Visualizing excitation waves inside cardiac muscle using transillumination

Voltage-sensitive fluorescent dyes have become powerful tools for the visualization of excitation propagation in the heart. However, until recently they were used exclusively for surface recordings. Here we demonstrate the possibility of visualizing the electrical activity from inside cardiac muscle via fluorescence measurements in the transillumination mode (in which the light source and photodetector are on opposite sides of the preparation). This mode enables the detection of light escaping from layers deep within the tissue. Experiments were conducted in perfused (8 mm thick) slabs of sheep right ventricular wall stained with the voltage-sensitive dye di-4-ANEPPS. Although the amplitude and signal-to-noise ratio recorded in the transillumination mode were significantly smaller than those recorded in the epi-illumination mode, they were sufficient to reliably determine the activation sequence. Penetration depths (spatial decay constants) derived from measurements of light attenuation in cardiac muscle were 0.8 mm for excitation (520 +/- 30 nm) and 1.3 mm for emission wavelengths (640 +/- 50 nm). Estimates of emitted fluorescence based on these attenuation values in 8-mm-thick tissue suggest that 90% of the transillumination signal originates from a 4-mm-thick layer near the illuminated surface. A 69% fraction of the recorded signal originates from > or =1 mm below the surface. Transillumination recordings may be combined with endocardial and epicardial surface recordings to obtain information about three-dimensional propagation in the thickness of the myocardial wall. We show an example in which transillumination reveals an intramural reentry, undetectable in surface recordings.     

Probing field-induced tissue polarization using transillumination fluorescent imaging

Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2-6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model.     

Behavior of rats kept under conditions of a shifted light/dark regimen and receiving melatonin

We studied the effect of shift in the natural light/dark regimen (desynchronosis) and treatment with melatonin on behavioral characteristics of rats with different activity in the open-field test. Experiments were performed on 172 Wistar rats kept under conditions of the natural or shifted light/dark regimen. Some animals were intraperitoneally treated with 1 ml physiological saline or melatonin in doses of 1 and 2 mg/kg, while others did not receive the injections. Desynchronosis altered the normal rhythm of locomotor activity and abolished the differences between daytime and nighttime activity rats not receiving the injections. The influence of melatonin on locomotor activity of rats maintained under normal or shifted light/dark conditions depended on its dose, time of treatment, and initial behavioral characteristics of animals. Our results indicate that the use of melatonin for treatment of disturbances produced by a shift in the light/dark conditions should be performed taking into account individual behavioral characteristics of the organism.     

Extracting surface activation time from the optically recorded action potential in three-dimensional myocardium

Optical mapping has become an indispensible tool for studying cardiac electrical activity. However, due to the three-dimensional nature of the optical signal, the optical upstroke is significantly longer than the electrical upstroke. This raises the issue of how to accurately determine the activation time on the epicardial surface. The purpose of this study was to establish a link between the optical upstroke and exact surface activation time using computer simulations, with subsequent validation by a combination of microelectrode recordings and optical mapping experiments. To simulate wave propagation and associated optical signals, we used a hybrid electro-optical model. We found that the time of the surface electrical activation (t(E)) within the accuracy of our simulations coincided with the maximal slope of the optical upstroke (t(F)*) for a broad range of optical attenuation lengths. This was not the case when the activation time was determined at 50% amplitude (t(F50)) of the optical upstroke. The validation experiments were conducted in isolated Langendorff-perfused rat hearts and coronary-perfused pig left ventricles stained with either di-4-ANEPPS or the near-infrared dye di-4-ANBDQBS. We found that t(F)* was a more accurate measure of t(E) than was t(F50) in all experimental settings tested (P = 0.0002). Using t(F)* instead of t(F50) produced the most significant improvement in measurements of the conduction anisotropy and the transmural conduction time in pig ventricles.     

Impulse activity of neurons in the lateral hypothalamus during microionophoretic administration of melatonin and noradrenaline in rats

Microionophoretic administration of melatonin into the perineuronal space of lateral hypothalamic neurons in WAG and Fischer-344 rats decreased the firing rate and regularized activity of the cells. Moreover, the effects of melatonin completely blocked the activation of neurons and changes in their pulse activity produced by norepinephrine. The effects of melatonin on neuronal activity in behaviorally active stress-resistant WAG rats were more pronounced than in behaviorally passive stress-predisposed Fischer-344 rats. These data suggest that stress-protective activity of melatonin is associated with inhibition of the pulse activity of neurons in emotiogenic structures of the brain and changes in neuronal sensitivity to norepinephrine.     

Effect of interleukin-1beta and interleukin-4 on serum albumin parameters in rats with various behavioural characteristics

We studied the effects of pro-inflammatory cytokine interleukin-1beta and antiinflammatory cytokine interleukin-4 on serum albumin parameters in rats with various behavioural characteristics in the open-field test. Under control conditions, the total concentration of serum albumin in active animals was higher than in passive those. However, the ratio of the effective-to-total concentration of albumin (i.e., binding capacity of this protein) was greater in passive rats. Administration of interleukins was accompanied by a decrease in the total content and effective concentration of albumin in passive and, particularly, in active rats. The initial intergroup differences in the ratio of the effective-to-total concentration of albumin were not found after injection of immunomodulating agents. It was mainly related to a more significant increase in this parameter in active animals. An increase in the binding capacity of albumin after cytokine treatment could be associated with conformational changes in the protein molecule. We believe that interleukin-1b and interleukin-4 exert similar effects on the properties of binding sites of serum albumin in rats with various behavioural characteristics.