoctorMany laboratory studies require oral administration of drugs. Dietary administration in food or water is useful, but is not always the best method. Orogastric gavage can be stressful. Here, we describe in detail a relatively stress-free technique that can be applied to multiple daily administrations using a palatable food item. This method was successfully used to administer water or methylphenidate 3 times daily to young pair-housed adolescent rats.Abbreviations:  NCTR, National Center for Toxicological Research; PND, postnatal day. Received: 18 Sep 2008. Revision requested: 22 Oct 2008. Accepted: 30 Oct 2008.Division of Neurotoxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas. *  Corresponding author. Email:  293 Food wafers for oral treatment in rodents were placed in the front and rear of the shelf in close proximity to the cage. The technician then tapped the cup on the top of the wire cage lid to gain the attention of the rat and emptied each wafer piece into the appropriate side of the cage without removing the cage lid. Dividers were removed approximately 5 min after each treatment time. Methylphenidate or water treatment.  Before the first treatment time (that is, 0830) each day, rats were weighed. Those data were transferred to the NCTR animal data collection software which calibrated the volume, based on body weight, necessary for each rat to receive 3 mg/kg of methylphenidate or 1 mL/kg of water at each of the 3 treatment times. The technician then placed a wafer piece into each of the 3 polypropylene cups for the first rat and, using the hand-pipettor, dispensed an identical volume onto each of the 3 pieces, 1 for each of the 3 subsequent treatment times. Each cup then was placed into the tray in the appropriate position. In the same way, 3 additional wafer pieces were treated for the second rat in that cage. Figure 3 shows the room setup and an example of a technician dispensing the volume onto the wafer pieces.Redding, CA) each labeled with the cage number and subject identification. Each of 3 aluminum trays (Figure 1) held the cups for 1 of the 3 daily treatment times; thus, there was a tray for the 0830 treatment, 1 for the 1130 treatment, and 1 for the 1430 treatment. Trays were configured for the NCTR cage-rack system, in which there are 4 shelves per rack, and each shelf contains 4 cages. Thus, each tray had 4 columns and 8 rows of cup holders. Because rats were pair-housed, the first row of 4 cups contained wafer pieces for the first rat in each of the 4 cages on the first shelf. The second row of 4 cups contained wafer pieces for the second rat in each of the 4 cages on the first shelf, and so on, until the eighth row, which contained wafer pieces for the second rat in each of the 4 cages on the last (fourth) shelf. Trays were stackable for space-saving purposes.The usual rat housing cages were modified slightly by creat-ing in each cage a slot that was slightly off center. Immediately prior to each treatment time, a clear acrylic divider (0.32 cm thick) was placed in each cage to physically separate the 2 rats in each cage (Figure 2). This divider fit tightly into the cage slot and could not be moved by the rats. Animal procedures.  All animal procedures followed the Guide  for the Care and Use of Laboratory Animals 13  and were approved in advance by the NCTR Institutional Animal Care and Use Committee. Anecdotal observations on a different group of ani-mals indicated that young rats found cheese crackers (Goldfish, Pepperidge Farm, Campbell Foods, Camden, NJ) especially palatable, but they were not particularly absorbent for the liquid drug. These observations also indicated that pieces of graham cracker (Honey Maid, Nabisco), although absorbent, seemed to  be less desirable than the pieces of vanilla wafers (Mini Nilla Wafer, Nabisco). In preliminary testing of the pipetting systems, absorbency appeared to be greatest on the back of the wafer pieces. Therefore, all further procedures used vanilla wafer pieces. The manufacturer’s nutritional information indicated that each full wafer contained 7 calories, or 1.75 calories in each quarter piece. Thus, each rat received approximately 5.25 ad-ditional daily calories from this treatment regimen. At no time were rats food-deprived; all were provided with ad libitum food [5LOH (pelleted irradiated diet), Purina Mills, St Louis, MO] and water.The 3-wk treatment period was postnatal days (PND) 29 to 50, and wafer training began with the arrival of the Sprague–Dawley rats (42 male, 42 female; derived from Crl: COBS CD BR outbred rats, Charles River, Wilmington, MA) from the NCTR Breeding Colony at weaning (PND 21). These rats were pair-housed with a same-sex, same-treatment nonsibling in standard polycarbonate cages with hardwood chip bedding in a room maintained on a 12:12-h light:dark cycle (lights, 0700 to 1900) at 22 ±  1 ° C (mean ±  SEM) and 45% to 55% humidity. On that day, several (3 to 5) wafer pieces were placed in the cage to habituate rats to the taste and smell of the wafers. Beginning on PND 22, body weights were collected each morning, and the 2 rats in each cage were separated (see below) and given untreated wafer pieces as described below for treated wafer pieces. Dur-ing these first few days, technicians observed the rats to ensure that each piece was consumed entirely. After approximately 3 d, all rats consumed pieces in their entirety within 2 to 3 min at all 3 treatment times.To physically separate the rats at each of the 3 treatment times (0830, 1130, and 1430), the technician placed a divider in each cage, ensuring that the first rat (tail tattoo 1 in each cage) was in the front half of the cage and the second rat (tail tattoo 2 in each cage) was in the rear. The appropriate tray containing wafer pieces was brought to the rack, and the cups for each cage Figure 1.  A sample tray with cups. This tray is for 1 of the 3 daily treat-ment times, and the first 2 rows contain treated wafers for the 4 cages on the first shelf (the first row contains treated wafers for rat 1 in the cage; the second row contains treated wafers for rat 2 in the cage). Both the tray and cups are labeled and color-coded for easy identification. The tray measures 45.7 ×  30.5 ×  3.2 cm, and cup holes are 3.2 cm in diameter. Figure 2.  A typical cage showing the acrylic divider (although clear when in actual use, an X was placed on the divider for photographic purposes) in place, with rat 1 in the front of the cage (near the cage card), and rat 2 in the rear of the cage. The small slot that holds the divider can be seen.  294 Vol 96, No 3 Journal of the American Association for Laboratory Animal ScienceMay 2009 This system could easily be implemented with other test substances or experimental paradigms. For individually housed rodents, the acrylic dividers would not be necessary. For mice, the wafers could be divided into smaller pieces, which likely would still be large enough to accommodate the volume of methylphenidate used here. The methodology could even be used for animals housed in wire-bottom cages, because our observations indicated that each rat held the wafer piece in the forepaws and consumed it entirely without allowing it to touch the bottom of the cage. However, our methodology may not be applicable to all test substances that require oral administration. For example, compounds with an adverse taste or odor likely will not be consumed voluntarily, but the adverse taste or odor could potentially be masked by the addition of a palatable flavor (for example, fruit concentrates) to the solution, such as the preparations added to children’s medications. This method may be inappropriate for paradigms in which a large volume must be administered (for example, due to poor solubility of the test compound), because the wafer will hold only a limited volume before becoming soft. Overall, our experience has been that animal care technicians are enthusiastic about this proce-dure, there appears to be little observable stress to the animal, and the experimenters are confident about the accuracy of the dose administered. Acknowledgments The authors acknowledge Richard Rasmussen (Bionetics Corpora-tion) for his construction of the trays and cage dividers, Carol Cain (Bionetics Corporation) for design improvement of the dividers, Clyde Ulmer and Kathy Carroll (Z-Tech) for creation of the network algorithm, and Lee McVay (Bionetics Corporation) and the incredibly talented animal care staff of the Bionetics Corporation at NCTR.This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publica-tion. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA. References  1. Archer    JE,   Blackman   DE.  1971. Prenatal psychological stress and offspring behavior in rats and mice. Dev Psychobiol 4: 193–248. 2. Arnsten   AF,   Dudley   AG.  2005. Methylphenidate improves pre-frontal cortical cognitive function through α 2 adrenoceptor and dopamine D1 receptor actions: relevance to therapeutic effects in attention deficit–hyperactivity disorder. Behav Brain Funct 1: 2. 3. Balcombe    JP,   Barnard   ND,   Sandusky   C.  2004. Laboratory routines cause animal stress. Contemp Top Lab Anim Sci 43: 42–51. 4. Brown   AP,   Dinger   N,   Levine   BS.  2000. Stress produced by gavage administration in the rat. Contemp Top Lab Anim Sci 39: 17–21. 5. Darnaudery   M,   Maccari   S.  2008. Epigenetic programming of the stress response in male and female rats by prenatal restraint stress. Brain Res Rev 57: 571–585. 6. Dobrovolsky   VN, Boctor   SY, Twaddle   NC, Doerge   DR, Bishop   ME, Manjanatha   MG, Kimoto   T, Miura   D, Heflich   RH, Ferguson   SA.  2009. Flow cytometric detection of Pig-A  mutant red blood cells using an erythroid-specific antibody: application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats. Unpublished data. 7. Ferguson   SA,   Berry   KJ.  2007. Oral Accutane (13-cis-retinoic acid) has no effects on spatial learning and memory in male and female Sprague–Dawley rats. Neurotoxicol Teratol 29: 219–227. 8. Ferguson   SA,   Cisneros   FJ,   Gough   B,   Hanig    JP,   Berry   KJ.  2005. Chronic oral treatment with 13-cis-retinoic acid (isotretinoin) or all-trans-retinoic acid does not alter depression-like behaviors in rats. Toxicol Sci 87: 451–459. Results Latency to consume the wafer was measured in subsets of rats prior to removal for blood sampling. After 1 wk (PND 22 to 28) of receiving untreated wafers 3 times daily, the average latency (mean ±  SE) to consume the methylphenidate-treated wafer piece on PND 29 was 64.9 ±  3.7 s (range, 22.6 to 127.1 s) for 36 rats and did not differ between males and females (average latency: male rats, 62.7 ±  4.3 s; female rats, 67.1 ±  6.2 s). After 21 consecu-tive days (that is, PND 50) of receiving methylphenidate-treated wafers 3 times daily, the average latency had declined to 41.2 ±  2.0 s (range, 21.4 to 70.9 s) for 35 rats (data from 1 animal were not recorded) and did not differ between males and females (male rats, 41.5 ±  2.8 s; female rats, 40.8 ±  2.9 s). These latencies were very similar to those (mean, 39.2 ±  3.8 s; range, 16.0 to 60.5 s) of 12 control rats that received water-treated wafers 3 times daily for 21 consecutive days (that is, PND 50).The metal trays and acrylic dividers were easy to clean in the cage washers, and the polypropylene cups are inexpensive enough to be disposable. The rats quickly adjusted to the thrice-daily treatment, typically orienting themselves in the correct half of the home cage before placement of the divider. The morning  body-weight measurement and preparation of the wafer pieces were the most time-intensive portions of this procedure. Dis-pensing the wafers at the 3 designated times typically took less than 15 min for as many as 32 cages.Wafers were allowed to air-dry after treatment. An analysis at NCTR indicated that methylphenidate hydrochloride was highly stable in rodent chow for as long as 14 d. 16  Therefore, the stability or concentration of the methylphenidate was unlikely to have decreased in the wafers, which remained in the cups for as long as 8 h. Serum levels of methylphenidate were well within our targeted range. 6 Discussion A pump system, metal trays with cup holders, and acrylic dividers were used to treat pair-housed rats orally with meth-ylphenidate on palatable wafers 3 times daily. Because social housing is beneficial for rats, this method of oral treatment allowed rats to be pair-housed with minimal separation each day (less than 20 min, or the minimum necessary to consume the wafers). Use of the pump system enabled us to dispense accurate volumes of the methylphenidate solution onto each wafer piece, and the trays with cup holders ensured that each subject was given the correct wafer piece. This method was time-efficient and simple to implement. Figure 3.  The room setup showing the automated pump system, com-puter, and body weight scale. A technician is using a push-button hand-pipettor to apply methylphenidate solution to each of the 3 wa-fer pieces for the 3 daily treatments for a single rat.  295 Food wafers for oral treatment in rodents 9. Ferguson   SA,   Flynn   KM,   Delclos   KB,   Newbold   RR,   Gough   BJ.  2002. Effects of lifelong dietary exposure to genistein or nonylphe-nol on amphetamine-stimulated striatal dopamine release in male and female rats. Neurotoxicol Teratol 24: 37–45. 10. Flynn   KM,   Ferguson   SA,   Delclos   KB,   Newbold   RR.  2000. Effects of genistein exposure on sexually dimorphic behaviors in rats. Toxicol Sci 55: 311–319. 11. Gioiosa   L,   Fissore   E,   Ghirardelli   G,   Parmigiani   S,   Palanza   P.  2007. Developmental exposure to low-dose estrogenic endocrine disruptors alters sex differences in exploration and emotional responses in mice. Horm Behav 52: 307–316. 12. Laviola   G,   Gioiosa   L,   Adriani   W,   Palanza   P.  2005. D-amphet-amine-related reinforcing effects are reduced in mice exposed prenatally to estrogenic endocrine disruptors. Brain Res Bull 65: 235–240. 13. National Research Council.  1996. Guide for the care and use of laboratory animals. Washington (DC): National Academy Press. 14. Palanza   PL,   Howdeshell   KL,   Parmigiani   S,   vom Saal   FS.  2002. Exposure to a low dose of bisphenol A during fetal life or in adult-hood alters maternal behavior in mice. Environ Health Perspect 110   Suppl 3: 415–422. 15. Reznikov   AG,   Nosenko   ND,   Tarasenko   LV.  1999. Prenatal stress and glucocorticoid effects on the developing gender-related brain.  J Steroid Biochem Mol Biol 69: 109–115. 16. Siitonen   P.  Stability of methylphenidate in rodent chow. Unpub-lished data. 17. Varma   M,   Chai    JK,   Meguid   MM,   Laviano   A,   Gleason    JR,   Yang   ZJ,   Blaha   V.  1999. Effect of estradiol and progesterone on daily rhythm in food intake and feeding patterns in Fischer rats. Physiol Behav 68: 99–107. 18. Varma   M,   Meguid   MM,   Hammond   WG,   Gleason    JR.  1999. Lack of influence of hysterectomy on meal size and meal number in Fischer 344 rats. Physiol Behav 66: 559–565. 19. Weinstock   M.  2001. Alterations induced by gestational stress in  brain morphology and behaviour of the offspring. Prog Neurobiol 65: 427–451. 20. Widholm    JJ,   Villareal   S,   Seegal   RF,   Schantz   SL.  2004. Spatial alternation deficits following developmental exposure to Aroclor 1254 and/or methylmercury in rats. Toxicol Sci 82: 577–589. 21. Zhu   N,   Weedon    J,   Dow-Edwards   DL.  2007. Oral methylphenidate improves spatial learning and memory in pre- and periadolescent rats. Behav Neurosci 121: 1272–1279.
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