Pilot Experiment on Non-Invasive Non-Thermal Disintegration of Human Mucinous Breast Carcinoma Ex Vivo Using Boiling Histotripsy.

We present the results of a pilot study demonstrating the feasibility of non-invasive non-thermal disintegration of human mucinous carcinoma of the breast ex vivo using sequences of high-intensity focused ultrasound pulses in boiling histotripsy regimen. The target volume was sonicated by focusing ultrasound pulses (n=20) of 1.5 MHz frequency, 10-msec duration and 1-sec pulse repetition period, 517 W acoustic power within the pulse, and 103 MPa shock front amplitude at the focus into each node of a volumetric grid 4×4×1 mm. Sonication was visualized and controlled using B-mode ultrasound imaging, total time of the treatment was 21 min. Histological hematoxylin and eosin and Masson's trichrome staining revealed the absence of tumor elements in the treated region confirming destruction of cancer cells and their nuclei after boiling histotripsy procedure.

Pilot ex vivo study on non-thermal ablation of human prostate adenocarcinoma tissue using boiling histotripsy.

Focused ultrasound technologies are of growing interest for noninvasive ablation of localized prostate cancer (PCa). Here we present the results of the first case study evaluating the feasibility of non-thermal mechanical ablation of human prostate adenocarcinoma tissue using the boiling histotripsy (BH) method on ex vivo tissue. High intensity focused ultrasound field was generated using a 1.5-MHz custom-made transducer with nominal F#=0.75. A sonication protocol of 734 W acoustic power, 10-ms long BH-pulses, 30 pulses per focal spot, 1 % duty cycle, and 1 mm distance between single foci was tested in an ex vivo human prostate tissue sample containing PCa. The protocol used here has been successfully applied in the previous BH studies for mechanical disintegration of ex vivo prostatic human tissue with benign hyperplasia. BH treatment was monitored using B-mode ultrasound. Post-treatment histologic analysis demonstrated BH produced liquefaction of the targeted tissue volume. BH treated benign prostate parenchyma and PCa had similar tissue fractionation into subcellular fragments. The results of the study demonstrated that PCa tumor tissue can be mechanically ablated using the BH method. Further studies will aim on optimizing protocol parameters to accelerate treatment while maintaining complete destruction of the targeted tissue volume into subcellular debris.

Functional Activity of Non-Proliferating Mesenchymal Stromal Cells Cultured at Different Densities.

We analyzed the state of intracellular compartments and production of cytokines in MSC depending on the culture density. MSC were growth-arrested with mitomycin C and seeded at a density of 300-7000 cell/cm2. MSC in low-density cultures had 2-fold higher levels of transmembrane mitochondrial potential (MitoTracker Red) and endogenous ROS (CMH2DCFDA), lysosomal compartments were less acidified (LysoTracker Green DND26), the production of immunoregulatory and angiogenic mediators VEGF, IL-6, IL-8, MCP-1, TGF-ß was more intensive. It was assumed that culture density can be an effective tool for phenotypic polarization of MSC providing directional changes in their properties.

[A novel method for non-invasive mechanical ablation of prostate tumors using pulsed focused ultrasound].

AIM: of the study: demonstrate the feasibility of non-invasive mechanical disintegration of human prostate tissue using pulsed high-intensity focused ultrasound (pHIFU), a method termed boiling histotripsy. MATERIALS AND METHODS: An ultrasound experimental system was developed for producing localized mechanical lesions in ex vivo biological tissue samples under ultrasound guidance. A series of experiments was carried out to create small single-focus lesions (volume < 2 mm3) and one large lesion (volume > 50 mm3) in ex vivo prostate tissue samples. After irradiation, two samples were bisected to visualize the region of destruction; the other tissue samples were examined histologically. RESULTS: During pHIFU irradiation under B-mode ultrasound guidance, a region of increased echogenicity caused by formation of vapor-gas bubbles was visualized in the target region. After exposure, small and large lesions filled with a suspension of liquefied tissue were observed. Histological examination confirmed that the prostate tissue in the focal region was disintegrated into subcellular fragments. CONCLUSION: A pilot study showed the feasibility of using boiling histotripsy as a non-invasive method for treating prostate diseases.

[The role of ATP-dependent potassium channels and nitric oxide system in the neuroprotective effect of preconditioning]. / Rol' ATF-chuvstvitel'nykh kalievykh kanalov i oksida azota v zashchitnom effekte prekonditsionirovaniya mozga.

AIM: To study a role of ATP-dependent potassium channels (K+ATP) in the neuroprotective effect of ischemic (IP) and pharmacological (PP) preconditioning and evaluate the dynamics of blood nitric oxide (NO) metabolites in cerebral ischemia. MATERIAL AND METHODS: A model of ischemic stroke induced by the electrocoagulation of a middle cerebral artery (MCA) branch was used in male rats (n=86). Glibenclamide, a selective inhibitor of ATP-sensitive K+ channels, and diazoxide, a potassium channel activator, were used. IP and PP were performed 24 h before MCA occlusion. Blood concentrations of NO, NO3- and NO2-were measured 5, 24 and 72 h after occlusion. RESULTS: IP decreased a lesion area by 37% (p<0/05) and the preliminary introduction ofglibenclamide levelled the effect of IP. A protective effect of PP was similar to that of IP. A decrease in oxygenated R-conformers of Hb-NO and a reverse increase in non-oxygenated T-conformers as well as NO3- и NO2-were noted 5h after MCA occlusion. In the first 24 h after MCA occlusion, contents of NO3- and NO2- returned to normal values. There were changes in the concentrations of Hb-NO complexes as well, with the predominance of R-conformers and minimal contents of T-conformers. Moreover, the correlations between K+ATP channel blockade and the decrease in serum NO3- and NO2 were found (p<0/03). CONCLUSION: The neuroprotective effect of preconditioning is caused by the activation of K+ATP channels. An analysis of NO metabolite concentrations in the blood of rats with IP suggests that Hb-NO complexes belonging to R-conformers deposit and carry NO in tissues releasing NO accumulated via R→T transfer in conditions of ischemia.

Changes in Sympathetic Innervation of Rat Caudal Artery in Experimental Myocardial Infarction. Effect of Semax Peptide.

Activation of the sympathetic nervous system aggravates the course of myocardial infarction. Semax peptide moderated the degree of this activation and prevented the increase in the density of sympathetic endings in rat caudal artery in 28 days after ischemia or ischemia/reperfusion. The peptide reduced the density of α-adrenoreceptors in the caudal artery of rats with myocardial infarction. Semax produced no effect on ß-adrenoreceptors in both experimental models. The experiments on isolated segments of the caudal artery revealed reduced vascular responsiveness to electrical stimulation and norepinephrine infusion in rats treated with Semax after ischemia/reperfusion injury.

[INFLUENCE OF 29-40 AND 65-76 MCP-1 FRAGMENTS ON MYOCARDIUM MORPHOLOGY IN RATS AFTER T9CTHFMIA-R'FPFRFTTON].

Monocyte chemotactic protein-1 (MCP-1) is a chemokine that stimulates monocytes and macrophage migration into the sites of acute of chronic inflammation. Our study shows morphological changes in ischemic myocardium followed by the administration of two synthetic structural fragments of MCP-1 that are monocyte/macrophage migration inductor peptide IX and peptide X an inhibitor. Results show that peptides can change time points of the inflammatory response in myocardium. Peptide IX administration leads to increased and accelerated inflammatory response, i. e. attracts an additional number of monocytes and macrophages into the inflammatory focus. The introduction of the peptide X observed prolonged inflammatory process with the overall gain signs of myocardial damage.

Effect of proinflammatory activation on F-actin distribution in cultured human endothelial cells under conditions of experimental microgravity.

We compared the state of actin cytoskeleton, morphology, and expression of VE-cadherin in endothelial cells of human umbilical cord vein under conditions of TNF-α-mediated activation and microgravity modeling and found that 3D-clinorotation for 24 h impaired the integrity of endothelial monolayer, altered cell morphology, induced cytoskeleton reorganization, and reduced the expression of VE-cadherin. The combination of experimental microgravity and proinflammatory activation led to more pronounced clearing of the perinuclear space from microfilaments and accumulation of depolymerized actin, which confirms additive effect of the studied factors on actin cytoskeleton of endothelial cells.

Comparison of mitochondrial fluorescent dyes in stromal cells.

Specific fluorescent mitochondrial dyes JC-1, MitoTracker Red FM, and MitoTracker Red CMXRos were studied on cultures of mesenchymal stromal stem cells from human adipose tissue. Static and dynamic experiments demonstrated the dependence of dye accumulation in the mitochondria and cytoplasm on mitochondrial potential in the presence of oxidative phosphorylation uncoupler CCCP. JC-1 dye demonstrated most pronounced bleaching upon repeated laser exposure even in very low doses, which should be taken into account when planning dynamic experiments on live cells. Moreover, this dye requires two-wave recording in red and green spectral bands. MitoTracker Red FM and MitoTracker Red CMXRos are resistant to bleaching, but for the latter, the intensity of fluorescence reflects not only the mitochondrial potential. MitoTracker Red CMXRos covalently binds to sulfhydryl groups in mitochondrial lipids and proteins, which can lead to misinterpretation of the fluorescence intensity-mitochondrial potential dependence.

Simple method of specimen preparation for scanning electron microscopy.

We compared conventional method for specimen (cell cultures, tissue specimens) preparation for scanning electron microscopy and a method without sputtering and critical-point drying. OTO-method (osmium-thiocarbohydrazide-osmium) with sample impregnation with hexamethyldisilazane followed by air drying was used as an alternative method. Excellent preservation of surface ultrastructures and electrical conductivity was proved. The method is easy to use and does not require additional costs for equipment.

[Structural features of cerebral arteriovenous malformations according to the data of optic microscopy and scanning electron microscopy of corrosion specimens].

The aim of this study was to investigate structure of cerebral arteriovenous malformations (AVM). We examined and treated 5 patients with AVMs of cerebral hemispheres. Treatment consisted of endovascular embolization with subsequent surgical resection of AVM. The series included 3 female and 2 male patients (age 19-44, mean - 28 years). Histoacryl (B. Braun, Germany) was used for embolization. Corrosion specimens were prepared according to standard technique and examined using optic and scanning electron microscopy. AVMs had similar structure. Casts were formed by distal segments of afferent arteries, fistulas, interjacent venous vessels, proximal segments of draining veins and vessels of angiomatous surrounding. The latter consisted of abnormal network of small arteries, veins, fistulas and dilated capillaries. The most variable length and formation of glomeruli was characteristic for interjacent venous vessels. AVMs extensively anastomosed with cerebral vessels. Big amount of aneurysms and aneurysmatic bulgings was present. Aneurysms of intranidal segment of afferent artery and angiomatous vessels are demonstrated.

[Influence of clinorotation on embryoid bodies morphology].

The compensative effects of gravitation at the first stages of embryonic development stages have been investigated using slow clinorotation of embryoid bodies from mouse R1 stem cell lines. Semithin sections (1-2 microm) analysis and electron microscopy study of embryonic bodies cells allowed to reveal morphological features of cells at different maturation stages. Significant decrease in the number of embryonic stem cells undergoing apoptosis as well as noticeable reduction of "lacunas" relative areas were found in clinorotated embryonic bodies compared to control. We propose that large cyst absence may be caused by initial differentiation and morphogenesis stages delay associated with autophagy processes in embryonic bodies.

[Comparative analysis of vasomotor responses and sympathetic innervation of feed arteries from locomotor and respiratory muscles in a rat].

The characteristics of feeding arteries of diaphragm and medial gastrocnemius (with a diameter of 200-250 micron) were studied. The registration of the mechanical activity of ring preparations under isometric conditions revealed that diaphragm arteries, like arteries of other muscles with a high content of slow muscle fibers, are highly sensitive to adrenoceptor agonists and acetylcholine. The differences in endothelium-dependent relaxation between diaphragm and gastrocnemius arteries are preserved in the presence of L-NAME and diclofenac. Responses to serotonin in diaphragm and gastrocnemius arteries are similar. At the same time, the high density of innervation is characteristic of diaphragm artery only, while in other slow muscles it is low. The density of adrenergic fibers plexus in the diaphragm artery is much higher than in the gastrocnemius artery. The results suggest that the properties of small arteries of diaphragm are determined not only by the oxidative capacity of diaphragm muscle fibers but also by the belonging of the diaphragm to respiratory musculature.

[Antistress and angioprotective influence of nitric oxide in epilepsy-prone rats of Krushinskii-Molodkina strain].

In rats of Krushinsky-Molodkina strain (KMR), the audiogenic stress induced epileptiform seizure and development of acute disturbances of cerebral circulation of hemorrhagic nature. The inhibitor of NO-synthase (L-NNA) increased the severity of clinical symptoms, mortality, and the intensity of intracranial hemorrhages. In contrast, L-arginine elevated the resistance of KMRs to acoustic stress. The intensity of nitrergic innervation was analyzed in preparations of the middle cerebral artery with the use of histochemical NADPH-diaphorase staining. In preparations of control KMRs, a net of NADPH-positive perivascular nerve fibers was found, while in experimental KMRs, in an hour after sound stimulation, such fibers practically were not revealed. Preliminary exposure of KMRs in hypoxic conditions (1 hour in hypobaric chamber at simulated altitude of 5000 m above the sea level) decreased the development of stress lesions. The protective effect of hypoxic training disappeared after the administration of NO-synthase inhibitor (L-NNA). The study demonstrated participation of nitric oxide (NO) in adaptive reactions of cerebral hemodynamics linked with the significant increase of cerebral blood flow.

Scanning electron microscopy of rat erythrocytes during chronic alcoholic intoxication combined with protein and vitamin deficit.

Electron microscopy revealed morphological changes in erythrocytes from rats with chronic alcoholic intoxication kept on protein- and vitamin B-deficient rations. All animals had anisopoikilocytosis (up to 50%). Most pronounced changes in erythrocyte population attesting to accelerated erythrocyte aging (stomato- and microcytosis, discocyte swelling, and spontaneous hemolysis) were found in alcohol-fed rats kept on deficient ration.

[Ultrastructural changes in the aortic arch intima in rats with various resistance to emotional stress]. / Izmeneniia ul'trastruktury intimy dugi aorty u krys s razlichnoi ustoichivost'iu k émotsional'nomu stressu.

Experiments were performed on 24 male Wistar rats weighing 210 +/- 14 g. Depending on the activity in the open field test, all rats were divided into behaviorally active and passive subgroups. Water-immersion stress (2 hrs) followed by 2-hrs rest period was used as the model of acute emotional stress. Tissue samples from aortic ascending arch were taken from control and stressed rats immediately after decapitation and used for further electron microscopy. The number of micropinocytosis vesicles along the free and basal layers of the cytoplasm and the area of mitochondria were determined. Emotional stress was shown to intensify metabolic processes involving micropinocytosis vesicles between the aortic lumen and its wall. Passive rats display the most severe alterations induced by stress (damages of aortic endotheliocytes, partial regeneration of the plasmalemma by membranes of micropinocytosis vesicles, and decrease in the average area of mitochondria). Morphological equivalents of the initial stages of emotional stress-induced formation of fibrous changes in the aortic arch intima are believed to be found. Such changes can lead to the appearance of atherosclerotic patches.

Telemedicine: capabilities of telecommunications in clinical practice in Russia.

Successful implementation of telemedicine services depends on an adequate telecommunications infrastructure. At present the main infrastructure in Russia and the countries of the former Soviet Union consists of analogue telephone lines. These are sufficient for e-mail and access to the World Wide Web but do not provide sufficient bandwidth for the transmission of realtime teleconsultations. ISDN lines and fibre optic cables are becoming more widespread in the major cities but have yet to be installed in the regions.

Simple PC-based system for morphometric analysis of synapses.

The computer system designed for synaptic morphometry of aksosomatic and aksospine synapses of brain is described in the present paper. It is based on an AT-comparable personal computer equipped with low-cost frame grabber. This hardware configuration allows to input images from any TV source such as TV camera, videorecorder for further processing. The appropriate software was written in Microsoft Quick Basic to measure the main morphometric parameters of axon ending, dendritic spines, total contact, active zone and mitochondria. Number of synaptic vesicles (total and active) were also counted using a mouse as a pointing device. The derivative parameters (vesicle density, mitochondrial density) are then calculated. All measured data are stored in ASCII format, allowing ease in editing and export into other application programs. Statistical evaluation and calculation of histograms is performed by associated program also written in Quick Basic. The advantages and disadvantages of this approach are discussed.

[The mapping of the local content of water and dry matter by using ultrathin frozen sections]. / Kartirovanie lokal'nogo soderzhaniia vody i sukhogo veshchestva s ispol'zovaniem ul'tratonkikh zamorozhennykh srezov.

The possibility and applications of quantitative dry mass and water content mapping using an image analysis of darkfield intensity digitalization were shown on ultrathin freeze-dried cryosections. The distribution map obtained may be further used both for direct measurements of local dry mass and water content under physiological state and pathological disorders and for calculation of the concentration values obtained by quantitative X-ray mapping on freeze-dried cryosections in terms of millimoles per litre of cellular water.