Acute and Sub-Chronic Intraperitoneal Toxicity Studies of the Elsholtzia ciliata Herbal Extract in Balb/c Mice.

Elsholtzia ciliata essential oil (E. ciliata) has been reported to have an impact on the cardiovascular system. However, its toxicity remains unknown. Therefore, the objective of this investigation was to evaluate the toxicological aspects of the E. ciliata extract. Male Balb/c mice were subjected to either acute (a single dose administered for 24 h) or sub-chronic (daily dose for 60 days) intraperitoneal injections of the E. ciliata extract. The mice were assessed for blood hematological/biochemical profiles, mitochondrial functions, and histopathological changes. Additionally, in vitro cytotoxicity assessments of the E. ciliata extract were performed on immobilized primate kidney cells (MARC-145, Vero) and rat liver cells (WBF344) to evaluate cell viability. The control groups received an equivalent volume of olive oil or saline. Our results demonstrated no significant detrimental effects on hematological and biochemical parameters, mitochondrial functions, cellular cytotoxicity, or pathological alterations in vital organs following the intraperitoneal administration of the E. ciliata extract over the 60-day sub-chronic toxicity study. In general, E. ciliata displayed no indications of toxicity, suggesting that the E. ciliata extract is a safe natural product with a well-defined therapeutic and protective index (found to be 90 and 54, respectively) in Balb/c mice.

Evaluation of the Cardiac Electrophysiological and Haemodynamic Effects of Elsholtzia ciliata Essential Oil on Swine.

The demand for the development of novel medicines with few side effects and no proarrhythmic properties is increasing. Extensive research on herbal extracts has been conducted with the expectation that the compounds will exert precise effects without harmful side effects. Elsholtzia ciliata (Thunb.) Hyl. essential oil (EO) possesses antiarrhythmic properties similar to those of class 1B antiarrhythmics, such as prolonging myocardial activation of the QRS complex and shortening the QT interval. In this study, we determined the kinetic profile of EO phytocompounds and the effects of EO on heart electrical activity and arterial blood pressure. For this study, we chose to use local breed pigs that were anaesthetized. The effects of an intravenous bolus of EO on ECG parameters, arterial blood pressure, heart rate variability, and blood levels of haematological and biochemical parameters were registered and evaluated. Following an intravenous injection of a bolus, EO exerted a vasodilatory effect, resulting in significant reductions in arterial blood pressure. EO also increased the heart rate and altered ECG parameters. The bolus of EO prolonged the QRS complex, shortened the QT interval, and nonmonotonically altered the PQ interval. After the administration of a bolus of EO, the activity of the autonomic nervous system was altered. This study confirms that EO possesses similar properties to class 1B antiarrhythmics and exerts a hypotensive effect; it reduces arterial blood pressure possibly by modulating peripheral vascular resistance.

Antiarrhythmic Properties of Elsholtzia ciliata Essential Oil on Electrical Activity of the Isolated Rabbit Heart and Preferential Inhibition of Sodium Conductance.

Elsholtzia ciliata essential oil (E. ciliata) has been developed in Lithuania and internationally patented as exerting antiarrhythmic properties. Here we demonstrate the pharmacological effects of this herbal preparation on cardiac electrical activity. We used cardiac surface ECG and a combination of microelectrode and optical mapping techniques to track the action potentials (APs) in the Langendorff-perfused rabbit heart model during atrial/endo-/epi-cardial pacing. Activation time, conduction velocity and AP duration (APD) maps were constructed. E. ciliata increased the QRS duration and shortened QT interval of ECG at concentrations of 0.01-0.1 µL/mL, whereas 0.3 µL/mL (0.03%) concentration resulted in marked strengthening of changes. In addition, the E. ciliata in a concentration dependent manner reduced the AP upstroke dV/dtmax and AP amplitude as well as APD. A marked attenuation of the AP dV/dtmax and a slowing spread of electrical signals suggest the impaired functioning of Na+channels, and the effect was usedependent. Importantly, all these changes were at least partially reversible. Our results indicate that E. ciliata modulates cardiac electrical activity preferentially inhibiting Na+ conductance, which may contribute to its effects as a natural antiarrhythmic medicine.

Optical mapping of the pig heart in situ under artificial blood circulation.

The emergence of optical imaging has revolutionized the investigation of cardiac electrical activity and associated disorders in various cardiac pathologies. The electrical signals of the heart and the propagation pathways are crucial for elucidating the mechanisms of various cardiac pathological conditions, including arrhythmia. The synthesis of near-infrared voltage-sensitive dyes and the voltage sensitivity of the FDA-approved dye Cardiogreen have increased the importance of optical mapping (OM) as a prospective tool in clinical practice. We aimed to develop a method for the high-spatiotemporal-resolution OM of the large animal hearts in situ using di-4-ANBDQBS and Cardiogreen under patho/physiological conditions. OM was adapted to monitor cardiac electrical behaviour in an open-chest pig heart model with physiological or artificial blood circulation. We detail the methods and display the OM data obtained using di-4-ANBDQBS and Cardiogreen. Activation time, action potential duration, repolarization time and conduction velocity maps were constructed. The technique was applied to track cardiac electrical activity during regional ischaemia and arrhythmia. Our study is the first to apply high-spatiotemporal-resolution OM in the pig heart in situ to record cardiac electrical activity qualitatively under artificial blood perfusion. The use of an FDA-approved voltage-sensitive dye and artificial blood perfusion in a swine model, which is generally accepted as a valuable pre-clinical model, demonstrates the promise of OM for clinical application.

Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart.

Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the ICaL and transient outward potassium current (Ito) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg2+ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K+ current (IK1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the ICaL, Ito, and IK1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.

Spectral characteristics of voltage-sensitive indocyanine green fluorescence in the heart.

Indocyanine green (ICG) fluorescent dye has been approved by the FDA for use in medical diagnostics. Recently, we demonstrated that ICG dye has voltage-sensitive properties with a dual-component (fast and slow) response in the Langendorff-perfused rabbit heart. Here, we extended our studies by showing the different spectral properties of both components for analysis of the fractional change in ICG fluorescence in response to voltage changes. We used light from four LEDs to obtain excitation; emission was measured using an EMCCD camera with band-pass filters and a spectrometer. We applied a graphical model with Gaussian functions to construct and evaluate the individual emission curves and calculated the voltage-sensitive portion of each component of the ICG fluorescence in the rabbit heart. The results revealed that each isolated component (fast and slow) emanates from a unique ICG pool in a different environment within the cell membrane and that each component is also composed of two constituents (ICG-monomeric and ICG-aggregated). We propose the existence of different voltage-sensitive mechanisms for the components: (I) electrochromism and field-induced reorientation for the fast component; and (II) field-induced dye squeezing that amplifies intermolecular interactions, resulting in self-quenching of the dye fluorescence, for the slow component.

Voltage-Sensitive Fluorescence of Indocyanine Green in the Heart.

So far, the optical mapping of cardiac electrical signals using voltage-sensitive fluorescent dyes has only been performed in experimental studies because these dyes are not yet approved for clinical use. It was recently reported that the well-known and widely used fluorescent dye indocyanine green (ICG), which has FDA approval, exhibits voltage sensitivity in various tissues, thus raising hopes that electrical activity could be optically mapped in the clinic. The aim of this study was to explore the possibility of using ICG to monitor cardiac electrical activity. Optical mapping experiments were performed on Langendorff rabbit hearts stained with ICG and perfused with electromechanical uncouplers. The residual contraction force and electrical action potentials were recorded simultaneously. Our research confirms that ICG is a voltage-sensitive dye with a dual-component (fast and slow) response to membrane potential changes. The fast component of the optical signal (OS) can have opposite polarities in different parts of the fluorescence spectrum. In contrast, the polarity of the slow component remains the same throughout the entire spectrum. Separating the OS into these components revealed two different voltage-sensitivity mechanisms for ICG. The fast component of the OS appears to be electrochromic in nature, whereas the slow component may arise from the redistribution of the dye molecules within or around the membrane. Both components quite accurately track the time of electrical signal propagation, but only the fast component is suitable for estimating the shape and duration of action potentials. Because ICG has voltage-sensitive properties in the entire heart, we suggest that it can be used to monitor cardiac electrical behavior in the clinic.

Evaluation of excitation propagation in the rabbit heart: optical mapping and transmural microelectrode recordings.

BACKGROUND: Because of the optical features of heart tissue, optical and electrical action potentials are only moderately associated, especially when near-infrared dyes are used in optical mapping (OM) studies. OBJECTIVE: By simultaneously recording transmural electrical action potentials (APs) and optical action potentials (OAPs), we aimed to evaluate the contributions of both electrical and optical influences to the shape of the OAP upstroke. METHODS AND RESULTS: A standard glass microelectrode and OM, using an near-infrared fluorescent dye (di-4-ANBDQBS), were used to simultaneously record transmural APs and OAPs in a Langendorff-perfused rabbit heart during atrial, endocardial, and epicardial pacing. The actual profile of the transmural AP upstroke across the LV wall, together with the OAP upstroke, allowed for calculations of the probing-depth constant (k ~2.1 mm, n = 24) of the fluorescence measurements. In addition, the transmural AP recordings aided the quantitative evaluation of the influences of depth-weighted and lateral-scattering components on the OAP upstroke. These components correspond to the components of the propagating electrical wave that are transmural and parallel to the epicardium. The calculated mean values for the depth-weighted and lateral-scattering components, whose sum comprises the OAP upstroke, were (in ms) 10.18 ± 0.62 and 0.0 ± 0.56 for atrial stimulation, 9.37 ± 1.12 and 3.01 ± 1.30 for endocardial stimulation, and 6.09 ± 0.79 and 8.16 ± 0.98 for epicardial stimulation; (n = 8 for each). For this dye, 90% of the collected fluorescence originated up to 4.83 ± 0.18 mm (n = 24) from the epicardium. CONCLUSIONS: The co-registration of OM and transmural microelectrode APs enabled the probing depth of fluorescence measurements to be calculated and the OAP upstroke to be divided into two components (depth-weighted and lateral-scattering), and it also allowed the relative strengths of their effects on the shape of the OAP upstroke to be evaluated.

Evolution of action potential alternans in rabbit heart during acute regional ischemia.

This study investigates the development of the spatiotemporal pattern of action potential alternans during acute regional ischemia. Experiments were carried out in isolated Langendorff-perfused rabbit heart using a combination of optical mapping and microelectrode recordings. The alternans pattern significantly changed over time and had a biphasic character reaching maximum at 6-9 min after occlusion. Phase I (3-11 minutes of ischemia) is characterized by rapid increase in the alternans magnitude and expansion of the alternans territory. Phase I is followed by gradual decline of alternans (Phase II) in both magnitude and territory. During both phases we observed significant beat-to-beat variations of the optical action potential amplitude (OAPA) alternans. Simultaneous microelectrode recordings from subepicardial and subendocardial layers showed that OAPA alternans coincided with intramural 2 : 1 conduction blocks. Our findings are consistent with the modeling studies predicting that during acute regional ischemia alternans can be driven by 2 : 1 conduction blocks in the ischemic region.

Optical mapping at increased illumination intensities.

Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5 mW/mm2, with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30%. At 660-nm excitation, the effect is ∼10%. These findings may have important implications for the design of optical mapping protocols in biomedical applications.