Electrically evoked compound action potentials recorded from the sheep spinal cord.

OBJECTIVES: The study aims to characterize the electrical response of dorsal column axons to depolarizing stimuli to help understand the mechanisms of spinal cord stimulation (SCS) for the relief of chronic pain. MATERIALS AND METHODS: We recorded electrically evoked compound action potentials (ECAPs) during SCS in 10 anesthetized sheep using stimulating and recording electrodes on the same epidural SCS leads. A novel stimulating and recording system allowed artifact contamination of the ECAP to be minimized. RESULTS: The ECAP in the sheep spinal cord demonstrates a triphasic morphology, with P1, N1, and P2 peaks. The amplitude of the ECAP varies along the length of the spinal cord, with minimum amplitudes recorded from electrodes positioned over each intervertebral disc, and maximum amplitudes recorded in the midvertebral positions. This anatomically correlated depression of ECAP also correlates with the areas of the spinal cord with the highest thresholds for stimulation; thus regions of weakest response invariably had least sensitivity to stimulation by as much as a factor of two. The choice of stimulating electrode location can therefore have a profound effect on the power consumption for an implanted stimulator for SCS. There may be optimal positions for stimulation in the sheep, and this observation may translate to humans. Almost no change in conduction velocity (∼100 ms) was observed with increasing currents from threshold to twice threshold, despite increased Aß fiber recruitment. CONCLUSIONS: Amplitude of sheep Aß fiber potentials during SCS exhibit dependence on electrode location, highlighting potential optimization of Aß recruitment and power consumption in SCS devices.

Translation of Methodology Used In Human Myocardial Imaging to a Sheep Model of Acute Myocardial Infarction.

INTRODUCTION: Pre-clinical investigation of stem cells for repairing damaged myocardium predominantly uses rodents, however large animals have cardiac circulation closely resembling the human heart. The aim of this study was to evaluate whether SPECT/CT myocardial perfusion imaging (MPI) could be used for assessing sheep myocardium following an acute myocardial infarction (MI) and response to intervention. METHODS: Eighteen sheep were enrolled in a pilot study to evaluate [(99m)Tc]-sestamibi MPI at baseline, post-MI and after therapy. Modifications to the standard MPI protocols were developed. All data was reconstructed with OSEM using CT-derived attenuation and scatter correction. Standard analyses were performed and inter-observer agreement was measured using Kappa (κ). Power determined the sample sizes needed to show statistically significant changes due to intervention. RESULTS: Ten sheep completed the full protocol. Data processed was performed with pre-existing hardware and software used in human MPI scanning. No improvement in perfusion was seen in the control group, however improvements of 15%-35% were seen after intra-myocardial stem cell administration. Inter-observer agreement was excellent (К=0.89). Using a target power of 0.9, 28 sheep were required to detect a 10-12% change in perfusion. CONCLUSION: This study demonstrates the suitability of large animal models for imaging with standard MPI protocols and its feasibility with a manageable number of animals. These protocols could be translated into humans to study the efficacy of stem cell therapy in heart regeneration and repair.

Super selective radio embolization of the porcine kidney with 90yttrium resin microspheres: a feasibility, safety and dose ranging study.

PURPOSE: We determined whether super selective radio embolization of the porcine kidney was technically feasible and evaluated histopathological changes in the treatment target zone (upper or lower renal pole), adjacent nontargeted kidney, and adjacent and distant organs after administering (90)Y labeled vs bland resin microspheres. MATERIALS AND METHODS: We performed super selective radio embolization with (90)Y resin microspheres in 1 kidney and with an equivalent number of bland microspheres in the corresponding pole of the contralateral kidney as a control. The aim was to achieve radio embolization of a target zone equivalent to approximately a third of the kidney volume. A pathologist independently graded macroscopic and microscopic changes in the kidney, and adjacent and distant tissue resulting from incremental increases (0.15 to 0.35 GBq) in implanted activity in 6 pigs. RESULTS: We recorded grade 4 histological changes in the treatment target zone (upper or lower renal pole) in 5 of 6 pigs after injecting (90)Y resin microspheres with evidence of nephron sparing effects in the adjacent renal tissue at the lowest activity. At activity greater than 0.3 GBq increasing damage was noted to adjacent renal tissue beyond the treatment target zone. No toxicity was evident in adjacent or distant organs. CONCLUSIONS: Delivery of highly targeted intra-arterial radiotherapy to the kidney is feasible and safe in the pig model. Further evaluation is warranted as a potential treatment for advanced renal cell carcinoma or for localized disease in patients who are not candidates for surgery.

Hand-assisted laparoscopic partial nephrectomy: a controlled study of bipolar inline radiofrequency ablation device in Swine.

BACKGROUND: Bipolar inline radiofrequency ablation (ILRFA) is an effective way to control bleeding during parenchymal organ transection. This was investigated in a study of laparoscopic hand-assisted partial nephrectomy in swine. METHODS: Nine Landrace pigs were divided between 2 groups; diathermy was employed in the control group for parenchymal transaction and ILRFA was applied in the experimental group through a hand-port and deployed into the resection plane. After complete coagulation, resection was performed using scissors. RESULTS: The mean intraoperative blood loss was 32 +/- 15 mL in the ILRFA group and 187 +/- 69 mL in the control group: a 82.9% reduction ( P = .015). The mean blood loss per resection area was 2.53 +/- 0.92 mL/cm(2) in the ILRFA group compared with 17.31 +/- 9.05 mL/cm( 2) in controls; the reduction was 85.4% (P = .005). CONCLUSIONS: ILRFA is effective in achieving reducing blood loss and provides a drier operative field for precise dissection.

InLine bipolar radiofrequency ablation device-assisted partial nephrectomy in a porcine model.

BACKGROUND: Nephron-sparing surgery has become an acceptable alternative to radical nephrectomy in the treatment of renal tumours. Control of intraoperative blood loss can be an important challenge during partial nephrectomy. InLine radiofrequency ablation (ILRFA) device has shown promising results of significant reduction of intraoperative blood loss in liver resection. In this study, we tested ILRFA-assisted partial nephrectomy in a porcine model. METHODS: Eight landrace pigs were used in this study. Every pig underwent 2-3 partial nephrectomies. The proposed line of parenchymal incision was circumferentially scored with a diathermy. Then, ILRFA was deployed into this resection plane. After complete coagulation, the resection was then simply carried out using the scalpel. For the control resection, we used diathermy to transect the other pole, further sutures were then used to secure the residual bleeding. RESULTS: The ILRFA deployments were set to 3 cm power algorithm. The average radiofrequency ablation coagulation time was 5 min. The mean intraoperative blood loss 35 +/- 7 mL in the ILRFA and 152 +/- 94 mL in the control, a 77.0% reduction (P = 0.024). The mean blood loss per centimetre of resection area was 2.09 +/- 1.41 mL/cm(2) in the ILRFA compared with 12.79 +/- 1.68 mL/cm(2) in controls; the reduction was 79.0% (P = 0.019). CONCLUSION: Our study indicates that ILRFA for partial nephrectomy in a porcine model is effective in reducing blood loss. Precoagulation before parenchymal transection appears to be a valid concept in nephron-sparing surgery.

Novel surgical management of renal trauma: InLine radiofrequency ablation coagulation.

BACKGROUND: Renal injury accounts for 10% of all abdominal trauma. Low-grade renal injuries can be managed without surgery. However, patients with grade IV or grade V injuries may require nephrectomy. In this study, InLine radiofrequency ablation device (ILRFA), which we developed for liver surgery was tested in the animal model of simulated renal injury. METHODS: A grade IV renal injury was induced in eight landrace pigs. Then treatment with ILRFA was compared to conventional diathermy and suture; totally 24 surgeries were carried out (12 ILRFA vs 12 control). RESULTS: No massive bleeding occurred and no animal died during the experiments. Immediately after surgery, the pigs were given euthanasia. The average of RFA coagulation time was 3.5 min. The mean intraoperative blood loss was 42 +/- 16 mL in the ILRFA and 195 +/- 58 mL in the control, a 78.5% reduction (P < 0.001). The mean blood loss per centimetre squared of resection area was 4.50 +/- 2.84 mL/cm(2) in the ILRFA compared with 18.73 +/- 6.89 mL/cm(2) in controls, the reduction was 76.0% (P = 0.001). CONCLUSION: InLine RFA is efficient for the management of haemorrhage in renal trauma in an animal model and deserves clinical evaluation.

Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking.

Cell therapies have been used to regenerate the heart by direct myocardial delivery, by coronary infusion and by surface attached scaffolds. Multipotent mesenchymal stem cells (MSC) with capacity to differentiate into cardiomyocytes and other cell lines have been predominantly trialled in rodents. However, large animal models are increasingly needed to translate basic research into new, safe regenerative therapies. Understanding the mode of action of cell therapies in the mammalian heart has been limited by cell tracking capability. This study examined the ability to track the fate of allogeneic MSC in sheep using various fluorescent dyes. MSC isolated from sheep bone marrow were grown in culture following extraction and flow cytometric characterisation. After labelling with fluorescent tracking dyes (e.g. CFSE and DiI) cells were tested for in vitro and in vivo signal up to six weeks. Labelling effect on cell division and differentiation was studied. Several dyes lost fluorescence and slowed cell division. However, the thiol reactive dye CM-DiI showed detectable in vivo fluorescence in labelled MSC six weeks after injection into sheep skeletal muscle and two weeks after implantation of an MSC coated biomaterial scaffold. CM-DiI labelled MSC differentiated in vitro showed label retention over four weeks. The fluorescent membrane dye CM-DiI tracks implanted sheep MSC and provides an alternative to traditional cell markers such as gene modified GFP.

The effects of general anesthesia on whole body and regional pharmacokinetics of local anesthetics at toxic doses.

BACKGROUND: Local anesthetic toxicity is often studied experimentally in anesthetized subjects, but clinical toxicity usually occurs in conscious patients. In this study, we determined the influence of general anesthesia on the pharmacokinetics of six local anesthetics administered i.v. at approximately the highest recommended doses. METHODS: Chronically instrumented ewes (approximately 45-50 kg, n = 18) were infused over 3 min with (base doses as HCl salts) bupivacaine (100 mg), levobupivacaine (125 mg), ropivacaine (150 mg), lidocaine (350 mg), mepivacaine (350 mg), or prilocaine (350 mg), on separate occasions when conscious and halothane anesthetized. Serial arterial, heart, and brain venous blood drug concentrations were measured by achiral/chiral high-performance liquid chromatography, as relevant. Whole body pharmacokinetics were assessed by noncompartmental analysis; heart and brain pharmacokinetics were assessed by mass balance. Drug blood binding, in the absence and presence of halothane, was assessed by equilibrium dialysis in vitro. RESULTS: Blood local anesthetic concentrations were doubled with anesthesia because of decreased whole body distribution and clearance (respectively, to 33% and 52% of values when conscious). Heart and brain net drug uptake were greater under anesthesia, reflecting slower efflux from both regions. Clearances of R-bupivacaine > S-bupivacaine and R-prilocaine > S-prilocaine, but, mepivacaine clearance was not enantioselective. Halothane did not influence blood binding of the local anesthetics. CONCLUSIONS: General anesthesia significantly changed whole body and regional pharmacokinetics of each local anesthetic as well as the systemic effects. General anesthesia is thus an important but frequently overlooked factor in studies of local anesthetic toxicity.

The effects of general anesthesia on the central nervous and cardiovascular system toxicity of local anesthetics.

BACKGROUND: Local anesthetic toxicity is often studied experimentally in acutely prepared, anesthetized laboratory animals. We determined the influence of halothane/O(2) anesthesia on cardiovascular and central nervous system (CNS) toxic responses to six amide-type local anesthetics administered i.v.. METHODS: Behavioral, cardiovascular, and pharmacokinetic responses were determined in previously instrumented ewes (approximately 45-50 kg, n = 18), on separate occasions when conscious and anesthetized, to bupivacaine (100 mg), levobupivacaine (125 mg), ropivacaine (150 mg), lidocaine (350 mg), mepivacaine (350 mg), prilocaine (350 mg), and saline (control) infused i.v. over 3 min. RESULTS: The local anesthetics caused convulsions in conscious sheep, but no overt CNS effects in anesthetized sheep. Negative inotropy and slight bradycardia without changes in arterial blood pressure occurred initially in conscious sheep, followed by positive inotropy, tachycardia, and hypertension at the abrupt onset of CNS excitotoxicity, along with widening of QRS complexes. Fatal cardiac arrhythmias occurred in, respectively, 3 of 11, 2 of 12, and 2 of 13 conscious sheep infused with bupivacaine, levobupivacaine, and ropivacaine; in 1 of 9 with prilocaine, electromechanical dissociation (followed by polymorphic ventricular tachycardia) caused death. In anesthetized sheep, cardiovascular depression, preexisting from the general anesthesia, was exacerbated by all local anesthetics, and increased QRS width was prolonged; concurrent blood local anesthetic concentrations were doubled. Nevertheless, all anesthetized animals survived. CONCLUSIONS: General anesthesia produced physiological perturbations, exacerbated local anesthetic-induced cardiovascular depression, and changed the pharmacokinetics of toxic doses of local anesthetics. However, cardiovascular fatalities from local anesthetics occurred only in conscious animals.

InLine radiofrequency ablation-assisted laparoscopic liver resection: first experiment with stapling device.

BACKGROUND: In liver surgery, the increase in advancement of laparoscopic equipment has allowed the feasibility and safety of complex laparoscopic liver resection. However, blood loss and the potential risk of gas embolism seem to be the main obstacles. In this study, we successfully used the InLine radiofrequency ablation (RFA) device to carry out laparoscopic hand-assisted liver resection in pigs. METHODS: Under general anaesthesia with tracheal intubation, pigs underwent InLine RFA-assisted laparoscopic liver resection. After installation of Hand Port and trocars, the InLine RFA device was introduced through Hand Port system and inserted into the premarked resection line. Then the generator was turned on and the power was applied according to the power setting. The resection was finally carried out using diathermy or stapler. For the control group, resection was simply carried out by diathermy or stapler. RESULTS: Eight Landrace pigs underwent 23 liver resections. Blood loss was reduced significantly in the InLine group (P<0.001) when compared with control group in both surgical methods (diathermy and stapler). CONCLUSION: In this study, we successfully carried out InLine RFA-assisted laparoscopic liver resection in both stapled and diathermy group. We showed that there was a highly significant difference between InLine and other liver resection techniques laparoscopically.

An experimental study of the treatment of liver injury with InLine RFA.

BACKGROUND: The liver is the most frequently injured abdominal organ after blunt injury; sometimes it may be very difficult to achieve haemostasis. In this study we examined the use of InLine radiofrequency ablation (RFA) for the coagulation and haemostasis of simulated liver injury. MATERIALS AND METHODS: Six pigs were tested in this study. We created two types of grade III to IV liver injury: peripheral and central. Then treatment with InLine RFA was compared to conventional diathermy and suture. A total of 32 surgeries were performed: peripheral injury (12 InLine versus 6 diathermy + suture); central injury (8 InLine versus 6 diathermy + suture). RESULTS: The reduction of blood loss was 63.88% in peripheral injury and 53.57% in central injury, respectively. There were significant differences in both types of injuries as regards blood loss (p<0.05) and blood loss per cm(2) (p<0.05). CONCLUSIONS: InLine RFA is efficacious in treating liver injuries in pigs and may have a significant potential for some human liver injuries.

Acute toxicity of local anesthetics: underlying pharmacokinetic and pharmacodynamic concepts.

The risk of accidental intravascular injection and consequent acute toxicity is ever-present with most neural blockade techniques. The severity of cardiovascular and central nervous system (respectively, CVS and CNS) toxicity is directly related to the local anesthetic potency, dose, and rate of administration. Nonetheless, although the anesthetic potency of ropivacaine and levobupivacaine is similar to that of bupivacaine, at usual clinical doses, ropivacaine and levobupivacaine are less likely than bupivacaine to cause convulsions or lethal dysrhythmias. Signs of CNS stimulation, ranging from tremors to convulsions and perhaps cardiac dysrhythmias, can be described in terms of a chaos-derived state change in which the local anesthetic appears to act as an initiator. Both CNS and CVS effects are rather poorly correlated with arterial drug concentrations but better correlated with concentrations in the respective regional venous drainage. Lung uptake reduces the maximum drug concentration by approximately 40%. Prolonging intravenous administration from 1 to 3 minutes results in a similar decrease in maximum concentration. This is an underlying tenet of dose fractionation, but the main advantage of dose fractionation is that the anesthesiologist is able to cease administration with less of the dose given if signs or symptoms of toxicity occur. Overall, it appears that the gains in safety from ropivacaine and levobupivacaine are due more to favorable pharmacodynamic enantioselectivity than to pharmacokinetic factors. This essay presents some pharmacokinetic aspects relevant to acute toxicity of local anesthetics, mainly using data from the authors' studies in a sheep model of simulated accidental intravenous administration.

Direct cardiac effects of coronary site-directed thiopental and its enantiomers: a comparison to propofol in conscious sheep.

BACKGROUND: Previous evidence from laboratory animal studies indicates that R-thiopental has a greater margin of safety than either the more potent S-thiopental or the clinically used rac-thiopental. Although thiopental can cause cardiovascular depression from direct myocardial effects as well as indirect central nervous system and peripheral effects, no studies have yet determined whether its myocardial effects are enantioselective. A lesser direct effect would provide further evidence supporting R-thiopental as a preferred single enantiomer replacement for rac-thiopental. METHODS: The direct myocardial effects of the thiopental enantiomers were compared to those of rac-thiopental and propofol, using a crossover design with small incremental doses infused over 3 min, on separate days, into the left coronary arteries of conscious sheep. Hemodynamic and electrocardiographic measurements were acquired, and serial blood samples were collected during the studies for drug analyses. RESULTS: All three forms of thiopental and propofol produced significant hemodynamic effects consisting of dose-related and rapid-onset decreases in left ventricular dP/dtmax and stroke volume, and increases in left coronary blood flow and heart rate. Cardiac output, mean arterial blood pressure, and central venous pressure remained unaltered. The effects did not differ significantly among rac-thiopental, enantiopure R- or S-thiopental, or propofol. Arterial blood drug concentrations were consistently less than those associated with systemic effects. CONCLUSIONS: Although previous evidence indicates that R-thiopental could make a suitable single-enantiomer replacement for rac-thiopental, the current study did not find a significant difference in direct cardiac effects among the thiopental enantiomers, racemate, or propofol.

Effects of imposed acid-base derangement on the cardiovascular effects and pharmacokinetics of bupivacaine and thiopental.

BACKGROUND: By changing physicochemical properties such as effective lipophilicity, changes in blood pH could alter the distribution, elimination, and effects of weakly ionizing drugs. The authors examined the outcome of imposed acid-base derangement on cardiovascular effects and myocardial and whole body pharmacokinetics of bupivacaine, a weak base, and thiopental, a weak acid. METHODS: Intravenous infusions of rac-bupivacaine HCl (37.5 mg) or rac-thiopental sodium (250 mg, subanesthetic dose) were administered over 3 min to previously instrumented conscious ewes with normal blood pH, acidemia imposed by lactic acid infusion, or alkalemia imposed by bicarbonate infusion. Hemodynamic and electrocardiographic effects were recorded; arterial and coronary sinus drug blood concentrations were analyzed by chiral high-performance liquid chromatography. RESULTS: Bupivacaine decreased myocardial contractility, coronary perfusion, heart rate, and cardiac output; however, cardiac output and stroke volume were not as affected by bupivacaine with acidemia. Thiopental decreased myocardial contractility and stroke volume and increased heart rate; acidemia enhanced the tachycardia and produced a greater decrease in stroke volume than with alkalemia. Taken as a whole, the cardiovascular changes were not systematically modified by acid-base derangement. Overall, the tissue distribution of bupivacaine was favored by alkalemia, but thiopental pharmacokinetics were essentially unaffected by acid-base derangement. Acid-base derangement did not influence the kinetics of either drug enantioselectively. CONCLUSIONS: At the doses used, the hemodynamic and electrocardiographic effects of bupivacaine and thiopental were not systematically modified by acid-base derangement, nor were there changes in regional or whole body pharmacokinetics of either drug that were clearly related to acid-base status.

Effects of CNS site-directed carotid arterial infusions of bupivacaine, levobupivacaine, and ropivacaine in sheep.

BACKGROUND: Previous preclinical safety studies in ewes have found intravenous levobupivacaine and ropivacaine to be less potent toward causing central nervous system (CNS) and cardiac toxicity than bupivacaine. Analogous cardiotoxicity has been demonstrated directly in various cardiac preparations ex vivo. Moreover, drug-related arrhythmogenicity has been demonstrated from direct CNS injection of local anesthetic agents in vivo, suggesting CNS-related cardiotoxicity. This study investigated whether CNS site-directed blood-borne drug administration (with minimal systemic recirculation) would demonstrate drug-related cardiotoxicity. METHODS: Direct CNS effects and indirect cardiotoxic sequelae were determined after bilateral carotid arterial infusions of levobupivacaine, bupivacaine, or ropivacaine in ewes. After pilot studies to validate the procedures, equimolar doses (24-96 micromol, approximately 7.5-30 mg) were infused over 3 min using a crossover design. Behavioral CNS signs, quantitative electroencephalographic (EEG), cardiovascular, and electrocardiographic effects were recorded. Drug blood concentrations in superior sagittal sinus and aorta were measured serially. RESULTS: Blood drug concentrations in the superior sagittal sinus were 5-10 times those concurrently in the aorta, confirming highly selective CNS delivery with minimal systemic recirculation. Dose-dependent CNS excitatory behavior and EEG changes, with increased mean arterial blood pressure, heart rate, cardiac output, and myocardial contractility, were found, consistent with sympathetic nervous system stimulation. The overall rank order of potency for these effects was ropivacaine < levobupivacaine < bupivacaine. Nonfatal cardiac arrhythmias were observed, but the type or frequency did not differ between drugs. CONCLUSIONS: Although CNS site-selective drug delivery produced quantitative differences between bupivacaine, levobupivacaine, and ropivacaine in some CNS effects and cardiac sequelae, no differences were found in their arrhythmogenic potential.