PERIVASCULAR INNERVATION OF CEREBRAL ARTERIES AND VASA NERVORUM: CHANGES IN DEVELOPMENT AND DISEASE KUMUD KUMAR DHITAL BSc., BM.BCh., FRCS. Submitted for the degree of Doctor of Philosophy to the University of London 2000 1 ProQuest Number: 10609109 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609109 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT Using fluorescence histochemical, and chemical assays, this thesis aims to study the perivascular innervation of cerebral arteries and that of nerve-trunk vasculature in both cranial and peripheral nerves. The first part of the thesis concerns the innervation of blood vessels within nerve trunks (vasa nervorum). A novel whole-mount preparation for the visualization of these vessels is described. Perivascular nerves supplying the vasa nervorum of various nerves in different species have been shown to contain noradrenaline, 5- hydroxytryptamine, substance P, vasoactive intestinal polypeptide, neuropeptide Y and calcitonin gene-related peptide. This is followed by a study of the changes in innervation seen in experimental and human diabetic nerves. Eight weeks following induction of diabetes, there was an increase in the density of NA-containing fibres innervating the vasa nervorum in the sciatic, vagus and paravertebral sympathetic nerve trunks. This was in marked contrast to a lack of NA-containing fibres within the optic nerve sheaths in the same animals. A lack of NA-containing perivascular nerves supplying vasa nervorum in a limited number of diabetic human sural nerve biopsies is also described. The second part of the thesis examines the innervation of the major cerebral arteries during development and hypertension. Both these studies were confined to rat models. Following an initial post-natal increase in the density of innervation, it is shown that in the aging rat there is a decrease in the number of nerves containing 2 vasoconstrictor neurotransmitter substances and an increase in the those containing vasodilator neurotransmitter substances. In the spontaneously hypertensive rat, there is an increased density of perivascular nerves containing NA and NPY at four weeks of age. The possibility that such an increase in innervation, before the onset of marked hypertension and medial hypertrophy, may be important in the development and maintenance of hypertension is discussed. 3 ACKNOWLEDGEMENTS The work presented in this thesis could not have been achieved were it not for the support and encouragement I have received from many colleagues at the Department of Anatomy and Embryology and Centre for Neuroscience at University College London, as well as others from outside institutions who have in one way or another helped me in this journey. I would first of all, like to extend my warmest thanks to my supervisor, Professor Geoffrey Bumstock for his guidance, friendship and enormous patience. I thank Professor Otto Appenzeller not only for initially introducing me to the study of the innervation of the vasa nervorum, but also for his seemingly endless energy which was gratefully infectious. In the laboratory, I extend special thanks to Drs. Jill Lincoln and Pam Milner for carrying out the biochemical assays, to Marina Mione for the collaboration in looking at the innervation of cerebral arteries in development and aging, and Annie Evans for her ever stoic support both with her secretarial skills and warm friendship. I also thank Dr. Renato Gerli and Professor Weber from Siena, Italy for providing the samples from hypertensive rats, and to Dr Watkins (King’s College Hospital, London) and Professor Thomas (Royal Free Hospital School of Medicine, London) for supplying the human sural nerve biopsies. This work was kindly supported by a Departmental Scholarship (Dept, of Anatomy, UCL) and an Overseas Research Studentship award. Finally, I wish to thank Jane, Bal and Uma for all their love and encouragement in helping me complete this thesis. 4 CONTENTS Page Abstract 2 Acknowledgments 4 Contents 5 Publications arising from thesis 8 Preface 10 Chapter 1: General Introduction 1.1 Historical Background 13 1.2 Structure of the vascular neuroeffector junction 17 1.3 Pattern of perivascular innervation 19 1.4 Neuromodulation and Cotransmission 23 1.5 Perivascular Nerve Types 1.5.1 Sympathetic nerves 26 1.5.2 Parasympathetic nerves 31 1.5.3 Sensory-Motor nerves 36 1.5.4 Intramural nerves 37 1.5.5 Purinergic Transmission 38 1.5.6 Serotonergic Transmission 40 1.5.7 Peptidergic nerves 43 1.5.8 Nitric oxide Transmission 50 5 1.6 Nerve Trunk Circulation 1.6.1 Introduction 60 1.6.2 Anatomy of nerve trunk circulation 61 1.7 Hypertension 68 1.8 Diabetes 74 Chapter 2: General Methodology 2.1 Histochemical demonstration of perivascular nerves 2.1.1 Localization of noradrenaline 84 2.1.2 Localization of peptides and 5-HT 87 2.2 Measurement of nerve density 90 2.3 Biochemical Assays 2.3.1 Detection of noradrenaline 94 2.3.2 Detection of NPY 96 2.4 Induction of diabetes 98 2.5 Preparation of solutions 99 Chapter 3: Innervation of Vasa Nervorum and Changes 103 in Development Chapter 4: Adrenergic innervation of vasa nervorum in 133 streptozotocin-diabetic rats and in human diabetes 6 Chapter 5: Developmental changes in the innervation of 153 major cerebral arteries by adrenergic, aminergic and peptidergic nerves in the rat Chapter 6: Differential changes in the expression of 184 noradrenaline and neuropeptide Y during development in the spontaneously hypertensive rat Chapter 7: General discussion 214 References 242 7 PUBLICATIONS RELEVANT TO THIS THESIS PAPERS: Appenzeller, O., Dhital K.K., Cowen T and Bumstock G (1984). The nerves to blood vessels supplying blood to nerves: the innervation of the vasa nervorum. Brain Res. 304. 383-386. Dhital K.K., Lincoln J, Appenzeller O and Bumstock G. (1986). Adrenergic innervation of vasa and nervi nervorum of optic, sciatic, vagus and sympathetic nerve trunks in normal and streptozotocin-diabetic rats. Brain Res. 267. 39-44. Dhital K.K., Gerli R., Lincoln J, Milner P., Tanganelli, P., Weber, G., Fruschelli R., and Bumstock G. (1988).Increased density of perivascular nerves to the major cerebral vessels of the spontaneously hypertensive rat: Differential changes in noradrenaline and nueropeptide Y during development. Brain Res. 444. 33-45. Mione, M.C., Dhital K.K., Amenta F and Bumstock G. (1988). An increase in the expression of neuropeptidergic vasodilator, but not vasoconstrictor cerebrovascular nerves in ageing rats. Brain Res. 460. 103-111. Anand, P., Gibson, S., Scaravilli, F., Blank, M., McGregor, G., Appenzeller, O., Dhital, K., Polak, J and Bloom, S. (1991). Studies of vasoactive intestinal polypeptide expression in injured peripheral neurons using capsaicin, sympathectomy and mf mutant rats. Neurosci.Lett. 118. 61-66. ABSTRACTS: Appenzeller, O., Dhital, K.K., and Bumstock, G. (1984).Vasa nervorum: Adrenergic, peptidergic and serotoninergic perivascular nerves. Neurology 34 (suppl. 11.181-2. Appenzeller, O., Dhital K.K., Lincoln J and Bumstock G. (1985). Adrenergic nerves of vasa nervorum and nervi nervorum in diabetes: Differential susceptibility of optic, autonomic and somatic nerves to streptozotocin. Neurology 35 (suppl. 1), 295. Dhital, K.K., Mione, M.C, Amenta, F. Bumstock., G. (1986). Developmental changes in the adrenergic, serotonergic and peptidergic innervation of rat cerebral arteries. In. Proc. Int. Workshop on Trends in Cluster Headache, Italy 1986. Alafaci, C., Dhital, K.K. Crockard, H and Bumstock, G. (1987). Serotonin- and calcitonin gene-related peptide-containing nerve fibres in the dura mater and pineal gland of rats and gerbils. J. Cereb. Blood Flow & Metab. 7 (suppl. 1). S596. 8 Crockard, H., Alafaci, C., Dhital, K.K., and Bumstock G. (1987). Noradrenergic innervation of blood vessels supplying human intracranial tumours. J. Cereb. Blood Flow & Metab. 7 (suppl. 1). S596 REVIEWS AND CHAPTERS Dhital, K. K. and Bumstock, G. (1987). Controle neurogene adrenergique et non- adrenergique de la paroi arterielle. In: Les Maladies de la Paroi Arterielle. Eds. J-P Camilleri, C. Berry, J-N Feissinger and J Bariety. Flammarion, Paris. pp77-105. Dhital K.K and Appenzeller, O. (1988).Innervation of the vasa nervorum. In: Non- adrenergic innervation of blood vessels. Eds. G Bumstock and S Griffith, CRC Press, Boca Raton, pp 191-211. Alafaci, C., Dhital, K.K. and Bumstock G. (1988). Purinergic mechanisms in cerebral circulation. In: Basic Mechanisms of Headache Eds. J. Olesen and L Edvinsson, Elsevier, Amersterdam, pp 163-175. Bumstock, G., Alafaci, C., and Dhital K.K. (1988). Neural transmitters in the cerebrovascascular bed. In: Cerebral circulation and neurotransmitters. From basic to clinical science. Proc. VII Int. Symposium on Morphological Sciences, Rome, 1988 Eds. C Cavallotti and L Fumagalli. EUR, Rome pp 7-26. Dhital, K.K. and Bumstock, G. (1989). Adrenergic and non-adrenergic neural control of the arterial wall. In: Disease of the arterial wall. Eds. J-P Camilleri, C Berry, J-N Feissenger and J Bariety. Springer, London, pp 97-126. Bumstock, G, Dhital K.K. and Alafaci C. (1989). Structural and chemical organisation of the autonomic nervous system. In:Autonomic Neuropathies. Ed. P.K. Thomas. New issues in Neuroscience J_, 277-289. 9 PREFACE The aim of this thesis is to study the perivascular innervation of two distinct circulatory beds supplying nervous tissue, namely that of vasa nervorum supplying blood to nerve trunks and of the major cerebral arteries.