Prototype Device for Computerized Blood Sampling and Data ...

Prototype Device for Computerized Blood Sampling and Data Collection in Freely Moving Swine J Hampsch1, S Peters1, D Mann1, R Guinn1, DL Matthews2, C Kissinger1, JN Marchant-Forde3, R Poletto3 1Bioanalytical Systems Inc., West Lafayette, IN 2Purdue University, West Lafayette, IN 3USDA-ARS, West Lafayette, IN

Abstract Collecting biofluid samples or physiological and behavioral data from animals presents challenges from excessive human intervention, and the stress of manual sampling. Our objective was to construct a device capable of protecting external leads and tubing used to facilitate automated sampling, dosing, and collection of physiological and behavioral data in freely moving swine. We constructed an octagonal, 1.2 m diameter pen with solid walls, plastic-coated perforated floor, selfcontained feed trough and in-built water supply. The most important feature built into the pen was its clockwise and counter-clockwise rotational capabilities. A pig fitted with a harness was connected via an umbilicus (a stiff, tightly coiled 1 m spring) to a sensor array above the center of the pen which detects twisting of the umbilicus past pre-established settings, causing the pen to rotate counter to the pig movement. The pig moves relative to the pen but remains stationary relative to the exterior space. A computerized instrument module for automated blood sampling was utilized to test both the pen platform and the ability to draw blood while keeping the sampling tubing untwisted and open. Instrument modules for preserving the blood samples, dosing, collecting physiological and behavioral data could be added also. Twelve pigs (Sus domesitica) including 4 Gottingen minipigs and 8 young conventional pigs (15-30 kg) were surgically implanted with jugular catheters. Catheters were implanted in the right external jugular vein with the tip in the anterior vena cava. The catheter was tunneled from the dorsal mid-neck region to the exposed vein, secured by sutures, vein ligated cranial to insertion. The pig was fitted with a harness, 2 to 4 days later installed in the device and tested for periods of up to 17 days, with 130 or more blood samples (1 - 2 mL) drawn without further handling of the animals. We observed no twisted or blocked tubing due to failure or malfunction of our device. Plasmas from blood samples were clear and non-hemolyized. We were able to protect tubing connected to a freely moving pig over an extended period of time, allowing for multiple, high-quality, automated blood samples to be taken without human intervention or physical manipulation of the pigs.

Introduction In many ways, the pig is a mirror of both metabolism and metabolic disease in man. The structural similarity of the heart and cardiovascular system, the liver and the kidney, make the pig a valuable screen for drug adsorption, distribution, metabolism, and elimination. However the usefulness of the pig to researchers in biomedical research has been limited by difficulties in handling and sampling. We have attempted to overcome these difficulties by developing an instrument that would allow convenient, simultaneous biofluid sampling and collection of physiological data in an awake, freely moving pig. Sampling and data collection could occur 24/7 and over a period of weeks, not just a few hours. Our objective was to construct a device capable of protecting external leads and tubing used to facilitate automated sampling, dosing, and collection of physiological and behavioral data in freely moving swine.

Materials and Methods We designed and constructed a prototype device with a 1.2 m diameter animal enclosure in an octagonal shape. The floor was plastic coated perforated steel and the side walls were solid, clear plastic with aluminum supports (Fig. 1, Fig. 2). A stationary waste basin under the floor captured and directed urine to an external collection point. A feeding and watering area were built into a wall panel to allow easy access and a functional watering system without external connections (Fig 3). The access door to the animal enclosure consisted of 2 sidewall panels that were hung on heavy duty hinges. When the

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access door was locked in the open position, it provided a secondary function by forming an enclosed area to restrict animal movement for manipulation or dosing (Fig 4, Fig. 5). The upper panels on the walls were removable to allow easier access to the pigs (Fig. 6). The animal enclosure was supported by a central bearing assembly that allowed it to rotate in either direction. A drive system consisting of an electric motor with gear reduction, a belt drive, and an adjustable speed motor controller allowed external control of clockwise and counterclockwise rotation of the enclosure (Fig. 7). The feedback control of the drive system consisted of a tether connecting the pig (via a harness) to an optical sensor array suspended above the enclosure. The sensor array converted the pig movement outside an allowed 270° arc into a signal that triggered the drive system to rotate the enclosure in the opposite direction of the pig movement (Fig. 8). This allowed the

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pig to remain relatively constant in relation to the room while allowing movement in the pen. Once the pig stopped moving, the drive system was triggered to stop rotating the enclosure. Acceleration and deceleration of enclosure rotation was controlled to occur in a gradual manner. This system also enabled automatic recording of time, direction, and duration of animal movement. The harness was an adjustable H design with additional strapping to control movement or slippage while on the pig (Fig. 9). A computer controlled, automated blood sampling system and an automated, refrigerated storage system for the blood samples was added to test the ability of the system to prevent twisting of the tubing or leads from a pig in the device during actual use.(Fig.10, Fig. 11, Fig. 12) Twelve pigs (4 Gottingen min-pigs and 8 young conventional pigs, all 15-30 kg) were surgically implanted with catheters in the right external jugular vein with the catheter tip in the vena cava. Briefly, the ventral and dorsal aspects to the neck were clipped and scrubbed in preparation for surgical implantation. The catheter was tunneled from a mid-neck dorsal incision to the ventral incision (the jugular vein exposed). The catheter tip was inserted into the vein, secured by sutures and ligated cranial to the insertion point. The pigs were allowed to recover for 2-4 days and then moved to the prototype device.

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Multiple blood samples (1-2 mL, EDTA) were obtained at programmed time points using the automated blood sampler while a subject was installed in the device for periods lasting up to 17 days. The blood samples were usually retrieved from the refrigerated collector once per day. Some of the samples were analyzed for cortisol and norepinephrine levels. Additionally, 2 larger conventional pigs (44 kg) were fitted with harnesses to determine the ability of the prototype device to handle larger animals. No surgery was required for these pigs.

Results To date the device has operated over 200 days with pigs connected without a major problem. Individual pigs have been connected continuously for up to 17 days without a device related problem. Pigs weighing up to 45 kg were accommodated with no adverse consequences. The optimum speed of rotation was determined to be approximately 7 rpm. To minimize animal imbalance, the acceleration was adjusted to require 4.5 seconds to ramp up to full speed or ramp down to a complete stop. In the course of testing the device and other experimentation, a combined total of more than 130 blood samples were taken from pigs with the automated blood sampling device. The separated plasmas were clear and of high quality. The tubing attached to the pig remained unkinked, untangled and usable during the time in prototype device. Some of the plasma samples obtained from pigs in the prototype device were analyzed for cortisol or norepinephrine (Fig. 13, Fig. 14, Fig. 15). Cortisol and norepinephrine concentrations were normal but higher immediately following connection to the prototype device due to the stress movement from the home pen, but plasma concentrations returned to normal within 2 – 4 hr after they were installed in the device. Both cortisol and norepinephrine concentrations were relatively constant and in the low normal range during the pig’s time in the device. Cortisol Concentrations With Automated Blood Sampling

Norepinephrine Concentrations with Automated Blood Sampling

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Discussion The intent of development of this device is to create a system that will allow pigs to be conveniently used in research. We see the device as a platform that allows for a wide range of biofluid collections (blood, plasma, serum, microdialysis and ultrafiltration collections, urine and feces) and gathering physiological data ( BP, temperature, ECG, heart rate, etc) over extended periods of time in animals that can move, eat, and respond to their environment. Other systems currently available can collect either blood samples or physiological data but generally not both at the same time. In addition to the automated blood sampler and collector used, other useful device modules are under development for use in pigs. Target device modules include automated dosing, a biometrics module for ECG, heart rate, BP and body temperature, ultrafiltration and microdialysis (brain and other tissues). The prototype device performed very well during this period and will form a basis for improvements for the next generation of the device. Improvements that are under revision include enhanced floor support, enhanced corrosion resistance, improvements to the motor control system and re-design of the instrument cabinetry. Overall the objectives of the prototype were met: it was completely functional, it was easy to use, the pigs were not stressed during its use and it allowed sample collection 24/7 for several weeks. This work was supported by Indiana 21st Century Research and Technology Fund Grant #118 and Grant Number R44RR022489 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.