Rysevaitė-Kyguolienė, Kristina

The aim of this study was to investigate the distribution, density, and morphological characteristics of small intensely fluorescent (SIF) cells within the ventricles of rat hearts and to evaluate their spatial relationship with cardiac neural structures. The study was performed using whole-mount ventricular preparations obtained from three Wistar rats (Rattus norvegicus). The preparations were immunohistochemically stained using antibodies against tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calcitonin gene-related peptide (CGRP). Fluorescence microscopy and panoramic imaging were used for visualization and qualitative assessment, while Fiji ImageJ, Microsoft Excel, and SPSS software were used for quantitative analysis.

SIF cells were identified based on TH immunoreactivity and were observed in all examined ventricular preparations. The cells demonstrated heterogeneous distribution patterns within the ventricular myocardium and were present both individually and in clusters. Most SIF cells exhibited round or oval morphology, although elongated cells with processes were also identified. SIF cells were commonly located near TH-positive nerve fibers and epicardial nerve bundles. Some SIF cell clusters were additionally associated with CGRP- and nNOS-positive nerve fibers, suggesting anatomical relationships with sensory and nitrergic components of the intrinsic cardiac nervous system.

Quantitative analysis demonstrated moderate inter-sample variability. The mean total number of SIF cells per heart was 191 ± 48.0, while the mean number of clusters was 13.7 ± 1.2. Round SIF cells represented approximately 71.7% of the total SIF cell population, whereas cells with processes accounted for 28.3%. Most SIF cell clusters were located near nerve fibers rather than within the myocardium.

The findings of this study demonstrate that SIF cells are integral components of the ventricular intrinsic cardiac nervous system and may contribute to local autonomic regulation within the heart.

Author: Edwin Paul Title: Changes in coronary vascular innervation in arterial hypertension. Aim: To investigate the impact of prolonged untreated arterial hypertension on nerve fibers in the perivascular area of coronary vessels, using spontaneously hypertensive rats of various ages, applying immunohistochemical analysis Objectives: 1.To assess differences in general (PGP9.5), peptidergic (CGRP-positive) and adrenergic (TH-positive) nerve fibers between young WKY (9-12 weeks) and old WKY (40-43 weeks) rats 2. To assess differences in general (PGP9.5), peptidergic (CGRP-positive) and adrenergic (TH-positive) nerve fibers between young and old SHR 3. To assess differences in general (PGP9.5), peptidergic (CGRP-positive) and adrenergic (TH-positive) nerve fibers between young WKY and SHR 4. To assess differences in general (PGP9.5), peptidergic (CGRP-positive) and adrenergic (TH-positive) nerve fibers between old WKY and SHR Material and methods: The study was conducted on male Wistar-Kyoto (WKY) rats, which was the normotensive group and spontaneously hypertensive rats (SHR) which was the hypertensive group. They were categorized into two age groups: young (8–12 weeks) and old (40–43 weeks). Animals were housed under standard laboratory conditions with a 12/12-hour light/dark cycle, controlled temperature (22 ± 2°C), and ad libitum access to food and water. After reaching the designated age, rats were euthanized in accordance with EU Directive 86/609/EEC and institutional ethical approval (No. G2-137). The heart tissues were collected then fixed in 4% paraformaldehyde then sectioned in a transverse way in order to obtain tissue samples of the right ventricle, left ventricle, and interventricular septum of rat specimens. Immunohistochemistry was performed to visualize tyrosine hydroxylase (TH) for sympathetic innervation, calcitonin gene-related peptide (CGRP) for sensory innervation, and PGP 9.5 as a pan-neuronal marker. These tissue samples were imaged using a Zeiss Axio Imager microscope with its accompanying Axiovision software, and morphometric analysis was performed with FIJI (ImageJ) software to quantify the percentage area of nerve fiber innervation in coronary vessels. Results: Analysis between Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) showed that CGRP (peptidergic) innervation was significantly higher in SHR rats across all cardiac regions compared to WKY control. The results we found in the interventricular septum indicated that CGRP levels showed an increase difference of 0.12 ± 0.03 in old WKY to 0.44 ± 0.07 in old SHR (p = 0.004), then in right ventricle (0.11 ± 0.06 vs. 0.40 ± 0.28, p = 0.284). In the LV, CGRP was higher in SHR but without statistical significance. TH-positive adrenergic fibers showed modest increased differences in SHR rats, especially in the RV of old animals (0.21± 0.18 in WKY vs. 0.59 ± 0.24 in SHR, p = 0.329). PGP9.5, representing total neural density, confirmed these findings: SHR rats had significantly higher PGP9.5 in the IVS (0.86 ± 0.01 vs. 0.46 ± 0.06, p = 0.048) and increased values in the RV and LV. CGRP levels in WKY rats showed a decrease between the young and old strain but such an observation was not made in SHR, suggesting strain- dependent preservation of sensory fibers. SHR rats showed consistent upregulation of both sensory and sympathetic innervation. All analysis was done using simple t-test and all statistical significance set at 95 percent (p<0.05). Conclusions:

1. SHR rats showed consistently higher PGP9.5 and CGRP-positive innervation than WKY rats, particularly in the interventricular septum and right ventricle. Adrenergic innervation was moderately elevated in SHR, indicating strain- related autonomic remodeling.
2. Young SHR rats had significantly more CGRP and PGP9.5 positive fibers than WKY, indicating early sensory remodeling. Adrenergic differences were minor, suggesting sensory changes precede sympathetic alterations in hypertension.
3. In SHR rats, CGRP expression slightly declined with age but remained higher than in WKY. TH and PGP9.5 innervation were preserved or mildly increased, suggesting age has a limited effect on autonomic remodeling in hypertensive hearts.
4. Old SHR rats maintained higher CGRP and TH innervation, particularly in the interventricular septum and right ventricle. PGP9.5 confirmed enhanced total innervation, supporting sustained autonomic remodeling in chronic hypertension.

Author: Dhruv Patil Title: Histochemical Alterations in the kidneys under long term hypertensive disease Aim: To investigate the changes of CGRP, CLR and AGT1R expression in the SHR and WKY rat kidneys, related to persistent hypertension. Objectives: 1. This study aims to assess the cumulative immunoreactivity for CGRP across various anatomical structures of the kidney, including the capsule, glomeruli, PCT, DCT, and stroma in the cortex, as well as the loop of Henle, collecting tubules, collecting ducts, and stroma in the medulla, in both prehypertensive and aged WKY rats and SHR.2. This study aims to assess the cumulative immunoreactivity for CLR across various anatomical structures of the kidney, including the capsule, glomeruli, PCT, DCT, and stroma in the cortex, as well as the loop of Henle, collecting tubules, collecting ducts, and stroma in the medulla, in both prehypertensive and aged WKY rats and SHR.3.This study aims to assess the cumulative immunoreactivity for AGT1R across various anatomical structures of the kidney, including the capsule, glomeruli, PCT, DCT, and stroma in the cortex, as well as the loop of Henle, collecting tubules, collecting ducts, and stroma in the medulla, in both prehypertensive and aged WKY rats and SHR. Material and methods: 8 Spontaneously hypertensive and 8 Wistar Kyoto rats of prehypertensive (8-9 weeks) and old (60-63 weeks) ages were sedated and euthanized to obtain their kidneys. Tissue samples were cut and frozen using cryomicrotome. Fluorescence immunohistochemistry, using antibodies against PGP 9.5, CGRP, CLRL, AGT1R and α-actin smooth muscle, were applied to the sections. Photos of the preparations were taken by LSM700 confocal microscope (Carl Zeiss, Gottingen, Germany), using the ZEN2010 program. Photos were morphometrically analyzed by FIJI Image J to determine the area fraction percent of each marker in the kidney, the data was collected and analyzed in MS EXCEL and compared the experimental group versus the control group. Results: For CGRP in the WKY rats old and young groups and SHR young group, the percentage of the sum of expression in the medulla was higher in comparison to its cortex while in the SHR old group the percentage of the sum of expression was higher in the cortex in comparison to its medulla. For CLR in all 4 groups (SHR old and young and WKY rats old and young) immunoreactivity was higher in the medulla in the blood vessels, smooth muscle and nervous fibers in comparison to its cortex and its structures. For AGT1R except for the WKY rat control old group cortex, all other groups expressed the highest immunoreactivity in their respective medulla. Conclusion: 1. When comparing within the same strain of rats, young SHR and young WKY rat groups expressed greater CGRP immunoreactivity in comparison to their old age groups of rats. When comparing between SHR and WKY strains of rats across age CGRP immunoreactivity is higher in WKY old and SHR young. 2. When comparing within the same strain of rats, young SHR and young WKY rat groups expressed greater CLR immunoreactivity in comparison to their old age groups of rats. When comparing between SHR and WKY strains of rats across age CLR immunoreactivity is higher in WKY old and young group 3. When comparing within the same strain of rats, young SHR and young WKY rat groups expressed greater AGT1R immunoreactivity in comparison to their old age groups of rats. When comparing between the SHR and WKY strains of rats across age AGT1R immunoreactivity is higher in SHR old and young group.

AIM: To investigate the differences between the kidney nerves and innervation of kidney artery of Spontaneously Hypertensive rats and control Wistar Kyoto rats. OBJECTIVES: (1) To investigate immunoreactivity of Protein Gene Product 9.5, Calcitonin Gene Related Peptide, neuronal Nitric Oxide Synthetase and Tyrosine Hydroxylase in kidney artery adventitia and kidney nerves in Spontaneously Hypertensive rats. (2) To investigate immunoreactivity of Protein Gene Product 9.5, Calcitonin Gene Related Peptide, neuronal Nitric Oxide Synthetase and Tyrosine Hydroxylase in kidney artery adventitia and kidney nerves in Wistar Kyoto rats. (3) To compare the differences between the immunoreactivity of Protein Gene Product 9.5, Calcitonin Gene Related Peptide, neuronal Nitric Oxide Synthetase and Tyrosine Hydroxylase in kidney artery adventitia and kidney nerves in Spontaneously Hypertensive rats and Wistar Kyoto rats.

Aim of the study: To investigate the differences between the innervation of adrenal glands of SHR and WKY rats by measuring and comparing immunoreactivity of CGRP, NOS, TH and VIP. Objectives: (i) To investigate immunoreactivity ofadrenergic, peptidergic, nitrergic and VIP neuromarkers in the adrenal medulla and cortex of SHR rats. (ii)To investigate immunoreactivity of adrenergic, peptidergic, nitrergic and VIP neuromarkersinthe adrenal medulla and cortex of WKY rats. (iii) To compare the immunoreactivity ofNOS, CGRP, TH and VIP in the adrenal medulla and cortex of SHR and WKY rats. Methodology: 3 WKY and 3 SHR rats were sedated and euthanized in order to obtain their adrenal glands. Tissue samples were frozen and cut using cryomicrotome. After the samples undergo anfluorescentimmunohistochemistry for antiPGP 9.5, CGRP, NOS, VIP and TH antibodies.Photos of the preparations were taken byAxioImage M2 fluorescent microscope (Carl Zeiss, Gottingen,Germany), using AxioVision software Rel. 4.8.2 program. Photos morphometrically analyzed by FIJI IMAGE J to determine the area fraction percent of each marker in each layer of the gland, the data were collected and analyzed in MS EXCEL and compared the experimental group versus the control group. Results: The results showed no significant difference between the amounts of the markers in different parts of the adrenal glands of WKY and SHR. However, the measurement did show a difference between the markers. The VIP in the outer layer is 1.13%, CGRP in the medulla of SHR is 0.31%.in WKY, in the outer layer of cortex VIP is 0.55%, in the inner layer of cortex TH is the highest (0.42%), in the medulla NOS is the highest marker (0.78%). The only marker who almost statistical significance (p=0.05) was NOS in the medulla. Conclusions: All markers are more present in the medulla of WKY over SHR. TH are higher in the inner layer of WKY, while VIP, NOS and CGRP are higher in SHR. In the outer layer of cortex, all markers except CGRP are higher in SHR.

Tyrimais, aprašytais šioje disertacijoje, siekta pagilinti mūsų žinias apie širdies vidinės nervinės sistemoms vystymąsi bei sandarą. Atsižvelgiant į žmogaus ir kiaulės širdies anatominius panašumus bei pastarosios rūšies populiarumą ksenotransplantacijos bei elektrofiziologiniuose širdies tyrimuose, tyrimų objektu pasirinkti naujagimio paršiuko bei suaugusios kiaulės prieširdžiai. Totalinių preparatų imunohistocheminio žymėjimo metodika leido pirmą kartą įvertinti širdinių nervinių ląstelių fenotipus bei dydį priklausomai nuo prieširdžių subrezginio, o dvi amžiaus grupės – aptikti amžiaus sąlygotus širdinių neuronų vystymosi požymius. Šie tyrimai iškelia idėją, kad naujagimių gyvūnų širdiniai neuronai dar nėra iki galo subrendę ir jų fenotipinis bei morfologinis vystymasis, o taip pat galimai ir neurogenezės procesas, tęsiasi postnataliniu laikotarpiu. Šie amžiniai pokyčiai apima sumenkusią simpatinių ir padidėjusią parasimpatinių neuronų dalį tarp širdinių nervinių ląstelių suaugusiose kiaulėse bei padidėjusią simpatinių neuronų dalį suaugusių gyvūnų dešiniajame prieširdyje. Taip pat aptikti galimi abipusiai kontaktai tarp simpatinių ir parasimpatinių neuronų bei peptidergių nervinių skaidulų kontaktai su parasimpatiniais neuronais. Taigi, šie morfologiniai rezultatai suteikia daugiau žinių apie žinduolių širdies nervinės sistemos vystymąsi bei amžinius pokyčius.

Aim of the study: The aim of our present study is to determine the innervation of cardiac ventricles in the ovine heart, using immunohistochemistry of nerve fibres and neurons located within intrinsic neural plexus. Objectives: To identify the size of nerves within the whole mount preparations obtained. To assess the ganglion size and neuron groups. To analyse neuron size within ganglia of the ovine ventricles. conclusion:

1. We noticed that the nerve sizes in the sheep ventricles varied with respects to locations. The total overall difference between the largest and the smallest nerves where 234.43µm.
2. It was found that the ganglion size was on average 231.42 2.69 µm2 .After grouping them based on neuron number we found the most abundant groups where those that contained 1-10 neurones and 11-20 neurones.
3. There was a big difference between the largest length of neuron within the ganglia and the smallest, showing great diversity in size. The overall mean length of neuron within a ganglia was 15.20.1 µm.

Radiodažninės abliacijos poveikis eksperimentinių avių širdžių skilvelių epikardiniams nervams. Medicinos studijų programos baigiamasis mokslinis darbas / mokslinė vadovė dr. Kristina Rysevaitė–Kyguolienė, Lietuvos Sveikatos Mokslų Universitetas, Medicinos akademija, Anatomijos institutas. Kaunas, 2017. Tyrimo tikslas. Imunohistochemiškai ištirti radiodažninės prieširdžių abliacijos poveikį avių širdies skilvelių epikardiniams nervams. Uždaviniai. 1. Ištirti radiodažninės abliacijos poveikį dorsalinio paviršiaus epikardiniams nervams. 2. Ištirti radiodažninės abliacijos poveikį ventralinio paviršiaus epikardiniams nervams 3. Palyginti epikardinių nervų pažaidos dydį tolstant nuo abliacijos vietos. 4. Ištirti skirtingo neurocheminio fenotipo skaidulų tankumą epikardiniuose nervuose, po atliktos prieširdžių abliacijos. Tyrimo metodika. Ištirtos devynios juodgalvių avių širdys, šesioms iš jų buvo atlikta prieširdžių radiodažninė abliacija. Imunochistocheminiu metodu buvo išryškinamos skirtingo neurocheminio fenotipo skaidulos, naudojant tris nervinių skaidulų žymenis (tirozino hidroksilazę (TH), cholino acetiltrasferazę (ChAT) ir baltymo geno produktą 9,5 (PGP9,5)). Panaudojant AxioVision 4.8.1 (Zeiss, Gottingen, Germany) programoje įdiegtus AutoMeasure matavimo modulius, apskaičiuotas nervinių skaidulų tankumas. Tyrimo rezultatai. Po atliktos prieširdžių RA dorsaliniame skilvelių paviršiuje statistiškai patikimai (p<0,001) sumažėjo TH, PGP 9,5, ChAT imunoreaktyvių (IR) skaidulų kiekis. TH–IR skaidulų sumažėjo 16,66, PGP 9,5–IR 15,98 ir ChAT skaidulų 0,411 santykiniais vienetais. Tolstant nuo abliacijos vietos skaidulų kiekio sumažėjimas išliko pastovus. Ventraliniame paviršiuje taip pat statistiškai patikimai (p<0,05) 7,9 santykiniais vienetais sumažėjo TH–IR skaidulų kiekis, 0,647 ChAT–IR skaidulų tankumas, PGP 9,5–IR skaidulų sumažėjimas statistiškai nereikšmingas. Palyginus avių po atliktos prieširždių abliacijos nervinių skaidulų tankumus tarp kairiojo ir dešiniojo priekinių skilvelių paviršių, nustatytas statistiškai reikšmingas adrenerginių skaidulų tankumo sumažėjimas 11,68 santykiniais vienetais ir bendrų nervinių skaidulų 7,93 santykiniais vienetais kairiajame skilvelyje. Išvados. 1. Radiodažninė prieširdžių abliacija pažeidžia dorsalinio skilvelių paviršiaus epikardinius nervus. 2. Radiodažninė prieširdžių abliacija pažeidžia ventralinio skilvelių paviršiaus epikardinius nervus. 3. Tolstant nuo abliacijos vietos, pažaidos dydis epikardiniuose nervuose nesumažėjo. 4. Po atliktos prieširdžių abliacijos, epikardiniuose nervuose sumažėjo TH–IR, ChAT–IR, PGP9,5–IR skaudulų tankumas.

Summary

Aim - The aim of the thesis is the investigate the impact of radiofrequency catheter ablation on the intrinsic nerve system of the myocardial layer of a sheep, by measuring and comparing three types of neuronal markers population using immune histochemical methods.

Objectives

1. To determine if there is difference in the nerve fibers population in all heart locations as a whole, between experimental group and control
2. To determine if there is difference in the nerve fibers population in dorsal heart between experimental group and control
3. To determine if there is difference in the nerve fibers population in ventral heart between experimental group and control
4. To determine if there is difference in the nerve fibers population in 4 different location (dorsal apex, dorsal middle, dorsal base, ventral) in the heart between experimental group and control

Methodology - 5 sheep undergo a catheter radiofrequncy ablation, three other sheep didn’t undergo the procedure and were used as a control group. After 5 months all sheep were euthanized, and their hearts were removed, and the hearts were cut into separate regions, tissue samples frozen and cute using criomicrotomy into samples, and then the samples undergo a immuno histochemstry for general, cholinergic and adrenergic neural markers using antibodies, photos of the samples were taken and analyzed by axiovesion program to determine the percent of the marker from the total area of each frame, the data were collected and compared the experimental group vs the control group.

Results – the study found a clear change in the levels of the neural markers, levels of PGP and TH Marker were lower in the experimental group, while the levels of ChAT were higher in the experimental group. The changes observed in 4 types of comparisons all regions of the heart as a whole, dorsal to dorsal, ventral and specific regions, though in some tables the changes the difference was not statistically significant.

Conclusions - catheter radio frequency ablation in the root of the pulmonary veins decrease the levels of nerve fibers in the myocardium and thus has a clear impact on the intrinsic system in the myocardium layer of the heart. The changes in the nerve fiber population is a decrease in the overall number of nerve fibers and decrease in the sympathetic nerve system fibers, while there is increase in the levels of the parasympathetic nerve fibers.