Bc. Natálie Lupienská: Nonverbal behaviour of elderly people during animal-assisted activity Abstract The aim of the study is to compare nonverbal behaviour of elderly people in three studied interactions: 1) with a therapeutic animal, specifically guinea-pig (Cavia aperea porcellus), 2) with a plush guinea-pig toy and 3) with an ordinary single-coloured cup. Twenty one elderly people (3 men and 18 women, average age of 85 years, SD 7,95 y) participated in the study. All data used for the purpose of the study (video recordings) were collected in a Prague residential house for elderly people in summer 2009. An ethogram composed of sixty elements was prepared. Consequently the Interact software was used for coding of those elements. The inter-subject comparison demonstrated that a guinea-pig significantly enhances an interest in an object and tactile behaviour of observed people. There was no significant difference between the three examined situations in terms of respondent's communication with a therapist, facial expressions or the overall activity. Keywords: nonverbal behaviour, animal-assisted activity, elderly, guinea-pig

To determine the origin of non-vagal afferent fibers innervating the heart of guinea pigs, capsaicin was injected into the ventricular myocardium to induce depletion of substance P (SP). The lower cervical, upper thoracic and lumbar spinal ganglia, as well as the left atrium and base of ventricles, were assayed for SP depletion by using the enzyme-linked immunosorbent assay (ELISA) and immunohistochemical procedures. Capsaicin affected spinal ganglia from the 3 regions differently. The substance P level in lumbar spinal ganglia remained fairly constant, while the level of SP from cervical and thoracic regions declined significantly. At the maximal depletion dosage (173 μg of capsaicin/kg), SP concentration decreased 72.3% in cervical spinal ganglia, 45.5% in thoracic ganglia and 56.1% in the atrium. The lack of SP depletion in lumbar ganglia indicates that capsaicin acted locally on cardiac afferents rather than systemically. Data from this study suggest that capsaicin-sensitive neurons of the heart have cell bodies in the lower cervical spinal ganglia as well as in the upper thoracic spinal ganglia.

Substance P (SP) evokes bradycardia that is mediated by cholinergic neurons in experiments with isolated guinea pig hearts. This project investigates the negative chronotropic action of SP in vivo. Guinea pigs were anesthetized with urethane, vagotomized and artificially respired. Using this model, IV injection of SP (32 nmol/kg/50 μl saline) caused a brief decrease in heart rate (-30 ± 3 beats/min from a baseline of 256 ± 4 beats/min, n = 27) and a long-lasting decrease in blood pressure (-28 ± 2 mmHg from baseline of 51 ± 5 mmHg, n = 27). The negative chronotropic response to SP was attenuated by muscarinic receptor blockade with atropine (-29 ± 9 beats/min before vs -8 ± 2 beats/min after treatment, P = 0.0204, n = 5) and augmented by inhibition of cholinesterases with physostigmine (-23 ± 6 beats/min before versus -74 ± 20 beats/min after treatment, P = 0.0250, n = 5). Ganglion blockade with chlorisondamine did not diminish the negative chronotropic response to SP. In another series of experiments, animals were anesthetized with sodium pentobarbital or urethane and studied with or without vagotomy. Neither anesthetic nor vagotomy had a significant effect on the negative chronotropic response to SP (F3,24 = 1.97, P = 0.2198). Comparison of responses to 640 nmol/kg nitroprusside and 32 nmol/kg SP demonstrated that the bradycardic effect of SP occurs independent of vasodilation. These results suggest that SP can evoke bradycardia in vivo through stimulation of postganglionic cholinergic neurons. Copyright (C) 1999 Elsevier Science Inc.

Effects of spantide ([D-Arg1,D-Trp7,9,Leu11]substance P) on coronary resistance vessels were studied in isolated guinea pig hearts perfused at constant rate with isotonic buffer containing 20 or 40 mM KCl. Spantide (1 μM) caused a 20-fold rightward shift of the substance P (SP) dose-response curve for vasodilation with no change in maximum (KB=5.3×10-8 M). Bolus injections of 0.25 to 250 pmol spantide had no effect, but higher doses caused a brief vasodilation followed by a larger, more prolonged vasoconstriction. Histamine produced similar changes in perfusion pressure. Antihistamines (H1 and H2) reduced or blocked responses to spantide and histamine. These findings indicate spantide is a competitive antagonist to SP in guinea pig coronary resistance vessels. In addition, high doses of spantide can cause prominent vascular effects which are mediated by histamine.

The parmacological effects of guinea pig vasoactive intestinal peptide (VIP) were studied in isolated perfused guinea pig hearts. Bolus injections of VIP produced a dose-dependent tachycardia that was not affected by atenolol. A decrease in amplitude of ventricular contractions occurred in response to all doses of VIP. This response was preceded by a small increase in amplitude in 3 of 6 hearts at the highest dose. VIP produced a decrease in perfusion pressure which was prominent after coronary tone was elevated with [Arg8]-vasopressin. The present findings support speculation that VIP may have a role in the regulation of heart rate and coronary blood flow.

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VTP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

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<p>Vitamin C inhibits the oxidation of biologically important molecules and may have a potential protective role against cancer, cardiovascular diseases, and aging. Clinically relevant models of vitamin C function are essential for understanding the role of the antioxidant in the pathogenesis of these complex diseases, and its therapeutic potential. In this thesis, we examine ascorbic acid synthesis and deficiency in animal models, and develop these animal models into powerful tools to examine specific questions of vitamin C function. This thesis first presents a review on the existing animal models for antioxidant function in human nutrition, focusing on their clinical relevance in chronic diseases. We concluded that equivocal proof of beneficial effects of high dose antioxidant supplementation has not been established, and further investigations of animal models of antioxidant function are needed to resolve outstanding questions.</p> <p>We then examined the feasibility and efficacy of an alternative vitamin C delivery method using gene therapeutic lentivirus vectors in a guinea pig model of vitamin C function. The guinea pig exhibits an inactivated gulonolactone oxidase gene (<em>Gulo</em>), which is required for endogenous ascorbic acid synthesis, and as such must acquire vitamin C from the diet. Using a lentivirus vector carrying the mouse <em>Gulo</em> under the murine cytomegalovirus (mCMV) promoter, which was previously developed as a part of my undergraduate thesis, we examined the ability of this gene therapeutic vector to mediate the expression of GULO and the production of ascorbic acid in guinea pigs. At a titre of 10¹⁰ viral particles per animal, the life of lentivirus-treated guinea pigs were prolonged by 35 days compared to the scorbutic control, which was given an ascorbic acid-free diet. Ascorbic acid was produced in the liver of the treated guinea pigs, but the amount produced was not sufficient to elevate plasma concentrations or fully correct the metabolic deficiency. We conclude that lenti-mCMV-<em>Gulo</em> is able to mediate the expression of GULO and endogenous production of vitamin C in guinea pigs.</p> <p>To test the role of vitamin C in cancer etiology and outcome, we are currently in the process of introgressing the <em>Gulo</em>^-/- inactivation mutation, developed by Maeda <em>et al.</em> in 2000, from the C57BL/6 strain background into the FVB/N strain background. The FVB/N strain is also the background for several models of <em>erbB2/neu</em> overexpression in human breast cancer, associated with increased metastasis and low patient survival rates. Taken together, this thesis develops two animal models of vitamin C function, which can be employed in future applications.</p> / Master of Science (MSc)

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