Samo Ribarič

Interactions between cardiac, respiratory and EEG-δ oscillations in rats during anaesthesia

We hypothesized that, associated with the state of anaesthesia, characteristic changes exist in both cardio‐respiratory and cerebral oscillator parameters and couplings, perhaps varying with depth of anaesthesia. Electrocardiograms (ECGs), respiration and electroencephalograms (EEGs) were recorded from two groups of 10 rats during the entire course of anaesthesia following the administration of a single bolus of ketamine–xylazine (KX group) or pentobarbital (PB group). The phase dynamics approach was then used to extract the instantaneous frequencies of heart beat, respiration and slow δ‐waves (within 0.5–3.5 Hz). The amplitudes of δ‐ and θ‐waves were analysed by use of a time–frequency representation of the EEG signal within 0.5–7.5 Hz obtained by wavelet transformation, using the Morlet mother wavelet. For the KX group, where slow δ‐waves constituted the dominant spectral component, the Hilbert transform was applied to obtain the instantaneous δ‐frequency. The θ‐activity was spread over too wide a spectral range for its phase to be meaningfully defined. For both agents, we observed two distinct phases of anaesthesia, with a marked increase in θ‐wave activity occurring on passage from a deeper phase of anaesthesia to a shallower one. In other respects, the effects of the two anaesthetics were very different. For KX anaesthesia, the two phases were separated by a marked change in all three instantaneous frequencies: stable, deep, anaesthesia with small frequency variability was followed by a sharp transition to shallow anaesthesia with large frequency variability, lasting until the animal awoke. The transition occurred 16–76 min after injection of the anaesthetic, with simultaneous reduction in the δ‐wave amplitude. For PB anaesthesia, the two epochs were separated by the return of a positive response to the pinch test at 53–94 min, following which it took a further period of 45–70 min for the animal to awaken. δ‐Waves were not apparent at any stage of PB anaesthesia. We applied non‐linear dynamics and information theory to seek evidence of causal relationships between the cardiac, respiratory and slow δ‐oscillations. We demonstrate that, for both groups, respiration drives the cardiac oscillator during deep anaesthesia. During shallow KX anaesthesia the direction either reverses, or the cardio‐respiratory interaction becomes insignificant; in the deep phase, there is a unidirectional deterministic interaction of respiration with slow δ‐oscillations. For PB anaesthesia, the cardio‐respiratory interaction weakens during the second phase but, otherwise, there is no observable change in the interactions. We conclude that non‐linear dynamics and information theory can be used to identify different stages of anaesthesia and the effects of different anaesthetics.

Monitoring the depth of anaesthesia.

One of the current challenges in medicine is monitoring the patients’ depth of general anaesthesia (DGA). Accurate assessment of the depth of anaesthesia contributes to tailoring drug administration to the individual patient, thus preventing awareness or excessive anaesthetic depth and improving patients’ outcomes. In the past decade, there has been a significant increase in the number of studies on the development, comparison and validation of commercial devices that estimate the DGA by analyzing electrical activity of the brain (i.e., evoked potentials or brain waves). In this paper we review the most frequently used sensors and mathematical methods for monitoring the DGA, their validation in clinical practice and discuss the central question of whether these approaches can, compared to other conventional methods, reduce the risk of patient awareness during surgical procedures.

Diet and Aging

Nutrition has important long-term consequences for health that are not only limited to the individual but can be passed on to the next generation. It can contribute to the development and progression of chronic diseases thus effecting life span. Caloric restriction (CR) can extend the average and maximum life span and delay the onset of age-associated changes in many organisms. CR elicits coordinated and adaptive stress responses at the cellular and whole-organism level by modulating epigenetic mechanisms (e.g., DNA methylation, posttranslational histone modifications), signaling pathways that regulate cell growth and aging (e.g., TOR, AMPK, p53, and FOXO), and cell-to-cell signaling molecules (e.g., adiponectin). The overall effect of these adaptive stress responses is an increased resistance to subsequent stress, thus delaying age-related changes and promoting longevity. In human, CR could delay many diseases associated with aging including cancer, diabetes, atherosclerosis, cardiovascular disease, and neurodegenerative diseases. As an alternative to CR, several CR mimetics have been tested on animals and humans. At present, the most promising alternatives to the use of CR in humans seem to be exercise, alone or in combination with reduced calorie intake, and the use of plant-derived polyphenol resveratrol as a food supplement.

Redox control of protein degradation

Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies.

The Pharmacological Properties and Therapeutic Use of Apomorphine

Apomorphine (APO) is an aporphine derivative used in human and veterinary medicine. APO activates D1, D2S, D2L, D3, D4, and D5 receptors (and is thus classified as a non-selective dopamine agonist), serotonin receptors (5HT1A, 5HT2A, 5HT2B, and 5HT2C), and α-adrenergic receptors (α1B, α1D, α2A, α2B, and α2C). In veterinary medicine, APO is used to induce vomiting in dogs, an important early treatment for some common orally ingested poisons (e.g., anti-freeze or insecticides). In human medicine, it has been used in a variety of treatments ranging from the treatment of addiction (i.e., to heroin, alcohol or cigarettes), for treatment of erectile dysfunction in males and hypoactive sexual desire disorder in females to the treatment of patients with Parkinsons disease (PD). Currently, APO is used in patients with advanced PD, for the treatment of persistent and disabling motor fluctuations which do not respond to levodopa or other dopamine agonists, either on its own or in combination with deep brain stimulation. Recently, a new and potentially important therapeutic role for APO in the treatment of Alzheimer’s disease has been suggested; APO seems to stimulate Aβ catabolism in an animal model and cell culture, thus reducing the rate of Aβ oligomerisation and consequent neural cell death.

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

Interactions Between Intrinsic Regulation and Neural Modulation of Acetylcholinesterase in Fast and Slow Skeletal Muscles

Summary1.Initiation of subsynaptic sarcolemmal specialization and expression of different molecular forms of AChE were studied in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat under different experimental conditions in order to understand better the interplay of neural influences with intrinsic regulatory mechanisms of muscle cells.2.Former junctional sarcolemma still accumulated AChE and continued to differentiate morphologically for at least 3 weeks after early postnatal denervation of EDL and SOL muscles. In noninnervated regenerating muscles, postsynapticlike sarcolemmal specializations with AChE appeared (a) in the former junctional region, possibly induced by a substance in the former junctional basal lamina, and (b) in circumscribed areas along the whole length of myotubes. Therefore, the muscle cells seem to be able to produce a postsynaptic organization guiding substance, located in the basal lamina. The nerve may enhance the production or accumulation of this substance at the site of the future motor end plate.3.Significant differences in the patterns of AChE molecular forms in EDL and SOL muscles arise between day 4 and day 10 after birth. The developmental process of downregulation of the asymmetric AChE forms, eliminating them extrajunctionally in the EDL, is less efficient in the SOL. The presence of these AChE forms in the extrajunctional regions of the SOL correlates with the ability to accumulate AChE in myotendinous junctions. The typical distribution of the asymmetric AChE forms in the EDL and SOL is maintained for at least 3 weeks after muscle denervation.4.Different patterns of AChE molecular forms were observed in noninnervated EDL and SOL muscles regeneratingin situ. In innervated regenerates, patterns of AChE molecular forms typical for mature muscles were instituted during the first week after reinnervation.5.These results are consistent with the hypothesis that intrinsic differences between slow and fast muscle fibers, concerning the response of their AChE regulating mechanism to neural influences, may contribute to different AChE expression in fast and slow muscles, in addition to the influence of different stimulation patterns.

The contribution of lumbar sympathetic neurones activity to rat's skin blood flow oscillations.

Skin blood flow on the rat’s paws using laser Doppler flowmeter, electrical activity of the heart (ECG) and respiration were measured simultaneously. The signals were recorded for 20 minutes, both before and after denervation, at core temperature 37°C and 38.5°C, that was maintained constant during the recordings. Spinal nerve fibres, at the level L3–L4, were transected. Experiments were performed on 15 adult Wistar rats under general anaesthesia. The oscillations in the measured signals were analysed in the time-frequency domain using wavelet transform. On the frequency region from 0.7Hz to 5Hz two characteristic peaks were observed in the skin blood flow spectrum. They correspond to the main peaks in the spectra of the ECG (around 3.3Hz) and respiration (around 1.3Hz). Several additional peaks were observed in the low frequency region, from 0.01 to 0.7Hz, in all measured signals. In this frequency region the relative energy contribution of the blood flow oscillations decreased after denervation only in the denervated left hind paw. This difference was not statistically significant at 37°C (p=0.098, Kruskal-Wallis test) but became statistically significant at 38.5°C (p=0.017). Relative energy contribution of the low frequency region, from 0.01 to 0.7Hz, decreased 2.5-fold in the blood flow of the denervated paw. Within this region the relative energy contribution decreased significantly in two intervals, from 0.01 to 0.08Hz and from 0.08 to 0.2Hz (p=0.023). In the higher frequency region, from 0.7 to 5Hz, o statistically significant differences were obtained in any paws when compared before and after denervation at the same core temperature. We conclude that the activity of lumbar sympathetic neurones contributes to low frequency skin blood flow oscillations.

3D Visualization and Measurement of Capillaries Supplying Metabolically Different Fiber Types in the Rat Extensor Digitorum Longus Muscle During Denervation and Reinnervation

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential. (J Histochem Cytochem 57:437–447, 2009)

The estimation error of skeletal muscle capillary supply is significantly reduced by 3D method.

Capillary supply of individual skeletal muscle fibers is usually evaluated from two-dimensional (2D) images of thin transverse sections by the number of capillary profiles around a fiber (CAF). This method is inherently inaccurate and the resulting capillary length measurement errors can be avoided by using an alternative three-dimensional (3D) approach where the mean length of capillaries around individual muscle fibers per fiber length (Lcap/Lfib) is measured from 3D images acquired by confocal microscopy. We quantified the error of the 2D method and its reduction by using a 3D approach in realistic geometrical models of muscle fiber capillary bed and in true muscle samples. In models we showed that Lcap/Lfib was sensitive to different arrangements of capillaries, while CAF underestimated capillarization since it could not detect the increased length of capillary bed. In true muscle samples, we detected statistically significant differences in the capillary supply of control and denervated rat soleus muscles by both 2D and 3D methods. Lcap/Lfib was larger than CAF in control muscles reflecting their more complicated capillary bed. Thus, 3D approach is more sensitive in agreement with the analysis of geometrical models. We conclude that the 3D method, though technically more demanding than 2D method, represents a more precise approach to evaluation of muscle capillarization. Moreover, the 3D method could be applied to other organs and we suggest potential medical applications.