All animal care and procedures were in accordance with the guidelines laid down by the National Health and Medical Research Council and approved by the Institutional Animal Ethics Committee (Department of Primary Industries, Victoria).
A total number of 15 crossbred sheep were used for this study with a mean weight of 55kg. Under general anesthesia (induction, 2 mg/kg propofol; maintenance, isoflourane 2% in oxygen), a 9 Fr vascular sheath was placed in a carotid artery and an 11 Fr sheath placed in a jugular vein. Limb isolation was performed using a percutaneous recirculation circuit (‘V-Vascular’, Osprey Medical, Minneapolis, USA), shown in . Under fluoroscopic guidance balloon catheters were positioned in the femoral vein and artery of the sheep via the carotid artery and jugular vein. Once in position a nitinol basket was placed within the femoral vein at the tip of the balloon catheter to provide support and prevent collapse of the vessel during recirculation. The system acts by capturing the femoral venous return, via the customized balloon catheter, which is oxygenated with a membrane oxygenator, the reoxygenated blood is then returned to the femoral artery via a roller pump.
Schematic of the V-Vascular system.
An initial pilot study with a 10 minute recirculation time was conducted in n=4 sheep to assess the safety of the system which is required to maintain adequate oxygen saturation without altering blood pH or increasing lactate within the limb. Blood was sampled from the arterial and venous arms of the circuit at baseline and at 2 minute intervals and analysed using the i-STAT system (Abbott Point of Care Inc.). Parameters assessed were O2 and CO2 saturation, lactate and blood pH. Arterial pressure was also monitored during recirculation. Histopathology of the artery/vein was also assessed following recirculation, samples were formalin fixed and embedded in paraffin, slides were cut and stained using haematoxylin and eosin (H&E) stain.
Gentamicin was administered using one of two methods, in both cases samples of the hind limb (bone, skeletal muscle and synovial fluid) were taken at 30 and 60 minutes post initiation of administration to determine Gentamicin levels. The control group received 4 mg/kg via the standard intra-venous route (n=6) while the second group received the antibiotic using percutaneous recirculation as described above for 30 minutes (n=5). In the recirculation group, once stable blood flow through the circuit was established, Gentamicin 4 mg/kg was delivered into the antegrade limb of the circuit over two minutes and recirculation continued for 30 minutes. To limit the systemic appearance of Gentamicin, femoral venous blood was collected for 2 minutes without recirculation at the conclusion of antibiotic administration, the catheters removed and recirculation allowed to return to normal for 30 min prior to the final samples of bone, skeletal muscle and synovial fluid (60 min post initiation of gentamicin delivery).
Following sampling at the 60 minute time point, animals were euthansed (potassium chloride 30 mk/kg, i.v) and samples from the liver, kidney and lung of the sheep obtained to examine Gentamicin levels.
Bone, skeletal muscle and synovial fluid samples from the limb, as well as kidney, liver and lung, were weighed and placed in saline. The tissue was ground, centrifuged and the supernatant removed for analysis of gentamicin levels using Flourescence Polarisation Immunoassay (Abbott AxSYM Gentamicin).
The study was conducted in a blinded fashion, analyses of the samples was conducted by researchers unaware from which group the data was obtained. Data are expressed as mean ± sem. Between or within group comparisons were performed using unpaired or paired t-tests where data was normally distributed, whilst non-normal data were analysed using rank sum test. A p value less than 0.05 was considered statistically significant.