ILAR Journal Online, Volume 43(3) 2002: Advanced Physiological Monitoring in Rodents

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ILAR Journal V43(3) 2002
Advanced Physiological Monitoring in Rodents

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Vascular Infusion

Methods in Vascular Infusion Biotechnology in Research with Rodents
Thomas E. Nolan and Hilton J. Klein

Thomas E. Nolan, D.V.M., is Vice President of Instech Solomon, Plymouth Meeting, Pennsylvania. Hilton J. Klein, V.M.D., M.S., is Senior Director of Comparative Medicine in the Department of Laboratory Animal Resources at Merck Research Laboratories, West Point, Pennsylvania.

Abstract

Infusion of experimental compounds into the vascular system of rodents and the need to collect blood and other biological fluids from small animals comprise an area of emerging importance to biomedical research and drug discovery and development. The advances in the development of transgenic rodents coupled with technical progress in the manufacture and commercial availability of various catheters, swivels, tethers, infusion pumps, and sample collection systems that are described have enabled biomedical scientists to miniaturize vascular infusion and sample collection systems previously used in animal species larger than the rat or mouse. Use of these advanced, miniature vascular infusion systems in rodents is possible only when careful planning of experimental design, expert surgical technique, adequate postoperative care, and fundamental animal welfare considerations are meticulously taken into consideration. Use of these vascular infusion systems in rodents promotes animal welfare and scientific progress through the reduction and refinement of animal models.

Key Words: animal alternatives; catheters; infusion pumps; miniaturization; rodents; surgical implants; tethers; vascular infusion

Animal Welfare and Study Design

Various vascular infusion and intravascular delivery systems have been used in rodents with varying degrees of success in biomedical research for many years (Fox and Beazley). Although these systems have been used successfully in larger animal species such as dogs, rabbits, and nonhuman primates, the same level of success has not been achieved with rodent species such as the rat and mouse. Because rodents are the most widely used animal species in biomedical research, it is reasonable to expect that the need for improving the methods of vascular infusion and delivery systems in rodents will continue especially as the use of transgenic mice and other rodent models increases in the future. Basic research in fields such as neuroscience, physiology, pharmacology, virology, immunology, oncology use large numbers of rodents (Cunliffe-Beamer 1993; NABR 1999) to assess the effects of biological and pharmacological active agents. Many of these studies (e.g., pharmacokinetic and/or pharmacodynamic studies) rely on vascular infusion technology to derive samples for the assessment of activity, biodistribution, and plasma duration.

Planning Considerations

The use of miniature vascular infusion delivery systems, automated pumps, swivels, and tethers, coupled with advances in aseptic surgical techniques, anesthesia, and perioperative care in rodents, has led to their more widespread use in research. These biotechnological advances contribute to the reduction in the use of dogs, rabbits, and nonhuman primates in biomedical research (USDA Annual Welfare Report 1999). The principles of reduction, refinement, and replacement (the "3Rs" of Russell and Burch [1959]) are further achieved based on these rodent vascular infusion technologies. The rapid advances of transgenic and genetically modified rodents using microsurgery further advance vascular infusion and delivery systems (Cunliffe-Beamer 1993).

Experimental design considerations are important planning aspects for the research team to ensure success with rodent vascular infusion studies. Before initiating an experimental study that involves vascular infusion or vascular delivery techniques in rats or mice, it is necessary for the principal investigator, the attending veterinarian, and other members of the institutional animal care and use committee to take into account several important considerations (NRC 1996; PHS 2000; Prentice and Oki 2000). All team members--and especially those involved in perioperative care--should have clearly delineated roles and responsibilities (Brown 1993). The research teams that design the experimental studies focus on the surgical plan should include pre- and postoperative care. Postsurgical care is an often overlooked and underemphasized aspect of ensuring the successful outcome of any aseptic rodent surgery and vascular infusion protocol (Cunliffe-Beamer 1993; Gentry and French 1994).

Investigators using rodents must resist the temptation to compensate for inadequate aseptic surgical or catheter maintenance techniques by increasing animal numbers to achieve statistical significance in their study. However, improper aseptic surgical techniques may invalidate a study. Serious adverse consequences and complications regarding the physiology, metabolism, and behavior of rodents with postoperative infections have been well documented (Bradfield et al. 1992; Cunliffe-Beamer 1993; Fox and Beazley 1975; Gentry and French 1994). Complications that have been reported for both mice and rats include cellulitis, phlebitis, ocular vascular lesions, amyloid deposits, thrombosis, and histopathology of major vessels and organs (Bradfield et al. 1992; Fox and Beazley 1975; Francis et al. 1992). Because these complications may cause pain and distress, we recommend using postsurgical analgesics.

Incorporating the information described above in the research protocol both assists the institutional animal care and use committee review and approval process and fulfills the recommended practices in the Guide (NRC 1996) and Public Health Service policy (PHS 2000). Alleviation of pain and distress reduces potential problems associated with alteration of physiology, metabolism, and behavior. Minimizing these variations increases reproducibility from animal to animal, reduces animal use, and is ethically justified for animal welfare and for improving study validity.

Technical Considerations

The vascular sites commonly used for catheter placement are the vena cava, tail vein, and femoral vein (Hagnuller et al. 1992). In the mouse, because of its small size (25-30 g), the jugular vein and vena cava provide relative ease of access (Horii 2000; VanWijk 2000). It is important to give serious consideration to the type of infusion vehicle. Certain vehicles (e.g., polyethylene glycol) used to solubilize and deliver drugs may have adverse side effects. These side effects may be minimized, but not avoided, by altering volumes and rates of vehicle infusion. For example, polyethylene glycol at concentrations ≥20% w/v may cause noisy inspiration, abnormal behavior such as altered grooming, depressed activity, and somnolence (Healing 2000). Other vehicles (e.g., Cremophor EL, which is a nonionic surfactant containing vehicle used in drug formulation) may cause adverse side effects in humans and animals (Gelderblom et al. 2001). Adverse effects include piloerection, hypoactivity, recumbency, and/or altered breathing and are usually dose dependent (L. B. Weekley, Merck Research Laboratories, West Point, Pennsylvania, personal communication, 2001).

Other important variables are the infusion volume and infusion rate. Improper selection of these variables may cause volume overload in the animals and may upset normal physiological homeostasis. With the rat as a model, infusion rates of 2 to 4 mL/kg/hr are commonly used without difficulty. Infusion rates as low as 0.1 to 0.5 mL/kg/hr have been used, but limitations in pump technology make these lower limits more variable and increase the likelihood of thrombotic occlusions of catheter systems (Healing 2000). Alternatively, dosing is best achieved using allometric scaling methods, especially in rodents because of their small body mass (Henness 1977).

Infusion System Components

The components of a rodent infusion system depend on the goals of the study. Primary factors include study length, study end point, access site, need for blood withdrawal, and periodic versus continuous access. For purposes of this discussion, the term acute is defined as those studies lasting up to 1 day in duration, and chronic is defined as those studies lasting longer than 1 day, during which time the animal is maintained in a normal physiological state. The need for chronic access invokes a different set of component requirements compared with an acute study.

Catheter/Cannula Composition and Physical Design

Significant advances in catheter technology have taken place in biomedical research. The term catheter denotes a flexible tubular device to infuse fluids into or collect biological specimens from vessels, hollow organs, or body cavities. A cannula is a rigid device placed into a hallow organ or body cavity. The rigidity of cannulas makes them unsuitable for vessels. However, a catheter allows for multiple improved modifications compared with a cannula. For example, a catheter might be supplied with specific tip geometry, specific predetermined length, and retention beads for stabilization in a vessel. Catheters are available in various composition states referred to as a biomaterial, which includes polyvinyl chloride ("PVC," Tygon), polyethylene ("PE"), silicone ("silastic"), polyurethane, polyamide, and polyethylethylketone ("PEEK").

The biomaterial of a catheter is important because it affects biocompatibility with tissue, adsorption, and absorption of test compounds, gas permeability, and sterilization methods. The durometer (hardness/flexibility) of a catheter has implications with regard to ease of handling by the surgeon and trauma to the vessel being implanted. Three types of catheter tip geometry (radius, straight, and bevel) are shown in Figure 1 and demonstrate various effects on the vessel lining (O'Farrell 1995).

Figure 1 Catheter tip geometry, illustrating the various geometric configurations commonly used in rodent catheters. Top to bottom: radius, straight, bevel.

A comparison of catheter material characteristics pertinent to the use of catheters in rodents appears in Table 1. Attempts at improving catheter thrombo-resistance have been made through the use of coatings on the surface of the catheter. Coatings have included TDMAC Heparin (STS Biopolymers Device Coatings, Henrietta, New York), Hydrocoat (Access Technologies, Skokie, Illinois), and, more recently, Carmeda Bio-Active Surface (CBAS¹) heparin coating (Instech Solomon, Plymouth Meeting, Pennsylvania). TDMAC was an early attempt at applying a heparin coating to the surface of catheters, but it failed to produce a long-term beneficial effect (J. R. Gehret, Merck Research Laboratories, unpublished observations.) Hydrocoat becomes very lubricous when wet, and it aids in inserting catheters that often must negotiate around several sharp turns and overcome frictional resistance. CBAS heparin coating has been shown in numerous human studies to improve resistance to thrombin formation and reduce the incidence of infection at the catheter site² (Appelgren et al. 1996; Arnander et al. 1987). Similar work in rats has revealed a significant increase in catheter patency with CBAS heparin coating compared with uncoated polyurethane catheters (P. Foley, University of Virginia, personal communication, 2001).

Catheter Access

The effectiveness of a catheter is directly linked to its ease of access to the vessel or nonvascular sites to collect blood, lymph, urine, synovial, gastric, intestinal, or cerebral spinal fluids. All target sites have the common catheter requirement for access to infuse or collect fluids. An important technology refinement is the use of implantable pumps, which allow the catheter and delivery system surgically placed within the body to provide a constant slow infusion. For other situations, access to the catheter is by externalizing the proximal end of the catheter or by surgically implanting vascular access ports to allow percutaneous catheter access. Externalized catheters have the disadvantage of possible local or systemic infections because they disrupt the epidermal integrity. External catheters may also elicit local inflammatory reactions such as fibrous tracts and granulomas. Elimination of these infections and inflammatory reactions usually involves removal of the catheter and rigorous antimicrobial therapy. Externalized catheters, which have the advantage of chronic tethered infusion systems, are discussed below.

With subcutaneous access ports, also known as vascular access ports (VAPs¹), it is possible to avoid catheter tract infections. The placement of a VAP between the fascia layer and the skin is shown in Figure 2. The VAP is composed of a chamber that communicates with the attached catheter through a hollow tube. The chamber is overlaid by a tightly packed silicone septum that, for access, is penetrated by a special Huber needle. The Huber needle shown in Figure 3 is designed to reduce coring of the skin and the silicone septum. Vascular access ports of good design may be used several hundred times before failure. Dalton (1985) has described pioneering work with vascular access ports.

Figure 2 Vascular access port (VAP) scheme. The design principle of the VAP system is shown. Huber needle is shown in position seated in the port implanted subcutaneously.

Figure 3 Huber needle. The needle tip design minimizes removal of plugs from tissue or ports minimizing contamination or port damage.

Even though the use of a port eliminates the permanent catheter exit point, careful attention to aseptic procedure is of utmost importance when accessing the port. VAPs are most useful for periodic infusion and sampling, although some investigators will use ports through a tethered arrangement for infusion of several hours. Ports also have the advantage of allowing multiple housing of rodents (vs. the single housing required with tethered animals). Ports of various design and composition are available from several sources.

Tethered Infusion Systems

Tethering of the test animal is done when long-term access to a catheter is required in a freely moving rodent. Early pioneers in the area of tethered infusion systems incorporated various "home-brew" systems (Guillery and Chodak 1984; Hagnuller et al. 1992; Hodge and Shalev 1992; Patijn et al. 1998; Paul and Chandrakant 1975; Yano and Yazawa 1979). A key element of any tether system is the swivel that allows for rotational movement of the tethered animal. In the late 1960s, Michael Loughnane, a biomedical engineer at Temple University, began to design and build swivels to meet the needs of research investigators for tethered infusion in rats. His continued efforts in this specialty area led to the commercialization of the swivel and many other well-engineered components and systems for tethered infusion, which are available from Instech Solomon.

Tethered infusion systems typically include a subcutaneous button, jacket or harness, tail cuff, or a head block apparatus placed near the catheter exit site. This restraint part of the system connects to a spring tether, which is attached to a swivel mounted to the animal's cage. Button tethers and head blocks require surgical placement and are used for long-term studies. They are fabricated of stainless steel, plastic, Dacron mesh, or silicone and are surgically implanted directly beneath the skin of the animal. Fixation of head blocks requires the use of dental acrylic, which attaches the device to the skull bones.

Jackets and harnesses require no surgical intervention and are commercially available in a number of compositions and sizes (e.g., Lomir Biomedical, Inc., Malone, New York; Kent Scientific Corporation, Litchfield, Connecticut; Alice King Chatham Medical Arts, Hawthorne, California). Jackets are reusable and are made of cloth or nylon in a vest-like conformation, with a reinforced area over the catheter exit site that attaches to the spring tether. Because of their cloth construction, jackets are prone to soiling and must be washed or replaced periodically. Harnesses have been used more recently as alternatives to jackets.

The Covance Harness (designed by a research technician at Covance Laboratories in Vienna, Virginia) is constructed of a molded elastomer saddle, with attached silicone bands that form a sling around the animal's body. They are commercially available for both rats and mice, are designed to be disposable, and do not require periodic cleaning (Instech Solomon). They present a much smaller contact area to the rodent's skin and are not as likely to interfere with thermoregulation. The saddle serves as the attachment point for the spring tether and as a protective covering for the catheter exit site.

Tail cuffs are constructed of stainless steel sutured around the coccygeal vertebrae, with stainless steel wire providing an attachment for the tether spring or other devices. The tail cuff system is less desirable than the other tethered devices because of potential surgical complications and tail restraint restrictions.

Selection of Attachment Devices

It is important to consider the following aspects of available products when selecting attachment devices: study duration, animal comfort and confinement, effect on animal temperature, cleanability and reusability, ease of use and surgical intervention requirement, protection of catheter from damage by animal and environment, and economic factors. Two common types of devices are spring tethers and swivels.

Spring Tethers

Spring tethers are designed to connect the animal with the swivel, provide passage and protection for the catheter(s), and provide for rotational torque from the animal to the swivel. Springs are available in various sizes depending on the number of catheters required to pass through them. Tether springs are generally 12 in long and are available in various other lengths that conform to cage size.

Swivels

Swivels are constructed of plastic, Teflon, or stainless steel and are connected to a spring tether that allows rotational movement for the animal. Swivels are available in single or multiple channel varieties, and the dual-channel swivels provide the most practical load limit. Three-channel swivels are impractical because of physical limitations in designing a swivel that will seal properly yet still have rotational torque low enough to be turned by the animal. A swivel is suspended approximately 12 in above the animal, and the mounting devices should be appropriate to the rodent's size and enclosure construction. Key factors with regard to the selection and use of swivels are rotational friction; seals and leakage; inlet and outlet tube gauge; dead volume; cleanability and sterility; reusable versus disposable characteristic; and mounting hardware. These factors are discussed briefly below.

Rotational friction. There is a direct relation between the number of channels and the frictional torque necessary to turn them. Frictional torque, which is measured in ounce inches (oz/in), varies with different swivel designs. Mice require a swivel with less than approximately 0.020 oz/in of torque for them to turn. Rats accommodate to swivels having up to approximately 0.300 oz/in of torque.

Seals and leakage. The quality of design of the seals in a swivel is the key factor to avoid leakage. The seals also have a direct effect on the rotational torque rating of a swivel.

Inlet and outlet tube gauge. Swivels for use in rodents range from 20 to 25 gauge, with internal bore diameters of 0.006 to 0.023 in. The gauge size is important for matching catheter sizes, and bore diameter affects the infusion volume and rate, pulse wave forms for pressure measurements, and potential for clot formation. Larger bore diameters tend to produce better pulse waveforms than smaller ones.

Dead volume. Dead volume (the space within the internal dimension of the tubes and associated components within a swivel) is normally not an issue with drug infusion and blood collection. However, dead volume does become important for very small-volume systems, such as in microdialysis studies.

Cleanability and sterility. Swivels must be cleaned after each use to prevent precipitation of test compound leading to obstruction of channels and eventual damage to the seals. Maintenance procedures involve flushing with an isopropyl alcohol solution followed by drying with room air through the channels of the swivel. Stainless steel and Teflon swivels can be sterilized by autoclaving, whereas plastic swivels require ethylene oxide techniques. Hydrogen peroxide gas plasma sterilization systems are not suitable for sterilization of these devices because the sterilizing gas does not reach the inside of tubing.

Reusable versus disposable. Stainless steel swivels are designed for long-term repeated use. Plastic disposable swivels are typically less durable and are meant for disposal after a single use. Thus, plastic swivels are useful in preventing cross-contamination between studies and when test articles must be discarded (e.g., in radioisotope studies).

Swivel and tether mounting hardware. The method for attachment of the swivel and spring tether assembly to the animal's primary enclosure depends on the weight of the system relative to the animal's size and the tension that may be transmitted to the assembly by the animal. For example, the weight of the assembly that is applied to a mouse must be carefully controlled so the animal is not burdened in its ability to move freely about the enclosure. The weight of the tether assembly on the animal can be easily alleviated by the use of a properly designed counterbalance system. The counterbalance is adjusted so that the arm of the device, rather than the animal, carries most of the weight. Counterbalance devices are required with rodents, but not with large laboratory animal species. An example of this type of system appears in Figure 4. The use of tether systems in larger species (e.g., dogs and nonhuman primates) requires durable mounting systems to prevent damage to them. Specifically, strain relief devices are required in these systems to prevent damage to the swivels from the strain the attached tether places on the animal.

Figure 4 Schematic of tethered mouse with syringe pump shows general design of pump-tether system. Note swivel and lever arm assembly permitting free movement of mouse.

Nonswivel Tethering Systems

There are currently two commercial systems available for tethering rodents for fluid and/or electrical signal access that function without the use of swivels (Swivelless Swivel, from Instech Solomon; and the Rat Turn, from Bio Analytical Systems, West Lafayette, Indiana). These systems rely on sensing the animal's movement and responding with either movement of the enclosure or of the tubing/wire conveyance mechanism surrounding the enclosure. The instruments are used primarily for microdialysis studies.

Infusion Pump and Collection Systems

As soon as an animal is prepared for infusion or sample withdrawal, infusion and withdrawal procedures can be accomplished using manual or automated methods. Manual infusion is performed with syringes attached to the catheters or pumps, which in turn can be attached to provide unattended infusion. Infusion pumps are implantable or ambulatory, and are also considered to be tethered or tetherless. Pump characteristics vary widely in terms of design types, flow rates, and accuracy. Design types include syringe, peristaltic, and piston. Syringe pumps (e.g., the Harvard Apparatus HA11 [Harvard Apparatus, Holliston, Massachusetts]) make use of a worm drive mechanism, which drives the plunger of a standard syringe at an adjustable rate. Syringe pumps tend to deliver low levels of flow (~0.01- .00 mL/hr) with an accuracy of ±2%. Peristaltic pumps (e.g., the Instech P720 model) typically deliver rates of 1 to 100 mL/hr, with an accuracy of ±5 to 8%. Piston pumps (e.g., the Instech Solomon-Pegasus pump) use a stepper motor to drive a cartridge-based piston, which in turn uses a system of valves to direct the flow. Piston pumps delivery a flow accuracy of approximately ±2%.

To determine the true accuracy of a given pump, it is important to review the trumpet curve of the pump (Figure 5). A trumpet curve uniformly compares the flow accuracy of pumps over a time period of approximately 30 hr. The issue of flow rate variability becomes critically important in studies involving a series of intermittent short infusions, especially at low-level flow rates.

Figure 5 Trumpet curve. Comparison of infusion pump with trumpet curves for accuracy determination.

Implantable Infusion Pumps

The ALZA osmotic minipump is widely available, yet is limited in volume that can be infused (Alza Corporation, Palo Alto, California). These pumps are quite useful in rodent species for low volume and constant delivery requirements. Implantable infusion pumps for use in larger laboratory animals species, including dogs and nonhuman primates, are not commonly used. One device, the Esox Pump (Advanced Neuromodulation Systems, Inc., Plano, Texas), is an elastomeric implantable pump that has been adapted from the clinical market and can be utilized in rats >250 g and in larger species. (Esox pumps are available in sizes of 1, 7, and 25 mL and cost approximately between $150 and $800 each.) Pumps for Tethered Infusion

Syringe pumps are used for tethered infusion, where the pump is stabilized and attached to a tether arrangement (Figure 4). With this method, animals can be tethered for long-term infusion for several weeks to months. Pumps for Tetherless Infusion

Tetherless infusion involves a fully ambulatory animal with fewer restrictions compared with a tethered system. Primary criteria for the design of a tetherless pump system are the overall weight of the pump and its related components. The animal must be capable of carrying the weight of the pump as well as any associated electronics and infusion materials. Pumps for tetherless infusion in laboratory animals have been adapted from clinical sources. Such pumps have been used for precision infusion of clinical medications for patients requiring treatment such as insulin, parenteral nutrition, analgesia, and chemotherapy. These pumps are quite sophisticated and offer a variety of levels of programmability, including direct keyboard input, connection to a personal computer, and remote control using radiotelemetry or infrared telemetry.

The two most popular pumps for tetherless infusion are the CADD peristaltic (SIMS-Deltec, Inc., St. Paul, Minnesota) and Pegasus micropiston (Instech Solomon) units. The Pegasus pump has the advantages of greater accuracy in delivery and smaller size and weight. Some of the characteristics of these pumps are listed in Table 2.

Sample Collection Methods

Most sample collection studies require dual catheters: one for delivery of the test article and the other for collection of blood. This procedure is accomplished in tethered rodents using dual channel swivels. Automated blood collection systems have recently become commercially available. The two systems, Culex from Bioanalytical Systems, Inc., and the Automated Blood Sampler from Instech Solomon, are operated from a computer interface that automatically collects serial samples for up to 24 hr. The Culex system is available for use in rats only. The Instech system can be used for rats and larger laboratory animal species, including dogs and nonhuman primates. Specifications for these systems are available on each company's web site. The major advantages of an automated collection system are accuracy of sample collection, reduced stress from handling of animals, and less technician time involved in collecting samples.

Summary and Conclusions

The ability to infuse compounds into the vascular system and collect blood and other samples from animals is critical to drug research. Rodent models and especially transgenic mouse models have evidenced the need for improved technologies to accomplish this objective. Devices such as the infusion swivel, catheters, implantable pumps, battery operated infusion pumps, and other instrumentation have made notable contributions to biomedical research involving infusion studies. The challenge now facing innovators in this field is to reduce the size of these devices to accommodate the ever-increasing need for studies in transgenic mouse models. Miniaturization is the goal (Goode 2002). Further advances in these biotechnologies promote scientific advances and accomplish the principles of the 3Rs--especially the ethical criteria of reduction and refinement, which constitute animal welfare.

¹Abbreviations used in this article: CBAS, Carmeda bioactive surface; VAP, vascular access port.

²A CBAS bibliography of more than 200 articles is available from Carmeda, North America, 8038 Wurzback Road, St. 360, San Antonio, Texas 78229.

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