Electroporation induced by internal defibrillation shock with and without recovery in intact rabbit hearts

Defibrillation shocks from implantable cardioverter defibrillators can be lifesaving but can also damage cardiac tissues via electroporation. This study characterizes the spatial distribution and extent of defibrillation shock-induced electroporation with and without a 45-min postshock period for cell membranes to recover. Langendorff-perfused rabbit hearts (n = 31) with and without a chronic left ventricular (LV) myocardial infarction (MI) were studied. Mean defibrillation threshold (DFT) was determined to be 161.4 ± 17.1 V and 1.65 ± 0.44 J in MI hearts for internally delivered 8-ms monophasic truncated exponential (MTE) shocks during sustained ventricular fibrillation (>20 s, SVF). A single 300-V MTE shock (twice determined DFT voltage) was used to terminate SVF. Shock-induced electroporation was assessed by propidium iodide (PI) uptake. Ventricular PI staining was quantified by fluorescent imaging. Histological analysis was performed using Masson's Trichrome staining. Results showed PI staining concentrated near the shock electrode in all hearts. Without recovery, PI staining was similar between normal and MI groups around the shock electrode and over the whole ventricles. However, MI hearts had greater total PI uptake in anterior (P < 0.01) and posterior (P < 0.01) LV epicardial regions. Postrecovery, PI staining was reduced substantially, but residual staining remained significant with similar spacial distributions. PI staining under SVF was similar to previously studied paced hearts. In conclusion, electroporation was spatially correlated with the active region of the shock electrode. Additional electroporation occurred in the LV epicardium of MI hearts, in the infarct border zone. Recovery of membrane integrity postelectroporation is likely a prolonged process. Short periods of SVF did not affect electroporation injury.     

Numerical modeling of in vivo plate electroporation thermal dose assessment

Electroporation is an approach used to enhance the transport of large molecules to the cell cytosol in which a targeted tissue region is exposed to a series of electric pulses. The cell membrane, which normally acts as a barrier to large molecule transport into the cell interior, is temporarily destabilized due to the development of pores in the cell membrane. Consequently, agents that are ordinarily unable enter the cell are able to pass through the cell membrane. Of possible concern when exposing biological tissue to an electric field is thermal tissue damage associated with joule heating. This paper explores the thermal effects of various geometric, biological, and electroporation pulse parameters including the blood vessel presence and size, plate electrode configuration, and pulse duration and frequency. A three-dimensional transient finite volume model of in vivo parallel plate electroporation of liver tissue is used to develop a better understanding of the underlying relationships between the physical parameters involved with tissue electroporation and resulting thermal damage potential.     

MRI study on reversible and irreversible electroporation induced blood brain barrier disruption

Electroporation, is known to induce cell membrane permeabilization in the reversible (RE) mode and cell death in the irreversible (IRE) mode. Using an experimental system designed to produce a continuum of IRE followed by RE around a single electrode we used MRI to study the effects of electroporation on the brain. Fifty-four rats were injected with Gd-DOTA and treated with a G25 electrode implanted 5.5 mm deep into the striata. MRI was acquired immediately after treatment, 10 min, 20 min, 30 min, and up to three weeks following the treatment using: T1W, T2W, Gradient echo (GE), serial SPGR (DCE-MRI) with flip angles ranging over 5-25°, and diffusion-weighted MRI (DWMRI). Blood brain barrier (BBB) disruption was depicted as clear enhancement on T1W images. The average signal intensity in the regions of T1-enhancement, representing BBB disruption, increased from 1887±83 (arbitrary units) immediately post treatment to 2246±94 20 min post treatment, then reached a plateau towards the 30 min scan where it reached 2289±87. DWMRI at 30 min showed no significant effects. Early treatment effects and late irreversible damage were clearly depicted on T2W. The enhancing volume on T2W has increased by an average of 2.27±0.27 in the first 24-48 hours post treatment, suggesting an inflammatory tissue response. The permanent tissue damage, depicted as an enhancing region on T2W, 3 weeks post treatment, decreased to an average of 50±10% of the T2W enhancing volumes on the day of the treatment which was 33±5% of the BBB disruption volume. Permanent tissue damage was significantly smaller than the volume of BBB disruption, suggesting, that BBB disruption is associated with RE while tissue damage with IRE. These results demonstrate the feasibility of applying reversible and irreversible electroporation for transient BBB disruption or permanent damage, respectively, and applying MRI for planning/monitoring disruption volume/shape by optimizing electrode positions and treatment parameters.     

A novel ex vivo heart model for the assessment of cardiac pacing systems

BACKGROUND: Advances in endocardial device design have been limited by the inability to visualize the device-tissue interface. The purpose of this study was to assess the validity of an isolated heart approach, which allows direct ex vivo intracardiac visualization, as a research tool for studying endocardial pacing systems. METHOD OF APPROACH: Endocardial pacing leads were implanted in the right atria and ventricles of intact swine (n = 8) under fluoroscopic guidance. After collection of pacing and sensing performance parameters, the hearts were excised with the leads intact and reanimated on the isolated heart apparatus, and parameters again recorded. RESULTS: Atrial ex vivo parameters significantly decreased compared with in vivo measurements: P-wave amplitudes by 39%, slew rates by 61%, and pacing impedances by 42% (p < 0.05 for each). Similarly, several ventricular ex vivo parameters decreased: R-wave amplitudes by 39%, slew rates by 62%, and pacing impedances by 31%. In contrast, both atrial (4.4 +/- 2.8 vs 3.3 +/- 2.8 V; p = ns) and ventricular thresholds increased (1.2 +/- 0.7 vs 0.6 +/- 0.1 V; p < 0.05 for all). Three distinct phenomena were observed at the lead-tissue interface. Normal implants (70%) demonstrated minimal tissue distortion and resulted in elevated impedance and threshold values. Three implants (13%) resulted in severe tissue distortion and/or tissue wrapping and were associated with highly elevated pacing parameters. Tissue coring occurred in four implants (17%) where the lead would spin freely in the tissue after overtorquing of the lead. CONCLUSIONS: The utility of the isolated heart approach was demonstrated as a tool for the design and assessment of the performance of endocardial pacing systems. Specifically, the ability to visualize device-heart interactions allows new insights into the impact of product design and clinical factors on lead performance and successful implantation.     

Mechanisms of superiority of ascending ramp waveforms: new insights into mechanisms of shock-induced vulnerability and defibrillation

Monophasic ascending ramp (AR) and descending ramp (DR) waveforms are known to have significantly different defibrillation thresholds. We hypothesized that this difference arises due to differences in mechanisms of arrhythmia induction for the two waveforms. Rabbit hearts (n = 10) were Langendorff perfused, and AR and DR waveforms (7, 20, and 40 ms) were randomly delivered from two line electrodes placed 10 mm apart on the anterior ventricular epicardium. We optically mapped cellular responses to shocks of various strengths (5, 10, and 20 V/cm) and coupling intervals (CIs; 120, 180, and 300 ms). Optical mapping revealed that maximum virtual electrode polarization (VEP) was reached at significantly different times for AR and DR of the same duration (P < 0.05) for all tested CIs. As a result, VEP for AR were stronger than for DR at the end of the shock. Postshock break excitation resulting from AR generated faster propagation and typically could not form reentry. In contrast, partially dissipated VEP resulting from DR generated slower propagation; the wavefront was able to propagate into deexcited tissue and thus formed a shock-induced reentry circuit. Therefore, for the same delivered energy, AR was less proarrhythmic compared with DR. An active bidomain model was used to confirm the electrophysiological results. The VEP hypothesis explains differences in vulnerability associated with monophasic AR and DR waveforms and, by extension, the superior defibrillation efficacy of the AR waveform compared with the DR waveform.     

Tissue electroporation. Observation of reversible electrical breakdown in viable frog skin.

Experiments by others have used isolated cell or bilayer membrane preparations to study the dramatic phenomena associated with electroporation. The present study observes electroporation behavior in an intact tissue. Viable samples of frog skin (Rana pipiens) were exposed to short electrical pulses of varying width and magnitude under "charge injection" conditions. After a pulse, the transtissue potential decayed with two distinct time constants, one short (tau approximately 0.3 ms) and the other longer (tau L approximately 2 ms). Above thresholds for the pulse magnitude and for the pulse width tau L decreased significantly, with progressively smaller tau L as the pulse magnitude and width increased. The postpulse potential, delta Utissue (t), and resistance, Rtissue, also decreased progressively. The tissue subsequently recovered to its original resistance and open circuit potential, delta U tissue,oc, within 2-3 min after a pulse. At that time another pulse experiment could be carried out, demonstrating repeatability and reversibility. No significant permanent changes in Rtissue and delta Utissue,oc were found. This is interpreted as avoidance of significant tissue damage. Taken together, these dramatic phenomena are characteristic of the reversible electrical breakdown previously observed in charge injection experiments with artificial planar bilayer membranes and with isolated cell membranes by similar very short pulses. The present experiments therefore demonstrate that electroporation can be repeatedly caused and observed in a viable tissue without apparent damage.     

Quantification of volume of tissue damage in stroke using T2- weighed MRI images

We have developed a quantitative technique for scoring of the severity of ischemic damage of the brain using quantitative data of the T2-weighted MRI images of brain in stroke. The principle of the method is the assumption that T2 signal increases proportionally to the severity of ischemic damage of cerebral tissue up to the level equal to intraventricular liquor signal in the case of postinfarction cystic degeneration. Depicting the mean T2-signal from the intraventricular liquor region as Iliq, the signal from ischemic brain area as Iinsult, and from the intact brain as Inorm, obviously, the volume quota of damaged tissue in the total volume of the stroke region is represented by the ratio (Iinsult - Inorm)/(Iliq - Inorm). The total volume of damaged tissue (VDT, cub.cm) in the stroke region is then the following sum taken over all slices i, where the stroke damage can be: VDT = sigma i d.Si.[(Iinsult - Inorm)/(Iliq - Inorm)]i, where d is the slice thickness, Si--area of the ischemic region in the slice i. The quota of damaged tissue in the physical volume of the stroke region is henceforth the following ratio: Q = [sigma i d.Si.[(Iinsult - Inorm)/(Iliq - Inorm)]i]/[sigma i d.Si]. The technique was applied in retrospective analysis of routine MRI studies in 15 patients referred because of acute ischemic stroke. The studies were performed using low-field MRI tomograph Magnetom-Open (Siemens Medical) with field strength 0.22 T. In patients studied during the first day after occurrence of ischemic insult with the minimal degree of acute neurologic deficit, who later have demonstrated clinically full recovery, the VDT was below 20 cm3, and Q was below 10%. In cases with VDT > 25 cm3 and Q > 20% the full regress was not observed in any patient. Henceforth, the quantification of cerebral damage in stroke using quantitative indices based on measurement of T2-parameters over ischemic and intact zones of the brain are of independent prognostic clinical value and improve clinical usefulness of the MRI in ischemic brain stroke.     

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to surrounding healthy tissue, critical vascular structures, and/or adjacent organs.     

Spontaneous calcium oscillations during diastole in the whole heart: the influence of ryanodine reception function and gap junction coupling

Triggered arrhythmias due to spontaneous cytoplasmic calcium oscillations occur in a variety of disease conditions; however, their cellular mechanisms in tissue are not clear. We hypothesize that spontaneous calcium oscillations in the whole heart are due to calcium release from the sarcoplasmic reticulum and are facilitated by calcium diffusion through gap junctions. Optical mapping of cytoplasmic calcium from Langendorff perfused guinea pig hearts (n = 10) was performed using oxygenated Tyrode's solution (in mM): 140 NaCl, 0.7 MgCl, 4.5 KCl, 5.5 dextrose, 5 HEPES, and 5.5 CaCl? (pH 7.45, 34°C). Rapid pacing was used to induce diastolic calcium oscillations. In all preparations, pacing-induced multicellular diastolic calcium oscillations (m-SCR) occurred across most of the mapping field, at all pacing rates tested. Ryanodine (1 μM) eliminated all m-SCR activity. Low-dose caffeine (1 mM) increased m-SCR amplitude (+10.4 ± 4.4%, P < 0.05) and decreased m-SCR time-to-peak (-17.4 ± 6.7%, P < 0.05) and its temporal synchronization (i.e., range) across the mapping field (-26.9 ± 17.1%, P < 0.05). Surprisingly, carbenoxolone increased the amplitude of m-SCR activity (+14.8 ± 4.1%, P < 0.05) and decreased m-SCR time-to-peak (-11.3 ± 9.6%, P < 0.01) and its synchronization (-37.0 ± 19.1%, P < 0.05), similar to caffeine. In isolated myocytes, carbenoxolone (50 μM) had no effect on the frequency of aftercontractions, suggesting the effect of cell-to-cell uncoupling on m-SCR activity is tissue specific. Therefore, in the whole heart, overt m-SCR activity caused by calcium release from the SR can be induced over a broad range of pacing rates. Enhanced ryanodine receptor open probability and, surprisingly, decreased cell-to-cell coupling increased the amplitude and temporal synchronization of spontaneous calcium release in tissue.     

Effects of electroporation on optically recorded transmembrane potential responses to high-intensity electrical shocks

The outcome of defibrillation shocks is determined by the nonlinear transmembrane potential (DeltaVm) response induced by a strong external electrical field in cardiac cells. We investigated the contribution of electroporation to DeltaVm transients during high-intensity shocks using optical mapping. Rectangular and ramp stimuli (10-20 ms) of different polarities and intensities were applied to the rabbit heart epicardium during the plateau phase of the action potential (AP). DeltaVm were optically recorded under a custom 6-mm-diameter electrode using a voltage-sensitive dye. A gradual increase of cathodal and well as anodal stimulus strength was associated with 1) saturation and subsequent reduction of DeltaVm; 2) postshock diastolic resting potential (RP) elevation; and 3) postshock AP amplitude (APA) reduction. Weak stimuli induced a monotonic DeltaVm response and did not affect the RP level. Strong shocks produced a nonmonotonic DeltaVm response and caused RP elevation and a reduction of postshock APA. The maximum positive and maximum negative DeltaVm were recorded at 170 +/- 20 mA/cm2 for cathodal stimuli and at 240 +/- 30 mA/cm2 for anodal stimuli, respectively (means +/- SE, n = 8, P = 0.003). RP elevation reached 10% of APA at a stimulus strength of 320 +/- 40 mA/cm2 for both polarities. Strong ramp stimuli (20 ms, 600 mA/cm2) induced a nonmonotonic DeltaVm response, reaching the same largest positive and negative values as for rectangular shocks. The transition from monotonic to nonmonotonic morphology correlates with RP elevation and APA reduction, which is consistent with cell membrane electroporation. Strong shocks resulted in propidium iodide uptake, suggesting sarcolemma electroporation. In conclusion, electroporation is a likely explanation of the saturation and nonmonotonic nature of cellular responses reported for strong electric stimuli.     

Atrioventricular conduction with and without AV nodal delay: two pathways to the bundle of His in the rabbit heart

The electrophysiological properties of atrioventricular (AV) nodal dual pathways have traditionally been investigated with premature stimuli delivered with right atrial pacing. However, little is known about the functional characteristics of AV nodal inputs outside of this context. Superfused rabbit triangle of Koch preparations (n = 8) and Langendorff-perfused hearts (n = 10) were paced throughout the triangle of Koch and mapped electrically and optically for activation pattern, electrogram and optical action potential morphologies, stimulation thresholds, and stimulus-His (S-H) intervals. Optical mapping and changes in His electrogram morphology were used to confirm the activation pathway. Pacing stimuli >or=2 mm above the tricuspid valve caused fast-pathway activation of the AV node and His with a threshold of 2.4 +/- 1.6 mA. An area directly below the coronary sinus had high thresholds (8.6 +/- 1.4 mA) that also resulted in fast-pathway excitation (P < 0.001). S-H intervals (81 +/- 19 ms) for fast-pathway activation remained constant throughout the triangle of Koch, reflecting the AV delay. Stimuli applied <2 mm from the tricuspid valve resulted in slow pathway (SP) excitation or direct His excitation (4.4 +/- 2.2 mA threshold; P < 0.001 compared with fast pathway). For SP/His pacing, S-H intervals showed a strong dependence on the distance from the His electrode and were significantly lower than S-H intervals for fast-pathway activation. SP/His pacing also displayed characteristic changes in His electrogram morphology. In conclusion, optical maps and S-H intervals for SP/His activation suggest that AV conduction via SP bypasses the compact AV node via the lower nodal bundle, which may be utilized to achieve long-term ventricular synchronization.     

Differential apoptosis markers in human keloids and hypertrophic scars fibroblasts

We previously demonstrated the increased amyloid precursor protein (APP) immunoreactivity around the site of damage after traumatic brain injury (TBI). However, the function of APP after TBI has not been evaluated. In this study, we investigated the effects of direct infusion of an anti-APP antibody into the damaged brain region on cerebral function and morphological changes following TBI in rats. Three days after TBI, there were many TUNEL-positive neurons and astrocytes around the damaged region and a significantly greater number of TUNEL-positive cells in the PBS group compared with the anti-APP group found. Seven days after TBI, there were significantly a greater number of large glial fibrillary acidic protein-positive cells, long elongated projections, and microtubule-associated protein-2-positive cells around the damaged region in the anti-APP group compared with the PBS group found. Seven days after TBI, the region of brain damage was significantly smaller and the time to arrival at a platform was significantly shorter in the anti-APP group compared with the PBS group. Furthermore, after TBI in the anti-APP group, the time to arrival at the platform recovered to that observed in uninjured sham operation group rats. These data suggest that the overproduction of APP after TBI inhibits astrocyte activity and reduces neural cell survival around the damaged brain region, which speculatively may be related to the induction of Alzheimer disease-type dementia after TBI.     

Advanced Heart Block During Acute Myocardial Infarction Treated with an Electrode Pacing Catheter

The mortality rate is high from advanced atrioventricular block associated with acute myocardial infarction. There is reason to believe that if in these patients the hearts are electrically paced with an endocardial pacing catheter, the mortality rate can be considerably decreased. Five patients in second- and third-degree heart block associated with acute myocardial infarction were paced with a considerable lowering of the expected mortality rate. Twenty-three cases from the literature are also presented and discussed. A silastic bipolar electrode catheter was used in these five cases. Four of the five cases returned to normal sinus rhythm within the first 10 days. The average duration of pacing was 6.7 days. It is the opinion of the author that second- and third-degree heart block associated with acute myocardial infarction should have a pacing catheter introduced at the earliest possible moment for continuous or demand endocardial pacing.     

Interference of programmed electromagnetic stimulation with pacemakers and automatic implantable cardioverter defibrillators

A commercially available magnetic therapy system, designed for clinical application as well as for private use without medical supervision, was examined with respect to its potential for causing electromagnetic interference with implantable pacemakers (PMs) and automatic implantable cardioverter defibrillators (AICDs). A sample of 15 PMs and 5 AICDs were experimentally investigated. Each of the implants was realistically positioned in a homogeneous, electrically passive torso phantom and exposed to the magnetic fields of the system's applicators (whole body mat, cushion, and bar applicator). The detection thresholds of the implants were programmed to maximum sensitivity and both unipolar as well as bipolar electrode configurations were considered. The evaluation of possible interferences was derived from the internal event storages and pacing statistics recorded by the implants during exposure. Any "heart activity" recorded by the implants during exposure was interpreted as a potential interference, because the implant obviously misinterpreted the external interference signal as a physiological signal. Only cases without any recorded "heart activity" and with nominal pacing rates (as expected from the program parameter settings) of the implants were rated as "interference-free." Exposure to the whole body mat (peak magnetic induction up to 265 microT) did not show an influence on PMs and AICD in any case. The cushion applicator at the highest field intensity (peak magnetic induction up to 360 microT) led to atrial sensing defects in four PM models with unipolar electrode configuration. Under bipolar electrode configuration no disturbances occurred. The bar applicator led to sensing problems and consecutively reduced pacing rates in all tested PM models under unipolar electrode configuration and maximum field intensity (peak magnetic induction up to 980 microT). Bipolar electrode configuration resolved the problem. The investigated AICDs did not show malfunctions under any investigated condition. In conclusion, the examined PEMF therapy system did not interfere with the investigated implantable cardiac devices with bipolar electrode configuration. However, unipolar electrode configuration in pacemakers seems to be potentially hazardous during application of the examined PEMF therapy system.     

Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.     

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.     

A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure

Background  

Irreversible electroporation (IRE) is a new minimally invasive technique to kill undesirable tissue in a non-thermal manner. In order to maximize the benefits from an IRE procedure, the pulse parameters and electrode configuration must be optimized to achieve complete coverage of the targeted tissue while preventing thermal damage due to excessive Joule heating.     

Na+ channel distribution and electrophysiological heterogeneities in guinea pig ventricular wall

We sought to explore the distribution pattern of Na(+) channels across ventricular wall, and to determine its functional correlates, in the guinea pig heart. Voltage-dependent Na(+) channel (Na(v)) protein expression levels were measured in transmural samples of ventricular tissue by Western blotting. Isolated, perfused heart preparations were used to record monophasic action potentials and volume-conducted ECG, and to measure effective refractory periods (ERPs) and pacing thresholds, in order to assess excitability, electrical restitution kinetics, and susceptibility to stimulation-evoked tachyarrhythmias at epicardial and endocardial stimulation sites. In both ventricular chambers, Na(v) protein expression was higher at endocardium than epicardium, with midmyocardial layers showing intermediate expression levels. Endocardial stimulation sites showed higher excitability, as evidenced by lower pacing thresholds during regular stimulation and downward displacement of the strength-interval curve reconstructed after extrasystolic stimulation compared with epicardium. ERP restitution assessed over a wide range of pacing rates showed greater maximal slope and faster kinetics at endocardial than epicardial stimulation sites. Flecainide, a Na(+) channel blocker, reduced the maximal ERP restitution slope, slowed restitution kinetics, and eliminated epicardial-to-endocardial difference in dynamics of electrical restitution. Greater excitability and steeper electrical restitution have been associated with greater arrhythmic susceptibility of endocardium than epicardium, as assessed by measuring ventricular fibrillation threshold, inducibility of tachyarrhythmias by rapid cardiac pacing, and the magnitude of stimulation-evoked repolarization alternans. In conclusion, higher Na(+) channel expression levels may contribute to greater excitability, steeper electrical restitution slopes and faster restitution kinetics, and greater susceptibility to stimulation-evoked tachyarrhythmias at endocardium than epicardium in the guinea pig heart.     

Near-terminal creep damage does not substantially influence fatigue life under physiological loading

Cortical bone specimens were damaged using repeated blocks of tensile creep loading until a near-terminal amount of creep damage was generated (corresponding to a reduction in elastic modulus of 15%). One group of cortical bone specimens was submitted to the near-terminal damage protocol and subsequently underwent fatigue loading in tension with a maximum strain of 2000 με (Damage Fatigue, n=5). A second group was submitted to cyclic fatigue loading but was not pre-damaged (Control Fatigue, n=5). All but one specimen (a damaged specimen) reached run-out (10 million cycles, 7.7 days). No significant differences in microscopic cracks or other tissue damage were observed between the two groups or between either group and additional, completely unloaded specimens. Our results suggest that damage in cortical bone allograft that is not obvious or associated with a stress riser may not substantially affect its fatigue life under physiologic loading.     

Disruption of the Cox-1 gene slows repair of microscopic lesions in the mouse gastric epithelium

Cyclooxygenase-1 (Cox-1) contributes to gastric defense of healthy tissue, but the role in the protection of the gastric epithelium after minor, acute damage has been difficult to study in vivo. Using 710-nm two-photon light absorption to create microscopic gastric damage in anesthetized mice with the gastric mucosal surface surgically exposed and perfused on the microscope stage, the acute response of surface cells to injury could be monitored using in vivo microscopy within seconds after injury. Using exogenous (Cl-NERF) and endogenous fluorophores, extracellular pH and cell death were monitored in real time during the entire damage and repair cycle. Two-photon damage was initiated by scanning approximately 200 microm(2) of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-1(+/-) and Cox-1(-/-) mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2-5 min, with larger maximal damage noted in Cox-1(-/-) mice. The maximal damage size seen in Cox-1(-/-) mice could be reduced by exogenous dimethyl-PGE(2). All damaged cells exfoliated, and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-1(+/-) (12 +/- 2 min, n = 12) than in Cox-1(-/-) (24 +/- 4 min, n = 14) mice. Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3-6 min). A smaller peak pH change was noted in Cox-1(-/-) mice (DeltapH = 0.3 +/- 0.04) than in Cox-1(+/-) mice (DeltapH = 0.6 +/- 0.2). Recovery to normal surface pH took longer in Cox-1(-/-) mice (27 +/- 5 min) than in Cox-1(+/-) mice (12 +/- 1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of microlesions.