Incremental benefit of a novel signal recording system during mapping and ablation.

AIMS: Current electrophysiology signal recording and mapping systems have limited dynamic range (DR) and bandwidth, which causes loss of valuable information during acquisition of cardiac signals. We evaluated a novel advanced signal processing platform with the objective to obtain and assess additional information of clinical importance. METHODS AND RESULTS: Over 10 canines, we compared intracardiac recordings within all cardiac chambers, in various rhythms, in pacing and during radiofrequency (RF) ablation across two platforms; a conventional system and the PURE EP™ [(PEP); Bio Sig Technologies, Inc., Los Angeles, CA, USA]. Recording cardiac signals with varying amplitudes were consistently and reproducibly observed, without loss of detail or introduction of artefact. Further the amplitude of current of injury (COI) on the unipolar signals correlated with the instantaneous contact force (CF) recorded on the sensing catheter in all the animals (r2 = 0.94 in ventricle). The maximum change in the unipolar COI correlated with the change in local electrogram amplitude during non-irrigated RF ablation (r2 = 0.61 in atrium). Reduction in artefact attributable to pacing (20 sites) and noise during ablation (48 sites) was present on the PEP system. Within the PEP system, simultaneous display of identical signals, filtered differently, aided the visualization of discrete conduction tissue signals. CONCLUSION: Compared to current system, the PEP system provided incremental information including identifying conduction tissue signals, estimates of CF and a surrogate for lesion formation. This novel signal processing platform with increased DR and minimal front-end filtering may be useful in clinical practice.

Intracellular and Extracellular Antifreeze Protein Significantly Improves Mammalian Cell Cryopreservation.

Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP-ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at -196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells.

Short Communication: effect of sodium butyrate, monensin, and butyric acid on the viability of Eimeria bovis sporozoites and their degree of damage to a bovine epithelial cell line.

This study aimed to determine the viability of sporozoites from Eimeria bovis when exposed to sodium butyrate (SB), monensin (MON), or butyric acid (BA), and to determine the effects of SB on sporozoite invasion of cells in comparison to MON as measured by the damage to a bovine epithelial cell line. To determine viability, isolated sporozoites were suspended in one of four treatments: control (CON) of cell culture medium alone, SB = 0.028 mg/mL suspended in control medium, MON = 0.01 mg/mL suspended in CON, and BA = 0.18 mg/mL suspended in CON. The number of live sporozoites was less for the MON and BA treatments compared to the CON and SB treatments. The number of dead sporozoites was similar regardless of treatment. There was a trend for treatment to affect the percent sporozoite viability. Control, SB and BA treatments were similar, while MON compared to control and SB had decreased percent viability. Results for MON, when compared to BA, were similar for percent viability. Lactate dehydrogenase (LDH) release was used to determine cellular damage to Madin Darby Bovine Kidney (MDBK) cells when exposed to E. bovis sporozoites in vitro. Cells were exposed to similar numbers of sporozoites and treated with: CON, SB = 0.028 mg/mL in control medium, MON = 0.01 mg/mL in control medium. Control LDH result (with sporozoites) was greater than both the SB and MON treatments while the LDH for SB and Mon and cells not exposed to sporozoites were similar. SB and MON were both shown to decrease cellular damage to MDBK cells as determined by decreased LDH release. SB has the potential to act as an anticoccidial alternative to MON.

Coccidial infections are a significant problem in food animals, including dairy cattle and although there are drug treatments available, these are banned in many countries. Potential alternatives to drugs like monensin include sodium butyrate (SB) and butyric acid (BA). These were evaluated to determine their effect on Eimeria bovis sporozoites in vitro. The number of viable sporozoites was similar between the control medium alone without monensin and SB treatments but reduced in monensin and BA treatments. The percent viable sporozoites were (percent viable is one measure) similar between the control and SB, and BA treatments while reduced in the monensin treatments. However, monensin and BA treatments were similar for percent viable sporozoites. SB and monensin similarly protected cells from damage caused by sporozoites. SB is likely to dissociate to sodium and butyrate as it passes through the digestive tract suggesting that sodium butyrate might be a viable anticoccidial for cattle.

Initial Experience with the BioSig PURE EP™ Signal Recording System: An Animal Laboratory Experience.

Current signal recording and processing systems have come a long way since their initial inception and use. There is, however, still ample scope for improvement, not only in the troubleshooting of their limitations, but also in the expansion of the boundaries in the recording of intracardiac signals. Here, we recount our experience with the use of the PURE EP™ signal recording system (BioSig Technologies, Inc., Minneapolis, MN, USA) in the animal laboratory.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in hamster aortic atherosclerosis: correlation with in-situ zymography.

Atherogenesis requires extracellular matrix (ECM) alterations, a process possibly mediated by matrix-degrading metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). The objective of this study was to examine the immunohistochemical expression patterns of MMPs-1, -2, -3 and -9 and their tissue inhibitors, TIMPs-1, -2, -3 and -4 during the three major stages of atherosclerotic lesion development in hypercholesterolemic Syrian Golden hamsters. Aortic atherosclerotic lesions (fatty streak, fibro-fatty and advanced) were histologically characterized in treated hamsters at 12, 24, and 49 weeks. The immunochemistry expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. MMP activity in control aortas and atherosclerotic lesions was characterized by in-situ zymography. Positive immunoreactivity for MMPs-2, -3, -9 and TIMPs-1, -2,-3, and -4 was observed in both control and atherosclerotic aortic arch segments, while MMP-1 was only observed in atherosclerotic lesions. Using in-situ zymography, we identified casein and gelatin degradation in fatty streak, fibro-fatty and advanced lesions. The immunohistochemical expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. In all lesion stages, substrate degradation was inhibited with 1,10-phenanthroline. Degradation of these substrates was not observed in control aortas. In addition, substrate degradation was inhibited with 1,10-phenanthroline. These findings suggested that in control segments, the net proteolytic balance was shifted in favor of MMP inhibition. Alternatively, despite the colocalization of MMPs and TIMPs in the treated segments, net proteolytic balance favored the catalytic MMPs.

Doxazosin, an alpha-1 antagonist, prevents further progression of the advanced atherosclerotic lesion in hypercholesterolemic hamsters.

The aim of this study was to examine the effect of doxazosin (DOX) on the further progression and regression of the advanced atherosclerotic lesion in the hypercholesterolemic hamster. Thirty-six, male F(1)B Golden Syrian hamsters, 10 weeks of age, were divided into 3 groups of 12 and fed a nonpurified hypercholesterolemic diet (HCD) containing 10% coconut oil and 0.1% cholesterol (wt/wt) for 9 months (HCD 9). One group of hamsters was euthanized at 9 months and their aortas were collected, fixed, and stored until analysis. The remaining hamsters were either maintained on the HCD for an additional 6 months (HCD 15) or fed the HCD plus 20 mg/kg/d DOX for the 6 months. At the end of the study (15 months), the DOX-treated hamsters had significantly lower plasma total cholesterol (TC) (-68%), low-density lipoprotein-cholesterol (LDL-C) (-73%), and triglycerides (TG) (-74%) compared with the HCD 15. The lumenal narrowing and intimal thickening atherosclerotic lesions were significantly less in the DOX-treated hamsters compared with the HCD 15 (-66% and -70%, respectively). These data suggest that DOX treatment prevents further progression of the advanced atherosclerotic lesion possibly by lowering plasma TC, LDL-C, and TG in hypercholesterolemic hamsters.

Decreased aortic early atherosclerosis in hypercholesterolemic hamsters fed oleic acid-rich TriSun oil compared to linoleic acid-rich sunflower oil.

Previous studies have demonstrated that low density lipoprotein (LDL) enriched in polyunsaturated fatty acids (PUFA) are more susceptible to oxidation (ex vivo) than those containing monounsaturated fatty acids (MUFA). To test whether this observation was associated with various parameters considered to be related with the development of early aortic atherosclerosis, hamsters were fed commercial hypercholesterolemic diets (HCD) containing either the PUFA, sunflower oil (SF) or the MUFA, TriSun oil (TS) at 10% with 0.4% cholesterol (wt/wt). LDL isolated from hamsters fed TS had significantly longer lag phase (30%, P < 0.05), a decreased propagation phase (-62%, P < 0.005), and fewer conjugated dienes formed (-37%, P < 0.007) compared to hamsters fed SF. Aortic vasomotor function, measured as degree of aortic relaxation, was significantly greater in the TS vs SF-fed hamsters whether acetylcholine or the calcium ionophore A23187 was used as the endothelium-dependent agonist. As a group, the SF-fed hamsters had significantly more early atherosclerosis than hamsters fed TS (46%, P < 0.006). When animals across the two diets were pair-matched by plasma LDL-C levels, there was an 82% greater mean difference (P < 0.002) in early atherosclerosis in the SF versus the TS-fed hamsters. While there were no significant associations with plasma lipids and lipoprotein cholesterol, early atherosclerosis was significantly correlated with lag phase (r = -0.67, p < 0.02), rate of LDL conjugated diene formation (r = 0.74, p < 0.006) and maximum dienes formed (r = 0.67, p < 0.02). Compared to TS-fed animals, aortic sections from hamsters fed the SF-containing diet revealed that the cytoplasm of numerous foam cells in the subendothelial space reacted positively with the monoclonal anti-bodies MDA-2 and NA59 antibody, epitopes found on oxidized forms of LDL. The present study suggests that compared to TS, hamsters fed the SF-diet demonstrated enhanced LDL oxidative susceptibility, reduced aortic relaxation, greater early aortic atherosclerosis and accumulation of epitopes found on oxidized forms of LDL.