Advancing Ethical Standards in Minipig Studies: The role of Vascular Access Buttons

Vascular Access Buttons (VAB) are increasingly becoming the preferred method for blood collection in rodent studies, offering a revolutionary approach that minimizes the need for frequent vein punctures. This technique represents a significant advancement in promoting the ethical conduct of animal research by providing a more convenient and less invasive method for intravenous drug administration and blood collection. In addition to their use in rodent studies, there is a growing interest in exploring the applicability of VAB buttons in other animal species. Charles River Laboratories Den Bosch in collaboration with Ellegaard Göttingen Minipigs A/S explored this application in Göttingen minipigs, conducting a study to compare the VAB with the traditional vena cava cranialis puncture. The study meticulously evaluated pharmacokinetic and clinical pathology parameters sampled via both methods, demonstrating their reliability and interchangeability. Furthermore, findings underscored the practicality, durability, and welfare enhancements associated with VAB employment in Göttingen minipigs, suggesting their potential as an ethical and reliable alternative for future research involving intravenous drug administration and blood collection in this species.

Vascular Access Buttons (VAB) are increasingly becoming the preferred industry standard for repeated blood collection in (extended) in vivo studies in rats and mice. Unlike with conventional vein punctures and vascular access ports (VAP), the VAB introduces a revolutionary approach featuring a self-sealing silicone membrane that eliminates the need for needle access. This sets it apart from VAPs, which relies on needle puncture of a septum-covered port and is therefore not often used as an alternative for conventional vein punctures. Consequently, the innovative VAB technology does allow animal technicians to access the vascular system without the need for frequent vein punctures, thereby minimizing stress and discomfort for the animals involved. 

By providing a more convenient and less invasive method for intravenous drug administration and blood collection, the use of VAB buttons aligns with the guiding principles of the 4 R’s—Reduce, Refine, Replace, Responsibility. For that reason, this advanced technique signifies a significant leap forward in ensuring the responsible conduct of animal research, emphasizing the ethical imperative of further exploration and investigation into potential applications of the VAB technology.

In addition to their use in rodent studies, there is growing interest in exploring the utility of VAB buttons in other animal species. In current in vivo studies involving Göttingen minipigs, stress remains a critical concern, particularly during the process of restraining, blood collection, and administering drug substances. The repetitive nature of venipunctures for blood collection, especially common in pharmacokinetic studies, exacerbates this issue and can compromise both animal welfare and data integrity. In severe cases, the stress-induced complications may even necessitate euthanasia, emphasizing the need for a more ethical and reliable alternative. Recognizing this need, Charles River Laboratories Den Bosch in collaboration with Ellegaard Göttingen Minipigs A/S have turned to VAB buttons as a potential solution.

A study was conducted at Charles River Laboratories Den Bosch to assess the feasibility, validity, and potential benefits of pre-implanted VAB buttons in Göttingen minipigs compared to the traditional method, the vena cava cranialis (VCC) puncture. By evaluating the reliability and usability of the VAB buttons, especially in the context of pharmacokinetic studies, the study aimed to mitigate stress and discomfort for minipigs while maintaining or, at best, enhancing the accuracy and reliability of study outcomes in future research.

Methods

Surgical Implantation of Vascular Access Buttons in Minipigs
For this study, a surgical procedure was performed on two Göttingen minipigs to implant the VAB button. The left external jugular vein was carefully dissected, and after ligation, two 3fr PU catheters were inserted at intervals of 7 and 9 cm. The catheters were securely fastened around the vessel using a modified Miller knot followed by square knots. Additional ties were placed cranially to the bead, and the catheter was further secured with the ends of the initial ligature. The incision was closed in three layers using PSDII, employing a continuous pattern, with the final layer intradermal. Subsequently, the catheters were tunneled to a subcutaneous pocket created behind the left ear and connected to the button. The implantation site was closed in three layers. Anesthesia was induced using a combination of zoletil, butorphanol, ketamine, and xylazine, and maintained with isoflurane. After discontinuation of anesthesia, the minipigs were allowed to recover. Following one week of post-operative care, including antibiotics and analgesia (a combination of butorphanol and ketamine), vascular access was tested. Meloxicam was administered before surgery and continued for three days post-surgery. The animals were then transferred to Charles River Laboratories Den Bosch.

Ensuring Optimal Proficiency: Training and Maintenance Protocol
At the onset of the study, Ellegaard Göttingen Minipigs provided comprehensive training sessions aimed at ensuring the proficiency of animal technicians in catheter maintenance, intravenous dosing, and blood collection techniques utilizing the VAB. This training regimen spanned a four-week period, during which the animal technicians familiarized themselves in the intricacies of these tasks. Emphasis was placed on the importance of regular catheter maintenance to uphold the optimal functionality of the VAB. This regular catheter maintenance involved weekly flushing using solutions such as Taurolock, heparin/saline, or heparin/glycerol  to effectively mitigate the risk of clot formation and to sustain the patency of the catheters. Moreover, daily practice sessions, excluding weekends, were dedicated to honing skills in blood sample collection and intravenous dosing (using saline with a maximum volume of 2.5 mL/kg). Throughout the entire duration of the study, spanning a 120-day period until necropsy, the usability and durability of the VAB was evaluated to ascertain its reliability and longevity in a research setting.

The Methodological Approach: How we compared the VAB to the VCC puncture
Following the initial phase of training, the study progressed to assess the effectiveness and validity of two distinct VAB buttons implemented in two separate Göttingen minipigs. In one minipig, both catheters of the VAB were surgically inserted into the jugular vein, whereas in the other, a hybrid approach was adopted, with one catheter positioned in the jugular vein and the other placed in the carotid artery [see Figure 1 for a visual representation]. This strategic variation in catheter placement aimed to explore the versatility and usability of the VAB across different anatomical contexts. 

Subsequently, dexamethasone, a widely used veterinary pharmaceutical agent, was administered via the VAB to both minipigs at a dose level of 0.3 mg/kg, followed by repeated blood collection for pharmacokinetic parameter analysis via both the VAB button and the traditional method, VCC puncture. It is important to note that blood collection via both methods was meticulously synchronized, ensuring simultaneous sampling to minimize potential confounding factors. This methodological precision was imperative for facilitating accurate comparisons within each minipig between the VAB and the traditional VCC method, as well as between the two distinct VAB buttons employed. Additionally, a comprehensive evaluation of clinical pathology parameters was conducted using blood samples obtained from both the VAB and the VCC for each minipig, prior to dosing, enabling a robust assessment of result consistency and methodological reliability within the study.

Results

Question I: Comparison of Pharmacokinetic Parameters between the VCC and VAB in Göttingen minipigs.

Result: Table 1 outlines the pharmacokinetic parameters of Dexamethasone in male Göttingen minipigs after a single intravenous (slow bolus) injection via the pre-implanted vascular access button (VAB). The exposure data for Animal No. 1 (with VAB Type I) and Animal No. 3 (with VAB Type II) yielded comparable results within each animal, regardless of the blood collection technique used [Table 1; Table 2]. For Animal No. 1, the sampling technique ratio between the VAB and the VCC was 1.07 for C0 and 1.05 for AUCtlast. For Animal No. 3, the sampling technique ratio between the VAB artery and the VCC was 1.19 for C0 and 0.88 for AUCtlast, while the ratio between the VAB vein and the VCC was 1.08 for C0 and 0.90 for AUCtlast. The consistency of the pharmacokinetic parameters of Dexamethasone across different blood collection techniques is also evident from the individual Concentration vs. Time curves in each Animal [Figure 2].

Thus, the pharmacokinetic parameters in Göttingen minipigs remained consistent across different blood collection techniques, as evidenced by comparable results within each animal. The slight variations observed can be due to normal variation but could also suggest small variations in the time of collection. Overall, the VAB proved to be a reliable method for intravenous drug administration and sampling. These findings support the utility of VAB for pharmacokinetic blood collection in in vivo studies with Göttingen minipigs.

Question II: Comparison of Clinical Pathology Samples from VCC and VAB in Göttingen minipigs

Result: This test aimed to investigate the comparability of clinical pathology samples obtained via the VCC and the VAB in Göttingen minipigs. Hematology, coagulation, and clinical chemistry values were measured and assessed for similarity between the two sampling methods [Table 3-5]. The comparison of clinical pathology parameters revealed consistent findings across most parameters, with only one notable exception. Hematological parameters, including red blood cell count, white blood cell count, and platelet count, showed negligible disparities between the techniques, all falling within clinically acceptable ranges1. Coagulation values, i.e., prothrombin time and activated partial thromboplastin time, exhibited similar results. Likewise, clinical chemistry values, encompassing electrolyte levels, liver function markers, and renal function indices, demonstrated comparable outcomes irrespective of the sampling technique employed for all but one parameter. One anomalous result was observed in one animal its clinical chemistry profile; notably elevated triglyceride levels when sampled via the VAB compared to VCC. This difference however is supported by literature that states how hugely triglycerides can vary depending on the location the blood is sampled; especially the conventional VCC tends to give an underestimate result for triglycerides2. For that reason, a crucial point to bear in mind is to standardize the site of collection throughout the study in order to decrease the potential for errors in data interpretation. The minor variations observed in all other clinical chemistry parameters were within acceptable limits and likely attributable to inherent biological variability.

In summary, while variations were observed between the two sampling locations, the overall congruity between samples collected via VCC and VAB suggests their interchangeability for clinical pathology analysis in Göttingen minipigs. The demonstrated equivalence between the VCC and VAB confirms that researchers can confidently utilize the VAB technique for blood sample collection for clinical pathology in Göttingen minipig studies. 

Question III: How practical is the use of the VAB button and for what duration can it be used?

Result: Both Göttingen minipigs exhibited no signs of disruption to their VAB button and/or catheter from (group)-housing conditions, nor was there any appearance of seromas surrounding the VAB button due to the surgical procedure. The animals were successfully housed together for a period of 53 days, which was made possible due to the protective aluminum cap on the button. It is important that this button is securely fastened to prevent loss in the cage.

Furthermore, the system improved the ease of blood collection by requiring only a PinPortTM injector attached to a syringe, allowing blood collection by a single animal technician without additional animal restraint. The VAB system remained patent throughout the entire 120-day study period for both animals. It is worth noting that the animal technicians applied positive pressure on the lock solution syringe during removal from the VAB septum to minimize the risk of clot formation. In cases of initial blood collection failure (four occurrences), an extra flushing procedure was performed, resolving the issue in all instances.

The results highlight the resilience and practicality of the VAB system in Göttingen minipigs, demonstrating its effectiveness in facilitating blood collection while ensuring the animals' welfare during group housing conditions. The catheters attached to the VAB prevented damage by the animals, due to their secure and self-contained design. Overall, we demonstrated that the VAB system offers a practical, durable, and efficient solution for blood sample collection and drug administration in Göttingen minipigs. Its ability to mitigate stress from the animals, reduce procedural time, and require minimal technical support positions it as a valuable technique for future research.

Conclusion

The Vascular Access Button (VAB) has generally proven to be a reliable and valuable alternative for intravenous drug administration and blood collection in Göttingen minipigs, particularly in research or clinical settings requiring frequent blood sampling. However, akin to any medical device, its reliability hinges on factors such proper placement, maintenance, and user proficiency. Adhering to manufacturer guidelines and best practices is crucial to ensure consistent and accurate blood collection. For instance, applying positive pressure to the lock solution syringe while disengaging it from the VAB septum minimizes the risk of clot formation. Moreover, utilizing sterile, pharmaceutical-grade lock and flush solutions, sterile catheters and supplies, along with maintaining aseptic technique during both surgical procedures and catheter maintenance must remain paramount. The use of non-sterile solutions can lead to patency issues at a minimum, or worse infections that could compromise the research integrity and the well-being of the animals.

Overall, the exploration of the VAB technology signifies a promising path toward elevating ethical standards in animal research. By prioritizing the welfare of research subjects and refining experimental methodologies, VAB buttons have the potential to revolutionize the field of in vivo studies across various non-rodent species. As such, continued research and innovation in this domain are essential to realizing the full benefits of this technology.

REFERENCES

1. Smith CN, Neptun DA, Irons RD. Effect of sampling site and collection method on variations in baseline clinical pathology parameters in Fischer-344 rats. II. Clinical hematology. Fundam Appl Toxicol. 1986 Nov;7(4):658-63. doi: 10.1016/0272-0590(86)90115-6. PMID: 3803760.
2. Neptun DA, Smith CN, Irons RD. Effect of sampling site and collection method on variations in baseline clinical pathology parameters in Fischer-344 rats. 1. Clinical chemistry. Fundam Appl Toxicol. 1985 Dec;5(6 Pt 1):1180-5. doi: 10.1016/0272-0590(85)90155-1. PMID: 4092880.