Patterned Magnetic Fields and Ageing Effects on Murine Mast Cells:
A Histological Investigation
By
Rafiq Rahemtulla
Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biology
School of Graduate Studies Laurentian University Sudbury, Ontario
© Rafiq Rahemtulla, 2012 Library and Archives Bibliotheque et Canada Archives Canada
Published Heritage Direction du 1+1Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-91648-3
Our file Notre reference ISBN: 978-0-494-91648-3
NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.
In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these.
While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada
Thesis Abstract
In the almost 150 years since the first known description of mast cells, the mechanism of their characteristic metachromasia, their mechanism of activation, and their role in allergy have been elucidated. Their potent preformed mediators as well as myriad other mediators which they can syntheise on demand make them powerful components in a variety of tissue. However, much remains to be learned about their role in normal and pathological physiology, particularly in the nervous system. The first two experiments in this document examined mast cells and assessed their state of degranulation in a rat model of multiple sclerosis and through the normal life course of rats ending near a state of 'natural death' . The third chapter considers a special response to seizure damage in the posterior thalamus, a region where mast cells tend to aggregate during development. Mast cells were assessed and quantified in syngeneic mouse melanoma samples to consider the role of the mast cell in this condition. Furthermore, the effects of weak magnetic fields on these cells will be examined through the first and last experiment. Biophysical models are explored. Acknowledgements
The data collected for studies contained herein were
from tissue samples from previous experiments conducted by the Laurentian University Neuroscience Research Group.
Thus, the author wishes to acknowledge the work of L.L.
Cook, Prof. C. Bloome, Dr. L. St. Pierre, G.F. Lafreniere, and Dr. C. Buckner.
The author is also grateful for the thorough readings and thoughtful comments during the preparation of this document provided by Dr. G.H. Parker and Dr. R. Lafrenie.
None of this work would have been possible without the encouragement, mentorship, and support of Dr. M.A.
Persinger. The author is extremely grateful for the privilege of working with and learning from such a bright mind and warm spirit.
iv Table o£ Contents
Thesis Abstract...... iii
Acknowledgements...... iv
List of Figures...... viii
Chapter 1 - Introduction...... 1
1.1 What are Mast Cells...... 1
1.2 A History of Research...... 3
1.3 Mast Cell Heterogeneity...... 9
1.4 Ontogenetic History...... 11
1.5 Phylogenetic History...... 15
1 . 6 Mast Cell Stimulators...... 17
1.7 Mast Cell Responses...... 20
1.8 Experimental Allergic Encephalomyelitis...... 24
1. 9 Mast Cells and Magnetic Fields...... 25
1.10 Lithium-Pilocarpine Epilepsy...... 26
1.11 Ageing...... 27
1.12 Melanoma...... 28
1.13 Staining Techniques...... 31
1.14 References...... 34
Chapter 2 - Nightly Exposure to Weak Time-Varying Magnetic
Fields Enhanced Brain Mast Cell Numbers: Testing Potential
Biophysical Models...... 46 2.1 Introduction...... 47
2.2 Specific Descriptions To Be Assessed...... 51
2 . 3 Methods...... 53
2.3.1 Specimens...... 53
2.3.2 History...... 53
2.3.3 Counting Procedures...... 54
2.3.4 Statistical Analyses...... 55
2 . 4 Results...... 56
2.5 Discussion and Conclusion...... 58
2.6 References...... 61
Chapter 3 - Mast Cell Numbers in Thalami of Very Old Rats...... 68
3.1 Introduction...... 69
3.2 Method...... 73
3.2.1 Specimens...... 73
3.2.2 Procedure...... 73
3.2.3 Statistical analyses...... 74
3.3 Results...... 75
3.4 Discussion...... 78
3.5 References...... 79
Chapter 4 - Diffuse Thalamic Alizarin Staining Following Cholinergic Seizures Overlaps With Major Mast Cell Location in the Rat Brain...... 85
4 .1 Introduction...... 86
4 .'2 Methods...... 90
4.3 Results...... 91 4.4 Discussion...... 93
4.5 References...... 96
Chapter 5 - Mast Cells in Mouse Melanomas Following Patterned Magnetic Field Exposures That Suppress B16 Cell Proliferation in Vitro...... 103
5.1 Introduction...... 104
5.2 Method...... 107
5.3 Results...... 109
5.4 Discussion...... 112
5.5 References...... 114
Chapter 6 - General Results, Discussion, & Conclusion 118
6.1 Brain Mast Cells Between Studies...... 118
6.2 Mast Cell Locations...... 120
6.3 Magnetic Field Effects on Mast Cells...... 122
6.4 Consequences of Brain Mast Cell Degranulation 125
6.5 Neuroinflammation...... 127
6.6 Conclusion...... 127
6.7 References...... 129
vii List of Figures
Figure 1.1. The number of PubMed listings responding to the search term mast cell by year of publication...... 8
Figure 1.2. The proportions of annual PubMed listings responding to the search term mast cell with respect to the entire database by year...... 8
Figure 1.3. The proportions of annual PubMed listings for Mast Cell responding to the search terms Heparin, Histamine, Tumor, or Allergy...... 9
Figure 2.1. Examples and criteria for mast cell degranulation classifications...... 55
Figure 2.2. The number of observed mast cells (vertical axis) by intensity of magnetic field exposure ...... 58
Figure 3.1. Scatterplot of number of thalamic mast cells by age...... 77
Figure 3.2. The mean relative proportion ± SEM of mast cells found along to the rostral-caudal axis ...... 77
Figure 4.1. Examples of the right posterior thalamus of rats killed between 1 and 50 days post seizure...... 92
Figure 5.1. Mean number of observed mast cells by exposure condition and photophase...... Ill
Figure 5.2. Proportion of mast cells in each of the five specified degranulation conditions for daytime and nighttime treated animals regardless of exposure of sham treatment...... Ill
Figure 6.1. The mean estimated total number of brain mast cells by study...... 119
Figure 6.2. The mean proportion ± SEM of mast cells in each of five predefined states of degranulat'ion between experiments...... 120
viii Figure 6.3. The proportions of mast cell orientation to the surface of melanoma samples...... 122
Figure 6.4. The mean proportion ± SEM of mast cells in each of five predefined states of degranulation from melanoma samples which were either exposed to real or sham magnetic fields regardless of photoperiod...... 125
ix Chapter 1 -Introduction
The cell is the basic unit of life as we know it. The cells of multicellular organisms differentiate into various forms resulting in a division of labour to sustain the entire organism. These cells organise into tissues and these tissues organise into organs. Therefore, the organ is an emergent property of the arrangement of multiple gestalts of organised cellular-based structures. A powerful strategy used by science is reductionism whereby a complex entity or phenomena is explained by its simpler components. The subsequent chapters in the document take this approach by examining the role of a single type of cell, the mast cell, in a variety of phenomena. This introduction will outline the basic biological, biochemical, and histological features of the mast cell and outline its potential relevance to the phenomena examined in the subsequent chapters.
1.1 What are Mast Cells
Mast cell are large (10-15 pm in diameter) granular cells. They are most frequently found in the connective tissue of a variety of organs as well as the serous membranes and intestinal mucosa. They are generally characterised histologically by their metachromatic granules under certain staining techniques (Selye, 1965). Under
1 standard light microscopy they resemble blood basophils.
Basophils differ from mast cells by three properties; they differentiate in the bone marrow; their mature form circulates in the blood; and they cannot divide (Galli,
1986) . Mast cells are dynamic; they are mobile within their tissue of residence and migrate in response to stimuli in addition to displaying a regular circadian periodicity in their abundance (Chen & He, 1986).
The electron-dense metachromatic granules of mast cells contain sulfated mucopolysaccharides (heparin), biogenic amines (histamine), and proteases. These molecules act to prevent blood coagulation, induce swelling, and re-shape connective tissue respectively (Bloom, 1984).
The mast cells are part of the immune system. They function as a part of both the innate immune response as well as the acquired immune response. The former is facilitated by the mast cell's potential to respond to bacterial products like lipopolysaccharide while the latter is mediated by immunoglobulin E (Wedemeyer et al., 2000).
2 1.2 A History of Research
The first known description of mast cells was completed in 1863 by Friedrick von Recklinghausen (Bloom, 1984).
However, the mast cell was named by Paul Ehrlich in his
1879PhD dissertation where he systematically described their appearance and locations under different stains, its metachromatic properties in particular. The name he selected for them, mastzellen, derived from the German for fattening, due to their large size and many cytoplasmic granules (Selye, 1965).
Mast cell research was initially slow until its unique biochemistry became evident. The number of mast cell publications listed on the American National Institute of
Health's PubMed database demonstrates this by showing a steady linear ten-fold increase in the number of annual publications between 1960 and 1990 (see figure 1.1).
Seyle's monograph listing all of the scientific reports on mast cells on all species in multiple languages was published in 1965 while working at the Universite de
Montreal (Selye, 1965). Though the number of yearly listings still increased from 1990 onwards, the rate of increase has slowed. This is particularly evident when considered with respect to the total number of yearly PubMed
3 listings where mast cell papers represented a maximum of about one in four hundred listings around 1990 and declined to about half of that proportion in 2011 (see figure 1.2).
Heparin was first isolated and thoroughly investigated biochemically due to its anti-coagulant properties beginning in the late 1910s at Johns Hopkins University (Jorpes,
1936) . Most of the work further elucidating the chemical structure and properties was performed at Carlsberg
Laboratories in Copenhagen and at Connaught Laboratories in
Toronto. Through these investigations it was found that toluidine blue would discolour towards reddish-purple when in contact with heparin (Jorpes, 1936) . In 1947 using mastocytomas from dogs, Oliver, Bloom, and Mangieri related the heparin content of tissues to the number, maturity, and metachromatic potential of mast cells (Oliver et al., 1947) providing clear evidence for the mechanism of mast cell metachromasia. The proportion of mast cell papers listed on the PubMed database that are retrieved with the search term heparin has represented less than 5% of the total yearly publications and has been in a steady decline since the early 1980s (see figure 1.3).
The discovery of compound 48/80, a potent mast cell degranulator by Paton in 1951 ushered in an era of interest in biogenic amines in mast cells (Bloom, 1984). This interest resulted in a rapid increase in PubMed listings of mast cell papers responding to histamine beginning in 1952 and peaking in 1985 where more than 40% of that year's mast cell listings responded to histamine as a search term (see figure 1.3). During this period 5-hydroxytryptamine was also found to be frequently contained in murine, but not human mast cells, and dopamine was occasionally found
(Bloom, 1984) .
As investigators were enthusiastically pursuing the biogenic amines in mast cell secretions a few others, noticing the powerful role of mast cells in urticaria pigmentosa began to consider the mast cell's role in the allergic response. This was demonstrated by sensitising guinea pigs to an antigen and eliciting an anaphylactic response and observing a drop in lung mast cell counts post mortem (Mota et al., 1956). The connection of mast cells to allergy is currently well established not only in the scientific literature, but also in public discourse. The proportion of mast cell research concerning allergy has demonstrated a gradual increase from the early 1950s to the present where it accounts for about one third of PubMed's mast cell listings (see figure 1.3).
5 Though Ehrlich did mention the propensity of mast cells to localise around tumours, the research on the role of mast cells in tumourgenesis and maintenance came much later.
Mastocytomas are common in domestic pets and have provided useful tissue for investigators. There is presently much controversy surrounding the pro- and/or anti-tumurogenic roles of mast cells in a variety of malignancies. Research on the roles of mast cells in tumour biology has and continues to gradually occupy a greater share of mast cell research (see figure 1.3). A critical point for this was
1971 when a model of tumour development from a small group of mutated cells was dependent on rapid angiogenesis
(Folkman, 1971).
With the increase in mast cell research intensity spanning mast cells in a variety of tissues, particularly the methods required to preserve mast cells in the gastrointestinal mucosa, a major controversy regarding the heterogeneity of mast cells as a function of location emerged. Leading up to the mid-1980s many investigators began the convention of employing the terms mucosal and connective tissue mast cells. A major international conference convened near Caledon, ON in 1986 by John
Bienenstock of McMaster University and Dean Befus of the
6 University of Alberta to review current knowledge of mast cell diversity and develop nomenclature recommendations.
This conference brought together, general histologists, pathologists, cell biologists and others and the proceedings were compiled in a single volume (Befus et al., 1986)-.
Research on brain mast cells was sporadic and mostly concerned the endocrine system or pathological conditions.
In the mid-1960s Campbell and Kiernan (1966) published a systematic search of hedgehog brains for mast cells and postulated neuroimmune interactions in the diencephalon.
Dropp (1976) extended this line of research to twenty-nine mammalian species. The developmental window of mast cell infiltration into the parenchyma of the diencephalon of the rat was demonstrated by Persinger (1981). In a review paper
Persinger (1977) outlined the existing postulates for brain mast cell functions such as regulation of vasculature permeability and also supplied some novel ones, in particular, leveraging the remarkable within species variations to suggest mast cell involvement in individual experience. Zuang and colleagues (1993) experimentally supported this hypothesis by demonstrating an increase in mast cells within the medial habenulae of male doves -engaged in courtship behavior. 1400
1200 A* jjp 1000 ♦ v( '■= 800 / X 600 A ! / 3 400 / i n n a a
200
0 w # * * * 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020
Year of Publication
Figure 1.1 The number of PubMed listings responding to the search term mast cell by year of publication.
_ 0.003 Uw « 0.0025
2 0.002 * /w+ 4 ♦ « * v 'S o.ooi c I 0.0005 ^ I 0 ¥ f 1940 1950 1960 1970 1980 1990 2000 2010 2020
Year of Publication
Figure 1.2 The proportions of annual PubMed listings responding to the search term mast cell with respect to the entire database by year.
8 £L 0.4500 0.4000
v a 0.2500 ♦ Heparin U <941 0 < /l 0.2000 « "O ■ Histamine 2 V 0.1500 V. O Year of Publication Figure 1.3.The proportions of annual PubMed listings for Mast Cell responding to the search terms Heparin, Histamine, Tumor, or Allergy. 1.3 Mast Cell Heterogeneity The most pronounced example of heterogeneity of mast cells is a function of location. The most notable schism is between what have historically been described as connective tissue mast cells and mucosal mast cells. The parallel titles of typical and atypical have been used respectively. Differences between these apparent classes of mast cells span differential staining properties, responses to different fixatives, different properties in culture, different histological appearance, and different granular contents (Befus et al., 1986). Mucosal mast cells are poorly visualised in formalin fixed samples, stain blue under alcian blue/safranin-o 9 staining, are smaller, and contain fewer, but more variable sized granules (Enerback, 1986). They express less sulfated chondroitin sulfate E rather than heparin. They also contain about one tenth of the histamine content per cell of connective tissue mast cells. Mucosal mast cells will divide rather than degranulate in response to compound 48/80. They also are more heavily ramified and thus believed to be more motile than connective tissue mast cells (Enerback, 1986) . They can be seen far more frequently within epithelial tissue. Mucosal mast cells can be persistently cultured with interleukin-3 (IL-3)(Enerback, 1986). The recognition that mucosal and connective tissue mast cells were artificial categories has made the classification of mast cells a major source of debate which culminated in a conference whose primary aim was to develop a series of recommendations. In the end, the consensus was that insufficient knowledge existed to develop an accurate classification system and that researchers should specify the species, tissue, and histological techniques of the mast cells they describe in vivo or the source and culturing techniques of mast cell data colle-cted in vitro (Befus et al., 1986). 10 Vesicular monoamine transporters (VMATs) allow for vesicular uptake of monoamines in a variety of secretory cells including neurons and mast cells. The actions of VMATs, whose actions can be blocked with the compound reserpine, can influence the number and size of vesicles (Colliver, et al., 2000). Furthermore, VMAT can allow mast cell granules to accumulate non-native biogenic amines found in its local microenvironment like dopamine via endocytosis (Enerback, 1986). A mast cell's granular state can also be influenced and varied by what has been described by Wilhelm (2005) as trans-granulation where mast cells and neurons can exchange vesicular contents. Therefore, individual mast cells in the same tissue can develop heterogeneous granular contents as a function of their immediate biochemical surroundings. 1.4 Ontogenetic History Despite Ehrlich's original suggestion that mast cells differentiate from local fibroblasts (Crivellato et al., 2003), mast cells derive from bone marrow hematopoietic stem cells, migrate through the blood, and mature on site.This was established by Kitamura and colleagues (1978) by experimentally returning the cutaneous mast cell density of the genetically mast cell deficient W/Wvmice to normal 11 levels with bone marrow transplants from C57/BL-6 mice. However, fibroblasts-derived factors are necessary to induce granule synthesis (Davidson et al., 1986). Maximow's observation of the inverse relationship between blood basophils and mast cells led him to hypothesise in 1906 that blood basophils were mast cell precursors (Enerback, 1986). While they both derive from hematopoietic stem cells, basophils differentiate and mature in the bone marrow while committed mast cell progenitor cells, which do not display the characteristic phenotypic features of mature mast cells, differentiate in the bone marrow and circulate in the peripheral blood until they localise in their target tissue where they eventually mature (Chen et al., 2005). Mature mast cells retain the ability to translocate to different tissue, even if the translocation involves being trafficked across the blood brain barrier (Silverman et al., 2000) . This effect likely mediates important developmental milestones in the whole organism due to the tight migration window of leptomeningeal mast cells into the thalamus of young rats between postnatal day 15 and 25 reported by Persinger (1981). Differential expression of proteases and other granule contents as well as staining properties have been reported 12 as a systematic function of location (Gurish& Boyce, 2002); thus, the local microenvironment likely has a subtle influence on the maturation process and fate of mast cells. Denburg and collegues (1986) hypothesised that the typical, connective tissue, and atypical, mucosal, mast cells are two distinct phenotypic fates for mast cell progenitors. An injection of cultured mucosal mast cells into dermis of mast cell deficient mice results in the initial disappearance of injected mast cells by conventional staining techniques for fifteen to twenty days until an estimated 400 to 800 thousand cells, well in excess of the initial injection, appeared and phenotypically resembled connective tissue mast cells. An explanation proposed for this effect by Kitmura and colleagues is that mucosal and connective tissue mast cells can de- and then re-dif ferentiate, or trans- differentiate into each other as a function of their local microenvironment (1986). The c-kit ligand stem cell factor (SCF) can induce proliferation of immature mast cells as well as maturation of those cells that resemble mature connective tissue mast cells (Tsai et al., 1991). Proliferation is far more common in the mucosal-type mast cells which resemble immature connective tissue-type mast cells than in the mature 13 connective tissue-type mast cells (Kitamura et al., 1986). A well characterised feature of helminth infection is a temporary T lymphocyte-dependent mucosal mast cell hyperplasia (Haig et al., 1986). The proliferative capacity of a mast cell is independent of its current state of granulation, and the phenotype of the daughter cells are dependent on the local environment, not that of the mother mast cell (Kuriu et al., 1989). In humans, the mucosal-type mast cells only express the protease tryptase while the connective tissue-type mast cells express chymase, carboxypeptidase, and cathepsin G in addition to tryptase (Metcalfe et al., 1997). Thus, the two major phenotypic flavours of mast cell may either be two divergent forms which can switch in response to environmental stimuli as postulated by Kitamura (1986), or they can reflect different stages along single, linear maturation process. The consequences of this ongoing debate may be relevant here since we are only seeking typical, connective tissue-type mast cells. Experimental effects may stunt the maturation process of mast cells thereby altering their bioactive constituents and ultimately, their effects on the surrounding tissue. 14 Connective tissue mast cells can survive for months in vivo with continued SCF stimuli, while SCF withdrawal in vitro results in apoptosis (Gurish, 2002). Conversely, the resolution of helminth-induced hyperplasia of mucosal mast cells is resolved within six weeks of helminth elimination. This is achieved through both apoptosis and the trafficking of mucosal mast cells into the gastrointestinal lumen for elimination (Gurish, 2002). 1.5 Phylogenetic History Although metachromatic materials can be demonstrated in invertebrates it is not likely that an invertebrate homologue of the mast cell exists (Selye, 1965 & Reite, 1965). Lower vertebrates, like fish, express a large number of mast cells and few blood basophils under histological evaluation (Selye, 1965); however, their residence tissues do not express any more histamine than similar tissue which does not contain any mast cells (Reite, 1965) . This suggests a different physiological role for mast cells in different members of the phylum. Among other lower vertebrate classes, amphibian mast cells have many processes and display unique shapes while reptilian mast cells visually resemble those of higher vertebrates (Selye, 1965). 15 Birds and mammals contain fewer mast cells per unit area in general when compared with lower vertebrates; however, they express a much higher concentration of histamine in their tissues (Reite, 1965). Homeotherms express much more histamine in their tissues than poikilotherms. Histamine, also appears to have no observable effects on poikilotherms while exerting a powerful cardiovascular effect on homeotherms (Reite, 1965). Within the mammals, a great diversity of patterns of tissues express mast cells, however, this seems to follow a species-specific pattern which does not display any direct relationship with phylogeny. Murine mast cells express the biogenic amine 5-hydroxytryptaime (serotonin) while human mast cells do not (Parratt & West, 1957).Ultrastructurally, systematic differences have been noted between murine and human mast cells. Human mast cells tend to be larger (diameter = 14-20 pm), have more uniformly sized granules, and express a series of regular laminar patterns along their granules, described as scrolls, gratings, and lattices (Dvorak, 1986). These patterns canbe attributed to a more concentrated and more organised intragranular matrix of proteoglycan (heparin). 16 1.6 Mast Cell Simulators One of the most characteristic responses of mast cells is anaphalactoid degranulation in response to immunoglobulin E (IgE). This response has been termed an immediate-type allergic reaction and has been classified as a type one hypersensitivity reaction (Sayed et al., 2008). Mast cells can express an IgE receptor (FceRI) surface density from 1200/pm2 for intestinal mast cells to 2800/pm2 for peritoneal mast cells (Befus et al., 1986). This response is not uniform and the various factors and cytokine secretion are influenced by many co-stimulatory and co- inhibitory signals (Sayed et al., 2008). The initial signal transduction event between allergen- bound IgE results from the crosslinking, rather than a conformational change of the membrane spanning FcERI receptors (Metcalfe et al., 1997).Menon and colleagues (1986) have been able to demonstrate histamine release with aggregation of about 0.03% of receptors and a maximum histamine release is reached with aggregation of 10% of total receptors. This cross-linking causes tyrosine phosphorylation activity within 5-15 seconds. Through a series of downstream signaling events phosphatidylinositol- 3-kinase activity becomes apparent resulting in the 17 hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate (PIP2 ) into the two second messengers inositol-1,4,5- trisphosphate (IP3) and diacylglycerol (DAG) (Melcalfe et al., 1997). IP3 activates intracellular calcium signals by opening channels on the endoplasmic reticulum membrane as well as on the plasma membrane. Due to the general nature of calcium intracellular calcium signaling along with the dual (endoplasmic reticulum and external) calcium sources a complex oscillation results. The resting [Ca2+] ranges from lOOnM to 500nM: within 30s of activation this concentration can reach 1200nM. The return to baseline can take up to 10 minutes (Melcalfe, 1997). DAG-PKC effects tend to be slower than those elicited by IP3. These include myosin phosphorylation, activation of transcription factors, activation of G proteins, and activation of the mitogen activation protein (MAP) kinase pathway. Inhibition of the latter results in a great reduction in exocytosis. The G proteins have been hypothesised to be necessary for the granule fusion stage of exocytosis. A degranulation event is also coupled with mitochondrial fission and migration towards the cell membrane. It has been postulated that mitochondria are an essential component of degranulation since uncoupling protein II can severely diminish mast cell degranulation (Theoharides, 2012). Connective tissue, but not mucosal, mast cells can also undergo degranulation independent of any specific surface receptor. Different classes of compounds exist, both natural (substance P) and synthetic (compound 48/80), but appear to follow a similar mechanism of action that can be blocked by benzalkonium chloride. Compound 48/80 enters the plasma membrane without completely passing through it and activates the G protein, Gi3 specifically (Aldenborg, 1987). Mucosal mast cells do not express this protein, and do not degranulate in response to compound 48/80 (Aldenborg, 1987). The mechanism of the proliferative, not secretory, response to compound 48/80 remains to be elucidated, however, it vanishes in athymic animals (Aldenborg, 1987). Mast cells can respond to a larger number of additional intrinsic agonists. Features like the exacerbation of allergic and autoimmune conditions by non-specific stress or the circadian periodicity of asthma attacks have led investigators to search for other biochemical pathways that activate or modulate the activity of mast cells. A recent review paper lists over seventy different receptors and their subtypes which have been demonstrated to be expressed on mast cell (Theoharides et al., 2012). 19 1.7 Mast Cell Responses The mast cell granule is rich with bioactive compounds. A dense proteoglycan matrix compartmentalises the granule. These proteoglycans are heparin and chondroitin sulfate E for connective tissue and mucosal mast cells respectively. The highly sulfated carbohydrate components of these compounds result in the high charge and unique histochemical staining properties of the granules. Heparin is also an anticoagulant (Oliver et al., 1947). Rat heparin can range in molecular weight from 750 to 1000 kDa as a function of the number of side chains. An estimated 4000 sulfate groups and 2000 carboxyl groups make heparin one of the most acidic macromolecules in the rat. Rat connective tissue mast cells contain between 10 and 20 gg of heparin per 106 cells (Stevens et al., 1986). Granules contain histamine in humans and rodents as well as serotonin in rodents (Parrat & West, 1957) . These amines act as neurotransmitters in the central nervous system. In addition histamine is involved with airway constriction, smooth muscle vasospasm, and regulating capillary permeability. Histamine is synthesised by the decarboxylation of the amino acid L-histadine and is quickly (in the order of minutes following release) deactivated by 20 methylation (Metcalfe, 1997). Connective tissue-type mast cells contain about 12pg/cell of histamine (Kruger, 1974). Serotonin, its name derived from serum tonic, is a vasoconstrictor and is an indoleamine synthesised from the amino acid tryptophan. In the rat intestinal mucosa, mast cells contain about one quarter of the serotonin with the rest being contained in the endochromaffin cells. Due to the action of mucosal mast cells during a helminth infection response, blood histamine levels can rise from a baseline of 40 ng/mL (15ng/mL plasma) to 200ng/mL (80ng/mL plasma) within 12 days (Enerback, 1986) . Mast cell granules have also been shown to contain other amines like dopamine; however, it is likely that they are a result of endocytosis from the local environment based on the low amounts of these other biogenic amines and inconsistency in their presence (Galli, 1986). Most of the protein in mast cell granules are protease enzymes (Metcalfe, 1997). These enzymes are varied both between and within species, but are typically classified with two major categories, chymases and typtases. In humans connective tissue mast cells are positive for both; whereas, mucosal mast cells are- only positive for typtase alone. These proteases are specific to mast cells and thus are a 21 frequent target for immunohistochemical staining. Tryptase isl34 kDa tetramer with a small and sterically hindered central active site making it specific for smaller proteins and insensitive to large inhibitors (Metcalfe, 1997). It is bound to and released with heparin, or analogous glucoseamineglycan. In vivo it can activate collagenase activity and break down neuropeptides (Metcalfe, 1997). Chymase is a 30 kDa monomer which is not bound to heparin. It is able to convert angiotensin I to II, degrade epithelial basement membranes, and degrade neuropeptides (Metcalfe, 1997). These proteases do not have a specific substrate and likely work on a number of proteins as a function of the current local tissue conditions (Pejler et al., 2010). In general, these proteases are likely involved with the mast cell's ability to regulate inflammatory conditions, reshape resident connective tissue and fight infection. In addition to the release preformed granular contents just discussed, mast cell activation also initiates de novo synthesis of mediators. These responses occur over hours while preformed granular mediators respond in minutes. On top of de novo synthesis of immunologic and inflammatory mediators, mast cells can recruit such mediators from other 22 immunoactive cells via chemotaxis (Theoharides et al., 2012). One group of de novo synthesised mediators are the arachidonic acid metabolites (Theoharides et al., 2012). Through cyclooxygenase activity prostaglandins and thromboxanes are produced. These compounds are involved in the initiation, maintenance and modulation of inflammation. Mast cell activation is not an all-or-none phenomenon. Mast cells can degranulate at different rates releasing different amounts of varied preformed mediators from their granules. The subsequent de novo synthesis of mediators can vary substantially as a function of the cell's current state, and the specific complement of activators which initiated the reaction (Theoharides, 2012). In fact, some activators can initiate only de novo synthesis of specific mast cell mediators without any apparent degranulation. For instance, mast cell activation by prostaglandin E2 in humans results in the release of vascular endothelial growth factor without degranulation (Abdel-Majid & Marshall, 2004). 23 1.8 Experimental Allergic Encephalomyelitis Experimental allergic encephalomyelitis (EAE) in Lewis rats is a model of autoimmune demyelination like the relapsing-remitting variety of human multiple sclerosis (Esquifino et al., 2006). This condition is induced with the injection of rat spinal cord homogenate suspended in Freud's complete adjuvant. The rat's immune system responds to myelin basic protein as its primary epitome and becomes sensitised to recognise this protein as foreign. Mast cells are necessary for the complete development of this condition, since mast cell-deficient mice do not develop the full severity of symptoms unless they are supplemented with exogenous mast cells (Gregory, et al., 2005). Mast cells are likely involved in two distinct phases during the progression of EAE. First, their ability to increase the permeability of the blood brain barrier could allow immune cell infiltration into the central nervous system. Second, through its release of cytokines like tumour necrosis factor alpha (TNFa) this is correlated with disease severity (Owans et al., 1995). Rats that have been induced with EAE display a three-fold increase in brain mast cell numbers than rats compared to controls. Furthermore, experimentally treating EAE inoculated animals with hydroxyzine, an antihistamine, 24 delayed the progression of EAE by 50%, and reduced the extent of mast cell degranulation by 70% (Dimitriadou, 2000). 1.9 Mast Cells and Magnetic Fields Cutaneous mast cell number has been experimentally increased by El Sayed and colleagues (1996) using various intensities and pulse frequency of laser stimulation. In addition Johansson and colleagues (2001) have found that mast cells migrate from deeper to more superficial dermal layers after two hours of exposure to television screen or computer monitors. Conversely, no changes in mast cell degranulation thresholds were found in culture following exposure to a 60 Hz, 5 mT magnetic field (Price & Strattan, 1998). Nocturnal exposure to a weak (15-60 nT) patterned magnetic field modeled on a sudden-onset geomagnetic storm (7 Hz) results in a suppression of symptoms of Lewis rats inoculated with EAE (Persinger et al., 1999). This suppression is only effective at the specific intensity and frequency combination (Cook & Persinger, 2000). The general increase in mast cells observed in Lewis rat brains following induction of EAE and the varied experimental exposures of these animals to magnetic field conditions 25 provides a good starting point for the investigation of the effects of weak, patterned magnetic fields on brain mast cells. Yamaguchi and colleagues found that magnetic pulse stimulation of a mouse model of melanoma (B16-BL6) resulted in treated groups developing tumours weighing only 54% than those of shams. They were not, however, able to extend this effect to B16 cells in culture and attributed this differential effect to the increased production of TNFa from a non B16 cell source (Yamaguchi et al., 2006). As mast cells have been known to secret this compound, they may be implicated in this effect. Hu and colleagues (2010) reported a decrease in mast cells in B16-BL6 mice exposed to three hours/day of a weak patterned magnetic field over shams in addition to a powerful decrease in overt tumour incidence rate (2010). 1.10 Lithium-Pilocarpine Epilepsy A model of partial temporal lobe epilepsy in rats is induced by an injection of the muscarinic agent pilocarpine. The sufficient dose of pilocarpine can be substantially reduced if it is preceded with an injection of a lithium synergist. This model is characterised by three stages: (a) an acute period lasting 24 hours, (b) a silent period 26 lasting from 4 to 44 days, and (c) a chronic period which persists for the duration of the animal's life. The acute period is characterised by the onset of status epilepitcus and electroencephalographic (EEG) abnormalities. The animal's EEG profile returns to normal during the silent period and the animal will begin to display spontaneous seizures as it moves into the chronic phase (Scorza et al., 2009). Since brain mast cells are a part of the central nervous system's intrinsic immune system, which is likely overwhelmed by the massive neuronal dropout resulting from status epilepticus, their role following this state warrants some investigation. Mast cells frequently accompany calcification in arthrosclerosis and have been hypothesised to contribute to its development (Jeziorska, et al., 1998). 1.11 Ageing Advanced ageing is often associated with a state of low-grade generalised inflammation. The myriad mast cell mediators involved in the inflammatory response as well as the overall increase in mast cell numbers with age (Payne, 2006) make them an attractive target for study. In addition to myelin basic protein, mast cells can be sensitised to beta amyloid protein, the basis of the characteristic 27 plaques of Alzheimer's disease (Niederhoffer et al., 2009). Mast cells, by number, comprise a miniscule proportion of cell types in the central nervous system or even the central nervous system's immune cells, microglia. Nonetheless, they are the first cells which respond to local insults. Furthermore, mast cells are capable of degradation of the neurovascular matrix in addition to activating microglia (Skaper et al., 2012) and inducing apoptotic responses in oligodendrocytes (Medic et al., 2010). Therefore, mast cells, though few in number, in addition to initiating neuroinflammation, exert powerful effects on neuroinflammatory processes by acting on the microglia, neurovascular matrix, and endothelial cells. 1.12 Melanoma Melanoma accounts for 2.8% of new human cancer cases with an incidence of 11.2 per 100 000 in Canada. It accounts for 1.2% of all cancer deaths (Melanoma Skin Cancer: Facts and Figures, 2009). Even within the skin cancers melanoma only account for about 4% of cases, however, it account for 80% of skin cancer deaths (Miller & Mihm, 2006). The current murine models to study melanoma in vivo can be placed in three general classes, (a) genetic 28 modification, (b) xenogenic transplantation, and (c) syngenic transplantation. The first is effective at exploring genesis events. The second is used to assess cell behaviour in general and metastatic features in particular. The third provides the best model to investigate immune- tumour interactions (Becker et al., 2009). The most frequently used syngenic model of melanoma is the B16/BL6 model where BL6 cells, derived from a spontaneous melanoma in a C57/BL6 mouse are injected subcutaneously into the dorsal surface of a C57/BL6 mouse. Several lines of research that target the immune system to prevent progression of this tumour have seen some efficacy (Becker et al., 2009), suggesting that this model may be a good representation of immune-tumour interaction. However, this model does not accurately represent the metastatic potential of human melanoma. General application of this model follows a reliable course. Untreated mice injected with approximately 2 x 105 B16 cells display visually apparent tumours within two weeks of injection. Animals can be sacrificed at anywhere from day seventeen to thirty-eight following injection to recover sufficiently palpable tumours for general histology (Yamaguchi, 2006; Hu, 2010) . 29 Mast cells have been shown to be both pro- and anti- tumurogenic (Theoharides et al., 2004). In the case of melanoma, mast cells are necessary for ultraviolet light immunosuppression, assist in the breakdown of local connective tissue to give a developing tumour room to grow, release melanoma cell mitogens like fibroblast growth factor-2, and secret vascular endothelial growth factor to promote tumour vascularisation. In addition, mast cell- deficient mice are not only less likely to develop an independent blood supply for a developing tumour, but are also less likely to develop melanoma metastases. Conversely, mast cells are still part of the immune system and can, via their chemotaxis factors, recruit neutrophils, eosinophils, monocytes, and CD4+ T lymphocytes to attack the developing tumour. Furthermore, common mast cell secretory products such as TNFa, interleukin 1 & 6, and interferon y have all been shown to be anti-tumerogenic (Ch'ng et al., 2006). Therefore, mast cells, likely as a function of their heterogeneity, play a complex role in the development and progression of melanoma. 30 1.13 Staining Techniques Toluidine Blue-0 stains mast cell granules metachromatically such that they appear purple with most other tissue appearing blue. For all tissue stained with toluidine blue herein, slides were deparaffinised in each of two changes of xylene for six minutes each, washed through two changes of 100% ethanol, and rehydrated by singular changes of 90% ethanol, 70% ethanol, and 60% ethanol for three minutes each. Slides were stained in toluidine blue solution (0.40g/200mL dH20) for three minutes, dipped in tap water and excess stain was removed in two changes of acetone for three minutes each. Finally, slides were cleared with ten dips in two changes of xylene and mounted with glass coverslips with Permount. Toluidine blue is part of the thiazine family of biological stains and thus it is a small stain (m.w. = 305.5) (Hotobin & Kiernan, 2002). A related thiazine employed is thionin (m.w. = 263.5). Thionin sections were deparaffinised in two changes of xylene for six minutes each, washed through two changes of 100% ethanol, and rehydrated through two changes of 95% ethanol, and one change of 80% ethanol for three minutes each. Slides were placed in dH20 for ten minutes prior to being stained in thionin solution (1.0g/200mL dH20, pH = 31 2.3) for thirty minutes. The excess stain was removed through two changes each of 95% and 100% ethanol for one minute each, and cleared through ten dips each in two changes of xylene and mounted with Permount. Alizarin red is a stain derived originally from textile dyes. The salt marketed under the name Alizarin Red-S (m.w. = 361) has been found to effectively visualise metallic accumulations in biological tissue, including calcium. This salt is poorly soluble in water and precipitates out of solution when it comes in contact with a variety of crystalised metal species (Puchtler et al., 1969). The McGee-Russell (1958) method was used whereby slides were deparaffinsed for six minutes each in two changes of xylene, washed through two changes of 100% ethanol, and rehydrated through two changes of 95% ethanol, and one change of 80% ethanol. Immediately prior to staining, slides were placed in dH20 for ten minutes. The staining solution was prepared by mixing 4.0 g of Alizarin Red-S in 200mL dH20 and calibrating the pH to 4.2 with glacial acetic acid and ammonium hydroxide. Excess stain was removed through two dips each in acetone, and an acetone : xylene (1:1) mixture and cleared - in ten dips each in two changes of xylene. Slides were mounted with glass coverslips using Permount. 32 Safranin-0 (m.w. = 351) was used as a non-thiazine mode of visualising mast cells. It belongs to the azine family and appears red in water. When acidified it is a very specific stain for highly acidic glucoseamineglycans like heparin which it stains orthochromatically. This preparation would result in virtually no background staining due to this specificity; therefore, samples stained with safranin herein included other stains to ensure that the entire tissue, rather than the mast cells alone could be easily viewed. Slides were deparaffinsed for six minutes each in two changes of xylene, washed through two changes of 100% ethanol, rehydrated through two changes of 95% ethanol, and one change of 80% ethanol. Immediately prior to staining slides were placed in dH20 for ten minutes. Then, they were stained for two minutes in Weigert's (iron) hematoxylin to visualise nuceli and rinsed in running tap water for ten minutes. Fast green (FGF) staining followed for two minutes to visualise connective tissue fibers and cytoplasm. The slides were placed for thirty seconds in 1% glacial acetic acid before staining for five minutes in 0.1% safranin-o solution. The excess stain was removed through two changes each of 95% and 100% ethanol for one minute each, and cleared through ten dips each in two changes of xylene and mounted using Permount. 1.14 References Abdel-Majid, R.M., & Marshall, J.S. (2004). Prostaglandin E2 induces degranulation-independent production of vascular endothelial growth factor by human mast cells. The Journal of Immunology. 172: 1227-1236. Aldenborg, F. (1987).Thymus dependence of compound 48/80- induced mucosal mast cell proliferation. International Achieves of Allergy and Immunology. 84(3): 298-305. Becker, J.C., Houben, R., Schrama, D., et al. (2009) . Mouse models for melanoma: a personal perspective. Experimental Dermatology. 19: 157-164. Befus, A.D., Bienenstock, J., Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Befus, A.D., Lee, T., Guto, R., et al. (1986). Histologic and Functional Properties of Mast Cells in Rats & Humans.In. Befus, A.D., Bienenstock, J., Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Bloom, G.D. (1984).A Short History of the Mast Cel.l. Acta Oto-Laryngologica. 414: 87-92. 34 Campbell, D.J., & Kiernan, J.A. (1966).Mast Cells in the Central Nervous System. Nature. 210(5037): 756-757. Ch'ng, S., Wallis, R.A., Yuan, L., et al. (2006). Mast cells and cutaneous malignancies. Modern Pathology. 19: 149- 159. Chen, C.C., Grimbaldeston, M.A., Tsai, M., et al. (2005). Identification of mast cell progenitors in adult mice. Proceedings of the National Academy of Sciences, USA. 102 (32) : 11408-11413. Chen, X.J., & He, Z.Y. (1989) .Quantitative Study of Circadian Variations in Mast Cells Number in Difference Regions of the Mouse.ActaAnatomica. 136: 222-225. Colliver, T.L., Pyott, S.J., Achalabun, M., &Eewing, A.G. (2000).VMAT-Mediated Changes in Quantal Size and Vesicular Volume. The Journal of Neuroscience. 20(14): 5276-5282. Cook, L.L., & Persinger, M .A. (2000). Suppression of experimental allergic encephalomyelitis is specific to the frequency and intensity of nocturnally applied, intermittent magnetic fields in the rats. Neuroscience Letters. 292(3): 171-174. 35 Crivellato, E., Beltrami, C.A., Mallardi, F., &Ribatti, D. (2003). Historical Review: Paul Ehrlich's Doctoral Thesis: A Milestone in the Study of Mast Cells. British Journal of Haematology. 123: 19-21. Davidson, S., Kinarty, A., Coleman, A., et al. (1986). Fibroblasts Are Required for Mast Cell Granule Synthesis. In. Befus, A.D., Bienenstock, J. , Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Denberg, J.A., Tanno, Y., SBienenstock, J. (1986).Growth Differentiation of Human Basophils, Eosinophils, and Mast Cells. In. Befus, A.D., Bienenstock, J., Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Dimitriadou, V., Pang, X., Theohardies, T.C. (2000). Hydroxyzine inhibits experimental allergic encephalomyelitis (EAE) and associated brain mast cell activation. International Journal of Immunopharmacology. 22: 673-684. Dropp, J.J. (1976). Mast cells in the mammalian brain. Acta Anatomica.' 94: 1-21. 36 Dvorak, A.M. (1986). Morphological Expressions of Maturation and Function Can Affect the Ability to Identify Mast Cells and Basophils in Man, Guinea Pig and Mouse. In. Mast Cell Differentiation and Heterogeneity. Befus, A.D., Bienenstock, J. , Denburg, J.A. ed. Raven Press: New York, NY. El Sayed, S.O., & Dyson, M. (1996). Effects of laser pulse repetition rate and pulse duration on mast cell number and degranulation. Lasers in Surgery and Medicine. 19: 433-437. Enerback, L. (1986). Mast Cell Heterogeneity: The Evolution of the Concept of A Specific Mast Cell. In. Mast Cell Differentiation and Heterogeneity. Befus, A.D., Bienenstock, J. , Denburg, J.A. ed. Raven Press: New York, NY. Esquifino, A.I., Cano, P., Zapata, A., et al. (2006). Experimental allergic encephalomyelitis in pituitary- grafted Lewis rats. Journal of Neuroinflammation.3: 20. Folkman, J. (1971). Tumor Angiogenesis: Therapeutic Implications. The New England Journal of Medicine. 285 (21) : 1182-1186. 37 Galli, S.J. (1986). Mast Cell Heterogeneity: Can Variation in Mast Cell Phenotype Be Explained Without Postulating the Existence of Distinct Mast Cell Lineages. In. Befus, A.D., Bienenstock, J., Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Gregory, G.D., Robbie-Ryan, M., Secor, V.H., et al. (2005). Mast cells are required for optimal autoreactive T cell responses in a murine model of multiple sclerosis. European Journal of Immunology. 35: 3478-3486. Gurish, M.F., & Boyce, J.A. (2002). Mast Cell Growth, Differentiation, and Death. Clinical Reviews in Allergy and Immunology. 22: 107-118. Haig, D.M., Menamin, C., & Jarrett, E.E.E. (1986). Mast Cell Development in the Rat.In.Befus, A.D., Bienenstock, J., Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Horobin, R.W., & Kiernan, J.A. ed. (2002). Conn's Biological Stains: A Handbook of Dyes, Stains and Florochromes for Use in Biology and Medicine. 10th ed. Bios Scientific Publishers: Oxford, UK. 38 Hu, J.H., St-Pierre, L.S., Buckner, C.A., et al. (2010). Growth of injected melanoma cells is suppressed by- whole body exposure to specific spatial-temporal configurations of weak intensity magnetic fields. International Journal of Radiation Biology. 86(2): 79- 88. Jexiorska, M., McCollum, C., & Woolley, D.E. (1998). Calcification in atherosclerotic plagues of human carotid arteries: associations with mast cells and macrophages. Journal of Pathology. 185: 10-17. Johansson, O., Gangi, S., Liang, Y., et al. (2001) . Cutaneous mast cells are altered in normal healthy volunteers sitting in front of ordinary TVs/PCs results from open-field provocation experiments. Journal of Cutaneous Pathology. 28: 523-519. Jorpes, E. (1935). On Heparin, its chemical nature and properties. Acta Medica Scandinavia. 88: 427-433. Kitamura, Y., Go, S., & Hatanaka, K. (1978). Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood. 52: 447-452. Kitamura, Y., Nakano, T., Sonoda, T., et al. (1986). Probable Transdifferentiation Between Connective Tissue and Mucosal Mast Cells. In. Befus, A.D., Bienenstock, J., Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Kruger, P.G. (1974). Demonstration of Mast Cells in the Albino Rat Brain. Experientia. 30(7): 810-811. Kuriu, A., Sonoda, S., Kanakura, Y., et al. (1989). Proliferative potential of degranulated murine peritoneal mast cells. Blood. 74(3): 925-929. Melanoma Skin Cancer: Facts and Figures. 02 March 2009. Public Health Agency of Canada. 26 June 2012. chttp://www.phac-aspc.gc.ca/cd- mc/cancer/melanoma_skin_cancer_figures- cancer_peau_melanome_figures-eng.php>. McGee-Russell, S.M. (1958). Histochemical methods for calcium. Journal of Histochemistry & Cytochemistry. 6: 22. Medic, N., Lorenzon, P., Vita, F., et al. (2010). Mast cell adhesion induces cytoskleletal modifications and programmed cell death in oligodendrocytes. Journal of Neuroimmunology. 218: 57-66. Menon, A.K., Holowka, W.W., & Baird, B. (1984). Small oligomers of immunoglobulin E cause large-scale clustering of IgE receptors on the surface of rat basophilic leukemia cells. Journal of Cell Biology. 98(2): 577-583. Metcalfe, D.D., Baram, D., & Mekori, Y.A. (1997).Mast Cells. Physiological Reviews. 77(4): 1033-1079. Miller, A.J., & Mihm, M.C. (2006). Mechanisms of Disease: Melanoma. New England Journal of Medicine. 355: 51-65. Mota, I., & Vugman, I. (1956) .Effects of Anaphylatic Shock and Compound 48/80 on the Mast Cells of the Guniea Pig Lung. Nature. 177(4505): 427-429. Niederhoffer, N., Levy, R., Sick, E., et al. (2009). Amyloid beta peptides trigger CD47-dependent mast cell secretory and phagocytic responses. International Journal of Immunopathology and Pharmacology. 22: 473- 483. Oliver, J., Bloom, F., & Mangieri, C. (1947). On The Origin of Heparin. Journal of Experimental Medicine. 86(2): 107-116. Owens, T., Sriram, S. (1995).The immunology of multiple sclerosis and its animal model, .experimental allergic encephalomyelitis. Neurological Clinics. 13: 51-73. 41 Parrat, J.R., & West, G.B. (1957) .5-Hyrdroxytryptamine and tissue mast cells. The Journal of Physiology. 137: 169- 178 . Payne, G.W. (2006) . Effect of Inflammation on the Aging Microcirculation: Impact on Skeletal Muscle Blood Flow Control. Microcirculation. 13: 343-532. Pejler, G., Ronnberg, E., Waern, I., & Wernersson, S. (2010). Mast cell proteases: multifaceted regulators of inflammatory disease. Blood. 115: 4981-4990. Persinger, M.A. (1977). Mast cells in the brain: Possibilities for physiological psychology. Physiological Psychology. 5(2): 166-176. Persinger, M.A. (1981). Developmental alterations in mast cell numbers and distributions within the thalamus of the albino rat. Developmental Neuroscience. 4(3): 220- 224 . Persinger, M.A., Cook, L.L., & Koren, S.A. (1999). Suppression of Experimental Allergic Encephalomyelitis in Rats Exposed Nocturnally to Magnetic Fields. International Journal of Neuroscience. 100: 107-116. Price, J.A., & Strattan, R.D. (1998).Analysis of the effect of a 60 Hz AC field on histamine release by rat peritoneal mast cells. Bioelectromagnetics. 19: 192- 198. Puchtler, H., Meloan, S.N., Terry, M.S. (1969). On the history and mechanism of alizarin and alizarin red s stains for calcium. Journal of Histochemistry & Cytochemistry. 17: 110. Reite, O.B. (1965). A Phylogenetical Approach to the Functional Significance of Tissue Mast Cell Histamine. Nature. 206 (4991): 1334-1336. Sayed, B.A., Christy, A., Quirion, M.R., & Brown, M.A. (2008) . The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance. Annual Review of Immunology. 26: 705-739. Scorza, F.A., Atida, R.M., Da Gragia, M. , et al., (2009). The pilocarpine model of epilepsy: what have we learned?. Annals of the Brazilian Academy of Sciences. 81(3): 345-365. Selye, H. (1965). The Mast Cells.Butterworth, Washington, DC. Silverman, A.J., Sutherland, A.K., Wilhelm, M., Silver, R. (2000). Mast Cells Migrate from Blood to Brain. The Journal of Neuroscience. 20(1): 401-408. 43 Skaper, S.D., Guisti, P., & Facci, L. (2012). Microglia and mast cells: two tracks on the road to neuroinf lamination. The Journal of the Federation of American Societies for Experimental Medicine. In Press, doi:10.1096/fj.11-197194 Stevens, R.L., Katz, H.R., Seldin, D.C., & Austen, K.F. (1986). Biochemical Characteristics Distinguish Subclasses of Mammalian MCs. In. Befus, A.D., Bienenstock, J. , Denburg, J.A. ed. (1986). Mast Cell Differentiation and Heterogeneity. Raven Press: New York, NY. Theoharides, T.C., Alysandratos, K.D., Angelidou, A., et al. (2012). Mast cells and inflammation. Biochemica et Biophysica Acta. 1822: 21-33. Theoharides, T., & Conti, P. (2004). Mast cells: the Jekyll and Hyde of tumour growth. Trends in Immunology. 25: 235-241. Tsai, M., Takashi, T., Thompson, H., et al. (1991). Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor. Proceedings of the National Academy of Sciences, USA. 88(14): 6382-6386. 44 Wedemeyer, J., Tsai, M., & Galli, S.J. (2000). Roles of mast cells and basophils in innate and acquired immunity. Current Opinion in Immunology. 12(6): 624-631. Wilhelm, M., Silver, R., & Silverman, A.J. (2005). Central nervous system neurons acquire mast cell products via transgranulation. European Journal of Neuroscience. 22 (9) : 2238-2248. Zuang, X., Silverman, A.J., & Silver, R. (1993). Reproductive Behavior, Endocrine State, and the Distribution of GnRH-like Immunoreactive Mast Cells in the Dove Brain. Hormones and Behavior. 27: 283-295. 45 Chapter 2 - Nightly Exposure to Weak Time-Varying Magnetic Fields Enhanced Brain Mast Cell Numbers: Testing Potential Biophysical Models Abstract To test which biophysical explanation was most congruent with numerical changes in the numbers of mast cells (MCs) within the thalamus of Lewis rats inoculated to produce experimental allergic encephalomyelitis these cells were counted for rats that had been exposed to 7 Hz or 40 Hz square wave magnetic fields whose intensities were less than 5 nT or ranged from 5 to 20 to 40 nT, 5 to 50 nT, or 5 to 500 nT over the course of the hourly, 6 min presentations. There was a strong threshold-like effect reflected by twice the number of MCs in the rats exposed to the three more intense fields compared to the <5 nT field. Of the three biophysical relationships employed to describe relationship between magnetic field strength and electromotive, a particular threshold current assuming a co-axial like model was most proximal. These results indicate that MC numbers respond to small changes in weak field intensities that are similar to those measured during geomagnetic activity. 46 2.1 Introduction A biophysical approach to demyelinating diseases such as multiple sclerosis in human beings or experimental allergic encephalomyelitis (EAE) in rodents has the potential to open new perspectives and treatments. EAE is the response to an immunological reaction (Goverman & Brab, 1996) to a decapeptide sequence found in myelin basic protein (MBP) (Fujinami & Oldstone, 1985) and in viral homologues (Jahnke et al., 1985) responsible for measles, hepatitis B and influenza A and B. Infiltration of mononuclear cells, particularly macrophages, T-lymphocytes and some B-lymphocytes (Huitinga et al., 1990), within the brain, and degranulation (Bo et al., 1991) of mast cells (MC) are coincident with or immediately precede the onset of clinical symptoms that include tail hypotonus and hind leg weakness. Compounds such as chromylan that prevent MC degranulation (Esposito et al., 2001), tumour necrosis- factor inhibitors (Monastra et al., 1991), alpha-1 adrenergic agents (prasozin) but not alpha-2 adrenergic agonists (Brosnan et al., 1985), and immunosuppressive or cortisol-elevating treatments such as restraint (Esposito et al., 2002) diminish the severity of the clinical symptoms. 47 Peripheral tolerance by oral consumption of MBP suppresses EAE by inducing peripheral tolerance (Chen, et al., 1994) while lesioning of the anterior hypothalamus attenuated the severity by actively inhibiting neurogenic controlled cell proliferation (Wertman et al., 1985). The specific timing of chemically-induced epileptic seizure can either suppress or facilitate the severity of EAE (Misshagi et al., 1992). However, all of these procedures are either invasive or blood-dependent processes. On the other hand we have found that whole body exposure of Lewis female rats for 6 min once per hour during the night to 7 Hz, amplitude modulated magnetic fields with intensities that increased from <5 nT to peak intensities of 50 nT produced marked amelioration of EAE symptoms (Cook & Persinger, 2000). The symptoms of rats exposed to the same temporal pattern of peak intensities at 500 nT or to 40 Hz fields at either 50 or 500 nT did not differ from sham-field controls. We employed the range (<5 nT, <5 nT to 50 nT, and <5 to 50 nT) rather than fixed intensities in order to simulate geomagnetic activity. For comparison AKMA micromagnets (60 mT) imbedded in the occipital skull potentiated EAE (Vidic et al., 1989). Laser pulses for 27 s over a 10,000 fold frequency range 48 with energy densities of 22 J-crrf2 increased the numbers of MCs in skin during tissue repair (El Sayed & Dyson, 1996). Enhanced degranulation was restricted to 20 Hz (45 ms pulse duration) and 292 Hz (3 ms pulse duration). Current densities of 0.5 mA'cm”2 of positive pulsed electrical stimulation reduced MC numbers in epithelial tissue by almost half that of controls or same-intensity negative polarity current (Reich et al., 1991). A direct effect of physiologically-patterned magnetic fields upon mast cell function might mediate the positive effects of this treatment. MCs, because of their perivascular location, have been considered the gateways to neuroimmunological dynamics (Persinger, 1977; Orr, 1988; Leon et al., 1994). Their copious granules, in the range of 0.2 to 0.6 pm, contain heparin, histamine, and a plethora of chemokines, nerve growth factors, and enzymes, such as cyclooxyigenase, that initiate the inflammatory chemical pathways (Schmauder-Chock & Chock, 1989). Release of MC granules peripherally can be evoked by antidromic stimulation of the cutaneous nerve (Kiernan, 1971) while enhancement of granule composition for MCs within the meninges follows electrical stimulation of the cervical superior ganglion (Ferrante et al., 1990). Incubation of 49 brain tissue with the degranulating agent C48/80 produces demeylination (Skaper et al., 1996) and neurotoxity due in large part from elaboration of NO by astrocytes from TNF- alpha (Salvemini et al., 1991). Mast cell degranulating peptide, a 22 amino acid sequence from bee venom, produces long-term potentiation (LTP) in hippocampal slices and results in formation of channels in asolectin bilayers that are particularly permeable to K+ (Ide et al., 1989). Numbers of thalamic MCs usually decrease during the development of EAE (Brenner et al., 1994). Given these data and the propensity for increased MCs around areas of deinnervation (Sanchez-Mejorada, 1992), which is interpreted as a reconstructing indicator, we counted the numbers of MCs and their degree of degranulation within the thalamus of female Lewis rats in which EAE had been induced. We predicted the equivalent energies and induced currents would be in the order of a pA. Whole cell currents of about 1 pA, without channel activity, was associated with inositol 1,4,5 triphosphate (Insl,4,5P3)-induced Ca++ influx in peritoneal MCs from C48/80 but only during membrane hyperpolarization (Matthews et al., 1989). 50 2.2 Specific Descriptions To Be Assessed According to Adey (1981), the threshold for discerning an electromagnetic field can be described by: V2 =k • T • B • Q (1) where k is the Boltzman constant (1.23- 1CT23 J ’K”1),! is the physiological temperature(310°K), B is the frequency band (7 Hz, or 40 Hz), and Q is resistance of an extracellular fluid (-300 Q-cm). The threshold value is about 10~9 V and when divided by the extracellular resistance indicates a current of 3-10“12 A or 3 pA, which is within the range reported by (Matthews, et al., 1989). If Faraday's law is applied for the lowest field strength (10-40 nT) to a cm2 area at 40 Hz, V would be in the order of 10~10 V but at 500 nT would be -2-10~9 V which would be above the thermal threshold. If this approach is optimal, then there should be an inflection point in MC numbers for the 500 nT exposed group compared to the lower intensity group. The second equation, assumes the elongated nature of the MC is similar to a coaxial cable described by: B=p0J(2na2)'1 r (2) Where I is the current, a is the radius of the cylindrical conductor (in the cell) and r is the magnetic field within 51 the small shell (membrane) around it. For J of 1 pA, the B is 8-1CT10 T, and assuming ~25 channel equivalents per cell surface in operation at any given time, this would integrate to 20 nT, the second intensity range for the exposures. If this approach is valid, then the MC change should occur for this group as well as those exposed to the higher intensities. The third equation involved the classic derivation by Liboff (1992) of the Larmor frequency resonance: mv2r'1=Bqv ( 3) and substituting v for 2nrf, results in: B=2nf kg-q'1 (4) For the mass of a proton, which is most likely involved with low current channels with marked pH sensitivity (Decoursey, 2002), the value for 7 Hz on the unit charge would be 220 nT but 1.2 uT for 40 Hz. If this is correct then an interaction between frequency of the applied field and intensity be would evident because the amplitude modulation of the 7 Hz field passes through this value as it slowly steps to 500 nT. 52 2.3 Methods 2.3.1 Specimens. Sectioned (10 vim) forebrains from 42 female Lewis rats were selected from our laboratory slide library. They were about 150 days of age when killed. 2.3.1 History. All rats had been injected subdermally into the left and right hand foot pad with 0.1 cc of fresh Lewis rat spinal cord (40 mg/kg) suspended in Freund's complete adjuvant. They were then placed into one of four plastic cages. Two of the cages were surrounded by Helmholtz coils (Persinger et al., 2005) while the two cages below this arrangement were not. Each of the coils was activated by a Commodadore computer connected to a Heath Schlumberger function generator that generated a amplitude modulated 7 Hz or 40 Hz square wave magnetic field for 6 min once per hour, starting at midnight, for 8 hours (onset of light cycle). The shape of the pattern has been published many times elsewhere (Persinger et al., 2005). The range of the amplitudes ranged from 1 nT to a peak intensity of either 50 nT or 500 nT for different groups. There was no discernible (<5 nT) field induced in the other cages when 50 nT was activated but a peak variable 10 to 40 nT field strength was present when 53 the 500 nT field was activated. This allowed for a unique opportunity to discern potential intensity threshold. At 20 days the rat brains, from approximately 10 different experiments, had been removed quickly and fixed in ethanol-formal-acetic acid. After processing they were embedded in paraffin and then sectioned at 10 pm by a microtome. Toluidine blue-0 stain had been used to identify any foci of mononuclear cells within the parenchyma. 2.3.3 Counting Procedures. There were between 9 and 14 sectioned brains per group for the 4 experimental groups. After additional staining with toluidine blue 0, MCs were counted at 400 X magnification within the entire thalamus in a total of 5 to 6 equally separated sections between the caudal and rostral boundaries of the thalamus. They were classified, from Type 1 to Type 5, according to progress in degrees of degranulation as shown in Figure 2.1. 2.3.4 Statistical analyses. The numbers of different types of MCs as a function of the frequency (7 Hz vs 50 Hz) and peak intensity (<5 nT, 20 nT, 50 nT, 500 nT) were analyzed by repeated measure analysis of variance (MANOVA) from SPSS PC 16 software. All post hoc and ancillary tests and procedures employed this program. 54 One - Intact, no degranulation Two - 1 to five discrete extra- cytoplasmic granules ■ Three - 6 to 10 discrete extra- cytoplasmic granules ■ Four - mass granule ejection with up to H of granular content found outside of the cytoplasm ■ Five - complete, or near complete degranulation with little or no apparent intracellular granules. 55 Figure 2.1 Examples and criteria for mast cell degranulation ■classifications. 2.4 Results The results of the three way analyses of variance with two between subject levels (intensity and frequency) and one within subject level (types of mast cells) demonstrated a statistically significant difference in the total numbers of mast cells as a function of magnetic field intensity [F(3,34)=6.81, p< 0.001] that accommodated more than one- third of the variance (eta2=0.36). Post hoc analysis indicated that the primary source of this main effect was due to the greater (almost twice the) number of mast cells within the thalami of rats that had been exposed to the peak intensity of 10-40 nT, 50 nT and 500 nT, compared to those exposed to experimental variations less than 10 nT (limits of measurement). These results are shown in Figure 2.2. In addition to the expected difference in the numbers of different types of mast cells [F(4,136)=184.86, p < 0.001], there was a significant interaction between type of mast cell and treatment intensity [F(12, 136)=3.06, p < 0.01] and a marginal three way interaction between intensity, frequency, and type of mast cell [F (12,13 6)=1.78, p < 0.06]. Post hoc analyses of the two-way interaction (partial eta2=0.21) indicated that there were more type 3 degranulated MCs in the thalami of rats exposed to the 10-40 56 nT fields compared to the reference (< 10 nT) group but not the 50 or 500 nT field. Polynomial analyses for the five types of MCs indicated that significant linear trends (all dfs=l,38) were significant for type 1 (F=9.23), 2 (F = 8.13), and 4 (F=7.43) MCs. There was no significant intensity dependence for type 5 (F<1) and marginally significant quadratic relation (peak at 50 nT) for type 3 (F=3.67) with a peak at the 10-40 nT and 50 nT intensities. Factor analyses of the five types of MC degranulation revealed two factors. The factor scores for the first factor (eigen value=2.25, explaining 45% of the variance), which was loaded significantly by type 1 (.89) and 2 (.84) MCs, was significantly higher for the 20, 50 and 500 nT groups compared to the <5 nT group. The means and SEMs for the scores for each group in increasing intensity were: -0.72 (.21), 0.43(.24), 0.24 (.37) and 0.41 (.28), respectively. Stated alternatively, the difference between the numbers of type 1 and 2 MCs in the thalamus between the <5 nT and other groups was separated by more than one standard deviation. 57 90 80 70 60 50 40 30 20 10 0 Figure 2.2 The toal number of observed mast cells in the thalamus (vertical axis) by intensity of magnetic field exposure (horizontal axis). 2.5 Discussion and Conclusion EAE is usually associated with reductions in brain MC numbers in the thalamus (Brenner et al., 2004). However in the present study the rats that had been exposed during the nocturnal period from the time of inoculation to a range between 1 nT and either 10 to 40 nT, 50 nT or 500 nT at 7 Hz or 40 Hz magnetic fields displayed significantly more MCs than those exposed to < 5 nT. The numbers of cells almost doubled and would be consistent with processes that could lead to amelioration of the clinical symptoms. However since our previous research (Cook & Persinger, 2000; 58 Kinoshameg & Persinger, 2004) showed that only those exposed to the 7 Hz, 50 nT fields displayed marked reductions in clinical symptoms, other mediating variables are likely involved. The increase in MCs occurred in brains of rats exposed to time-varying magnetic field intensities between 10 nT and 500 nT (the range of the study) . The increase showed a threshold like effect in that all groups above the 10-40 nT intensities displayed comparable numbers of thalamic mast cells. From the three explanations we explored, which are clearly not the only possibilities, the results are most consistent with the effect of the magnetic field acting upon the co-axial cable like properties of the mast cell and its membrane. Like a switch, once the threshold had been reached, there was no further effect. The only differential effect upon MC degranulation, rather than MC number, occurred in the thalami of rats exposed to the 10-40 nT fields. Although the effect was small it was significant statistically and is consistent with the second explanation of the co-axial like cable. Within the brain, as in most tissues, MCs often appear as elongated cells along a blood vessel. This particular geometry may facilitate this particular mechanism of 59 interacting with weak externally applied magnetic fields. Although we did not measure mitotic indices, it may not be coincidence that numbers of MCs in the groups exposed to the >10 nT fields were almost double the value of the group exposed to the <5 nT field. Calcium has been a classical candidate for mediating magnetic field effects since the eminent biophysicist Wolfgang Ludwig first described the equations in 1968 [34] and both Adey (1981) and Liboff (1992) verified its central role in multiple transmembrane cellular functions. Muehlen et al. (1991) showed that a triphosphate-activated GTP- binding protein, specifically by a G protein that couples to phosopholipase C, increased intracellular calcium in mast cells. Recent research by Buckner (2011) has verified that temporally-patterned magnetic fields, like the ones employed in this study, activate subtypes of calcium channels. 60 2.6 References Adey, W.R. (1981). Tissue interactions with nonionizing electromagnetic fields. Physiological Reviews. 61:435- 513. Bo, L., Olsson, T., & Nyland, H., et al. (1991). Mast cells in brains during experimental allergic encephalomyelitis in Lewis rats. Journal of Neurological Sciences. 105: 135-142. Brenner, T. Soffer, D. Shalit, M., et al. (1994). Mast cells in experimental allergic encephalomyelitis: characterization, distribution in the CNS and in vitro application of myelin basic protein in neuropeptides. Journal of Neurological Sciences. 122: 210-213. Brosnan, C.F., Goldmuntz, E.A., Cammer, W. , et al. (1985). Prazosin, an alpha-1 adrenergic antagonist, suppresses experimental autoimmune encephalomyelitis in the Lewis rat. Proceedings of the National Academy of Sciences, USA. 82: 5915-5919. Buckner, C. (2011). Effects of electromagnetic fields on biological processes are spatial and temporal- dependent. Laurentian University Ph.D. Dissertation (Biomolecular Sciences): Sudbury, ON. 61 Chen, Y., Kuchroo, V.K., Inobe, J. , et al. (1994). Regulatory T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science. 265: 1237-1240. Cook, L.L., & Persinger, M.A. (2000). Suppression of experimental allergic encephalomyelitis is specific to frequency and intensity of the applied, intermittent magnetic field in rats. Neuroscience Letters. 292: 171- 174 . Decoursey, T.E. (2002) Voltage-gated proton channels and other proton transfer pathways. Physiological Reviews. 83: 475-579. El Sayed, O.S., & Dyson, M. (1996).Effect of laser pulse repletion rate and pulse duration on mast cell number and degranulation. Lasers in Surgery and Medicine. 19: 433-437. Esposito, P., Chandler, N., Kandere, K., et al. (2002). Corticotrophin-releasing hormone and brain mast cells regulate blood-brain-barrier permeability induced by acute stress. The Journal of Pharmacology and Experimental Therapeutics. 303(3): 1061-1066. 62 Esposito, P., Jacobson, S., Connolly, R., et al. (2001). Non-invasive assessment of blood-brain barrier (BBB) permeability using a gamma camera to detect 99Tecthnetium-gluceptate extravasation in rat brain. Brain Research Protocols. 8: 143-149. Ferrante, F. Ricci, A. Felici, L., et al. (1990). Suggestive evidence for a functional association between mast cells and sympathetic nerves in meningeal membranes. Acta Histochemica Cytochemica. 23(5): 637-646. Fujinami, R.S. & Oldstone, M.B.A. (1985). Amino acid homology between the encephalitogenic site of myelin basic protein and viruses: mechanisms of autoimmunity. Science. 230: 1043-1046. Goverman, J. & Brab, T. (1996) . Rodent models of experimental allergic encephalomyelitis applied to the study of multiple sclerosis. Laboratory Animal Sciences. 46(5): 482-492. Huitinga, I., van Rooijen, N., de Groot, C.J.A., et al. (1990) Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages. Journal of Experimental Medicine. 172: 1025-1033. 63 Ide, T., Taguchi, T,, Morita, T., et al. (1989). Mast cell degranulating peptide form voltage gated and cation- selective channels in lipid layers. Biochemical and Biophysical Research Communications. 163(1): 155-160. Jahnke, U. , Fischer, E.H., & Alvord, E.C. (1985). Sequence homology between certain viral proteins and proteins related to encephalomyelitis and neuritis. Science. 229: 282-286. Kiernan, J.A. (1971) Degranulation of mast cells following antidromic stimulation of cutaneous nerves. Journal of Anatomy. Ill: 349-350. Kinoshameg, S.A., & Persinger, M.A. (2004). Suppression of experimental allergic encephalomyelitis in rats by 50 nT, 7 Hz amplitude-modulated nocturnal magnetic fields depends on when after inoculation the fields are applied. Neuroscience Letters. 370: 166-170. Leon, A., Buriani, A., Toso, R.D., et al. (1994). Mast cells synthesize, store, and release nerve growth factor. Proceedings of the National Academy of Sciences, USA. 91: 3739-3743. Liboff, A.R. Cyclotron resonance in membrane transport.in B. Norden and C. Ramel (eds), Interaction mechanisms of 64 low level electromagnetic fields and systems, pp. 130- 147, Oxford Press, Oxford, 1992. Ludwig, H.W. (1968). A hypothesis concerning the absorption mechanism of atmospherics in the nervous system. International Journal of Biometeorology. 12: 93-98. Matthews, G., Neher, E., & Penner, R. (1989). Second messenger-activated calcium influx in rat peritoneal mast cells. Journal of Physiology. 418: 105-130. Misshagi, B., Richards, P.M., & Persinger, M.A. (1992). Severity of experimental allergic encephalomyelitis in rats depends upon the temporal contiguity between limbic seizures and inoculation. Pharmacology, Biochemistry and Behavior. 43: 1081-1086. Monastra, G., Cross, A.H., & Raine, C.S. (1991).Prevention of experimental allergic encephalomyelitis by an inhibitor of tumor necrosis factor (TNF) synthesis. Journal of Neuropathology and Experimental Neurology. 50: 295. Orr, E.L. (1988). Nervous-system-associated mast cells. Drug Development Research. 15: 195-205. 65 Persinger, M.A. (1977). Mast cells in the brain: possibilities for physiological psychology. Physiological Psychology. 5: 166-17 6. Persinger, M.A., McKay, B.E. 0'Donovan, C.A., et al. (2005). Sudden death in epileptic rats exposed to nocturnal magnetic fields that simulate the shape and the intensity of sudden changes in geomagnetic activity: an experiment in response to Schnabel, Beblo and May. International Journal of Biometeorology. 49: 256-261. Reich, J.D., Mingyu, X. Cazzaninga, A.L., et al. (1991). Different polarity electrical stimulation can manipulate the number of mast cells during healing of superficial wounds", The Journal of Investigative Dermatology. 96(4): 574. Salvemini, D. Masini, E. Pistelli, A., et al. (1991). Nitric oxide: a regulatory mediator of mast cell reactivity. Journal of Cardiovascular Pharmacology. 17: S258-S2 64. Sanchez-Mejorada, G. & Alonso-DeFlorida, F. (1992). Changes in mast cell distribution in skeletal muscle after denervation. Muscle and Nerve. 15: 716-719. Schmauder-Chock, E.A., & Chock, S.P. Localization of cyclo- oxygenase and prostaglandin E2 in secretory granule of mast cell. The Journal of Histochemistry and Cytochemistry. 37: 1319-1328. Skaper, S.D., Facci, L. Romanello, S., et al. (1996). Mast cell activation causes delayed neurodegeneration in mixed hippocampal cultures via the nitric oxide pathway. Journal of Neurochemistry. 66: 1157-1166. Vidic, B., Marie, D. , & Jankovic, B.D. (1989).Potentiation of experimental allergic encephalomyelitis (EAE) by chronic exposure of the brain to magnetic fields. CNS Immunopharmacology and Immunotoxicology, No. 1104. von z. Muhlen, F., Eckstein, F. & Penner, R. (1991). Guanosine 5' [beta-thio]triphosphate selectively activates calcium signaling in mast cells. Proceedings of the National Academy of Sciences, USA. 88: 92 6-930. Wertman, E., Ovadia, H., Feldman, S., et al. (1985). Prevention of experimental allergic encephalomyelitis by anterior hypothalamic lesions in rats. Neurology. 35: 1468-1470. 67 Chapter 3 - Mast Cell Numbers in Thalami of Very Old Rats Abstract Brain mast cells may have multiple roles involved with inflammation and neuromodulation. Their numbers can be altered by behavioural measures such as handling and sexual activity as well as by pharmacological treatments. To discern if the numbers or state of degranulation of mast cells was altered in very old rats (>700 days) just before death from "natural causes", thalamic values were compared with three groups of younger animals. There were no significant change in the number of MCs with age; however, the degree of degranulation varied primarily in the middle- aged groups. These results suggest that thalamic mast cell function may be more related to the degrees of degranulation rather than raw numbers. From a general prespective, the very old "healthy" aged rat brain does not exhibit classic inflammatory or diffuse degenerative processes to which MCs respond. 68 3.1 Introduction Mast cells (MCs) are remarkably mobile cellular components of connective tissue that are typically found around blood vessels throughout the body (Kalesnikoff & Galli, 2008). Within brain space their location near blood vessels (Dropp, 1976a; Johnson and Krenger, 1992), proclivity to concentrate in regions of deinnervation (Olsson, 1968), and capacity to degranulate in response to neurogenic stimulation (Kiernan, 1971) places them in a unique position as gatekeepers to the brain parenchyma (Persinger, 1977; Theoharides, 1990) . The numbers of MCs within the brain are reduced by preweaning handling (Persinger, 1977), treatment by thyroid hormones (Sabria, et al, 1987) and stress induced by social isolation (Bugajski, et al., 1994). Mated mice displayed increased MC numbers (Yang et al, 1999) over the dorsal thalamus while light- deprived rats display a ten-fold increase in MC number within the lateral geniculate bodies (Mares and Brueckner, 1977). Brain MC membrane-limited (Aubineau and Dimitriadou, 1989) granules contain histamine, about 20 ng/cell for adults and 200 ng/cell in very young rats (Kruger, 1974), which accounts for at least 50% of this biogenic amine in 69 the young brain (Grzanna and Schultz, 1982) and perhaps as high as 90% in the thalamus (Goldschmidt et al, 1985) . MCs also stain for heparin (a highly sulfated glycosaminoglycan) , and (in the rat) serotonin which act in a coordinated fashion to induce local physiological responses. In the thyroid, for example, the release of granules from perifollicular cells produces increased vascular permeability due to the heparin and alterations in colloidal substance that precedes iodothyronine release due to serotonin. Comparable contributions to brain function by local MCs occurs as well (Purcell and Atterwill, 1995). Agents, such as theophylline or proxicromil, which inhibit activation of mast cells, block or markedly attenuate the development of experimental encephalomyelitis in Lewis rats. Degranulation of local MCs (Bo et al, 1991) is frequently observed coincident with or just prior to the wide spread edema and inflammation with which these diseases are associated (Orr, 1988). Mast cell degranulating peptide, a 22 amino acid sequence, induces convulsions and in small amounts induces long-term potentiation in the CA 1 region of the hippocampus (Ide, et al, 1989). Infusion of rats with purified peritoneal MCs resulted in MC appearance within about an hour around the blood 70 vessels which enter, traverse, and exit the velum interpositum between the dorsal thalamus and hippocampus of the rodent brain and represented between 2% and 20% of the local MC population (Silverman et al, 2000). The plethora of other compounds associated with MCs include cytokines, eicosanoids, substance P, chymotrypsin-like esterases, antigen-inducible TNF-alpha (tumor necrosis factor), nerve growth factor or NGF (Purcell and Atterwill, 1995) and antimicrobial peptides (Beaven, 2009). Localisation of cyclo-oxygenase, the first step in prostaglandin cascades, in the secretory granules of MCs could initiate the conditions associated with brain dysfunctions such as the dementias (Purcell and Atterwill, 1995) . A more complete list has been published by Gali et al (2005) . The many stimuli that can trigger granule release include synthetic compounds such as C-48/80, wasp venom (mastoparan), viruses, parasites, and classic immune receptor mediators, particularly IgE (Galli et al, 2008). Consequently the typical numbers of neurons within the brain as a function of ageing, particularly in very old mammals, may help elucidate the causes and correlates of delayed onset diseases and anomalies. A maximum of numbers of MCs within the normal rat brain occur around 13 to 15 71 days of age (Persinger, 1981; Lambracht-Hall et al, 1990), about the time 35S, an indicator for heparin sulfate, peaks and then begins to decline (Ibrahim and Koshayan, 1981). Kiernan (1976) indicated that MCs do not occur in the normal brains of most mammals, including humans. Others suggest marked species differences in the localization of MCs; for example copious MCs are found in the pineal organs and area postrema (Dropp, 197 6b) of human beings but not in rats. However, most mammals show copious MCs in the region of the subfornical organ and the dorsal thalamus (Cammermeyer, 1973; Dropp, 1973). In hamsters ageing (21 to 622 days) was associated with appearance of MCs in the choroid plexus of the lateral and third ventricles (Kelsall and Lewis, 1964). In rat brains over 1 month of age the total numbers of MCs, particularly within the thalamus where more than 98% occur, diminish slowly with geriatric progression (Dropp, 1976a). The primary age-span measure of MCs for rodents was conducted by Kelsall (1966) for hamsters between 35 and 844 days of age. Whereas none of the hamsters less than 200 days showed MCs, about half of the much older (>500 days) showed thalamic MCs. Given the potential potency of these cells and their modulation of pathology we examined their numbers in a population of very old rats over 700 days of age. 72 3.2 Method 3.2.1 Specimens A total of 35 female Wistar albino brains were measured. Eight of the brains were from rats that were allowed to live to the natural end point (701 to 826 days of age) before they displayed indicators that required euthanasia. The remaining brains were obtained from various experimental studies over the last 20 years. All of the rats while alive had been exposed to more or less the same caging, lighting and temperature conditions in our rat colony. 3.2.2 Procedure All rats had been killed by decapitation and the brains had been removed within less than 5 min and placed in ethanol-formalin-acetic acid (EFA). After processing and embedding in paraffin, the cerebrums had been sectioned at 10 pm between the posterior and the anterior commissures. In this study a total of 5 to 7 sections, more or less equally spaced, between the caudal and rostral boundaries of the thalamus, corresponding approximately to -2.3 mm to -5.6 mm relative to bregma or approximately 330, 10 um sections, were stained with toulidine blue 0 for each rat. The total 73 number of MCs per thalamus was determined by taking the mean numbers of MCs for each section and multiplying by the number of potential 10 pm sections (330) comprising thalamic space. The numbers of MCs per section in the thalamus were counted at 400 X magnification. They were easy to identify by their morphology and frank metachromasia. In addition, the state (type) of the MC degranulation was indicated according to the following scale: 1) intact, no extracellular granulation, 2) 1 to 5 discrete extracellular granules, 3) 6 to 10 granules, 4) mass injection, i.e., more than one-quarter of granules found outside the cell, and 5) complete nor near-complete degranulation. 3.2.3 Statistical analyses The basic design was a two way analysis of variance with one between (age group) and one within (type of MC) subject level. Post hoc analyses (Tukey's, p c.05) and all other analyses were completed by SPSS 16 PC software. 74 3.3 Results Two way analyses of variance with one between (age group) and one within (degree of degranulation) subject level for numbers of MCs demonstrated no statistically significant differences between age groups [F(3,31) <1.00]. However there were significant differences between the types of MCs and an interaction [F(12, 124) =2.48, p < 0.01, eta- squared=19%] between age group (90 to 100 days, 101 to 200 days, 200 to 700 days and 701 to 827 days) and degree of degranulation. Post hoc analysis indicated that the primary source of the interaction was due to the greater numbers of type 1 (no degranulation) cells in the youngest group of rats compared to the other four groups and for the greater numbers of type 3 degranulated MCs in the 200 to 700 day old group. The two way ANOVA for the proportion of each type of MC in order to accommodate for the range in raw numbers of cells as a function of age group and cell type verified the statistically significant interaction [ F (12,124)=3.67, eta- squared=27%]. The explained variance for the raw and proportioned values for the five types of MC morphologies ranged from 50 to 58%. Two way ANOVA as a function of age group and left or right side of the thalamus did not reveal any significant 75 main effects or interactions (all Fs <1). Factor analysis, after varimax rotation, for the five types of MCs revealed two factors. The first factor (eigen value=2.41) that explained 48% of the variance was loaded significantly (>0.5) by type 1 (-.96), type 4 (. 77) and type 5 (.69) classifications. The second factor (eigen value=1.32) was loaded by type 2 (.69) and type 3 (-.8 9) types. One way analyses of variance as a function of age group demonstrated significant age group differences for factor scores for factor 1 only [F(3,30)= 4.88, p <.01, eta2=33%]. Post hoc analyses indicated that the youngest and very oldest age groups did not differ significant but showed lower factor scores than the two middle groups. The proportion of distribution of MCs within thalamic space between -5 mm and -1 mm, referenced to the bregma, is shown in Figure 3.2. As expected the numbers of MCs peaked within the region of the caudal half of the thalamus. Subsequent polynomial analyses indicated that the relationship between age group and type 1 [F(1, 30)=14.64, p <. 001] and type 3 [F (1, 30) =8 . 39, p < . 01 ] MCs was quadratic with the primary decrease in type 1 and increase in type 3 MCs occurring in the middle two age groups. 76 3500 3000 2500 ♦ ♦ 2000 ♦ ♦ ♦ 1500 * ♦ * ♦ 1000 ♦ ♦ ♦ 500 * . ^♦ ♦ ♦ ♦ ♦ * ♦ ♦ 0 ♦ ♦ 0 100 200 300 400 500 600 700 800 900 Figure 3.1. Scatterplot displaying the calculated number of thalamic mast cells (vertical axis) by age in days (horizontal axis). 0.7 0.6 0.5 it ; 1 it « 1 o 1.5 2 2.5 3 3.5 4 4.5 5 Figure 3.2. The mean relative proportion ± SEM of mast cells per section for the entire sample found (vertical axis) at various distances from Bregma in mm (horizontal axis). 77 3.4 Discussion Although MC numbers can be influenced by a variety of behavioral and early ontogenetic variables, old age, at least in this sample did not appear to affect their quantity. To our knowledge this is one of very few studies that have examined very old rats that were about to die from "natural causes" from senescence. The numbers of MCs did not differ from younger rats, including those that could be considered "old" from many contexts (200 to 700 days of age) indicates that the very old rats did not display the type of inflammation and vascular conditions that are typically associated with MCs. The increased proportion of degranulating MCs for the mature (100 to 200 days) and old (200 to 700) rats compared to the younger rats and the very old rats might suggest that intermediate ages are more prone to the conditions that induce this degranulation. Any explanation that the very old rats displayed a profile similar to the youngest group must be tempered with the possibility that only the very healthy rats lived longest. What is clear is that the large quantitative differences in MCs one would expect in a very aged rat, if ageing is implicitly a disease and inflammatory process, were not observed. 3.5 References Aubineau, P., & Dimitriadou, V. (1989). Mast Cells in the Cerebral Circulation. Characterization and Possible Function.In Neurotransmission and cerebrovascular function II, J. Seylaz and R. Sercombe, ed. (Elsevier Science Publishers B.V. Biomedical Division), pp. 293- 307 . Beaven, M.A. (2009). Our perception of the mast cell from Paul Ehrlich to now. European Journal of Immunology. 39: 11-25. Bo, L., Olsson, T., Nyland, H., et al. (1991). Mast cells in brain during experimental allergic encephalomyelitis in Lewis rats. Journal of Neurological Sciences. 105: 135- 142 . Bugajski, A.J., Chlap, Z., Gadek-Michalska, et al. (1994). Effect on isolation stress on brain mast cells and brain histamine levels in rats. Agents and Actions. 41: 75-76. Cammermeyer, J. (1973). Mast cells and postnatal topographic anomalies in mammalian subfornical body and supraoptic crest. Zeitschrift fur Anatomie und Entwicklungsgeschichte. 140(3): 245-269. Dropp, J.J. (1973). Mast cells in the central nervous system of several rodents. The Anatomical Record. 174(2): 227- 237 . Dropp, J.J. (1976a). Mast cells in mammalian brain. I. Distribution. Acta Anatomica. 94: 1-21. Dropp, J.J. (1976b). Mast cells in the human brain.Acta Anatomica. 105: 505-513. Galli, S.J., Grimbaldeston, M., Tsai, M. (2008). Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nature Reviews: Immunology. 8(6): 478-486. Galli, S.J., Nakae, S., and Tsai, M. (2005). Mast cells in the development of adaptive immune responses.Nature Immunology. 6(2): 135-142. Goldschmidt, R.C., Hough, L.B., & Glick, S.D. (1985). Rat brain mast cells: contibutions to brain histamine levels. Journal of Neurochemistry. 44(6): 1943-1947. Grzanna, R., & Shultz, L.D. (1982). The Contribution of Mast Cells to the Histamine Content of the Central Nervous System: A Regional Analysis. Life Sciences. 30: 1959- 1964 . 80 Ibrahim, M.Z., & Koshayan, D.S. (1981).The mast cells of the mammalian central nervous system. VIII. Uptake of 35S and 3H-5-hydroxytryptophan inside and outside of CNS. Cell and Tissue Research. 220(3): 529-538. Ide, T . , Taguchi, T., Morita, T., et al. (1989). Mast cell degranulating peptide form voltage gated and cation- selective channels in lipid layers. Biochemical and Biophysical Research Communications. 163(1): 155-160. Johnson, D., & Krenger, W. (1992).Interactions of Mast Cells with the Nervous System - Recent Advances.Neurochemical Research. 17(9): 939-947. Kalesnikoff, J., & Galli, S.J. (2008). New developments in mast cell biology. Nature Immunology. 9(11): 1215-1222. Kelsall, M.A. (1966). Aging on Mast Cells and Plasmacytes in the Brains of Hamsters. The Anatomical Record. 154: 727-739. Kelsall, M.A., & Lewis, P. (1964). Mast cells in the brain. Federation Proceedings. 23: 1107-1108. Kiernan, J.A. (1971). Degranulation of mast cells following antidromic stimulation of cutaneous.nerves. Journal of Anatomy. Ill: 349-350. 81 Kiernan, J.A. (1976). A comparative survey of the mast cells of the mammalian brain. Journal of Anatomy. 121: 303- 311. Kruger, P.G. (1974). Demonstration of Mast Cells in the Albino Rat Brain. Specialia. 30(7): 810-811. Lambracht-Hall, M., Dimitriadou, V., & Theoharides, T.C. (1990) .Migration of mast cells in the developing rat brain.Brain Research.Developmental Brain Research. 56(2): 151-159. Mares, V., & Bruckner, G. (1977).Increased Number of Mast Cells in the Brain of Light Deprived Rats. Physiologia Bohemoslovaca. 26: 458-459. Olsson, Y. (1968). Mast Cells in the Nervous System. International Review of Cytology. 24: 27-7 0. Orr, E.L. (1988). Nervous-system-associated mast cells.Drug Development Research. 15: 195-205. Persinger, M.A. (1977). Mast cells in the brain: Possibilities for physiological psychology. Physiological Psychology. 5(2): 166-176. 82 Persinger, M.A. (1977). Preweaning body marking reduces brain mast cell numbers in rats. Behavioral Biology. 21(3): 426-431. Persinger, M.A. (1981). Developmental alterations in mast cell numbers and distributions within the thalamus of the albino rat. Developmental Neuroscience. 4(3): 220- 224 . Purcell, W.M., & Atterwill, C.K. (1995). Mast Cell in Neuroimmune Function: Neurotoxicological and Neuropharmacological Perspectives. Neurochemical Research. 20(5): 521-532. Sabria, J., Ferrer, I., Toledo, A., et al. (1986). Effects of Altered Thyroid Function of Histamine Levels and Mast Cell Number in Neonatal Rat Brain. The Journal of Pharmacology and Experimental Therapeutics. 246: 612- 616. Silverman, A.J., Sutherland, A.K., Wilhelm, M., Silver, R. (2000). Mast Cells Migrate from Blood to Brain. The Journal of Neuroscience. 20(1): 401-408. Theoharides, T.C. (1990). Mast Cells: The Immune Gate to the Brain. Life Sciences. 44: 607-617. 83 Yang, M.F., Chien, C.L., & Lu, K.S. (1999). Morphological, irranunohistochemical and quantitative studies of murine brain mast cells after mating. Brain Research. 846: 30- 39. 84 Chapter 4 - Diffuse Thalamic Alizarin Staining Following Cholinergic Seizures Overlaps with Major Mast Cell Location in the Rat Brain Abstract The brains of rats in which cholinergic (lithium/pilocarpine) seizures had been induced and were killed 1 to 50 days later were stained with Alizarin Red. Remarkably diffuse staining through the dorsal thalamus was evident by post-seizure day 10 and very obvious by post seizure day 25 but was no longer evident by day 50. There was a conspicuous spatial overlap in the broad area of this stain and the location of where most of brain's mast cell population had been during the periweaning age in this population of rats. This congruence could suggest a mast cell factor, whose numbers can be affected by behavioral and pharmacological stimuli, in the later contribution to seizure-induced damage. 85 4.1 Introduction Following the chemogenic induction of limbic epileptic seizures by 3 mEq/kg of lithium followed 4 to 24 hrs by 30 mg/g of pilocarpine (Persinger et al, 1988), catastrophic changes in the concentrations of glutamanergic and gamma aminobutyric acid (Walton et al., 1990) occur that are associated with several stages of electrical instability and markedly elevated glucose utilization (Handforth & Treiman, 1995). Later morphological damage is strongly correlated with those early stages of metabolic perturbation (Peredery et al., 2000; de Vasconcelos et al., 2002). During subsequent days and weeks (Dube et al., 2000; Peredery et al., 2000,) variable degrees of neuronal dropout and gliosis occur in about 100 different Paxinos and Watson (1986) identifiable nuclei within the subcortical telencephalon (piriform cortices, amygdala, and hippocampal formation), the substantia nigra, and particularly the thalamus (Persinger et al., 1993). Within the thalamus there is also a visually conspicuous accumulation of calcium-protein granules about two months post-seizure that is similar to the calcification found in conditions associated with rapid neuronal dropout (Lafreniere et al., 1992). The punctate location of these crystallizations is conspicuously similar 86 to the ontogenetically earlier location of mast cells (Persinger, 1977). The etiology of these primarily thalamic-concentrated calcium granules in seized rats weeks after the initial injury, while even more severely injured areas such as the amygdala and entorhinal cortices are devoid of these indicators, is unclear. The dorsal thalamus is relatively unique in the rat brain for its copious presence of mast cells before puberty. During the preweaning period, mast cells (MC) contribute about half of the brain's histamine concentrations, long before the maturation of the synthesizing enzyme histidine decarboxylase (Schwartz, 1975) . MCs are mobile and remarkably versatile cytological elements whose metachromatic granules are sources of histamine, heparin, and serotonin (in the rat). Upon stimulation MCs can also synthesise an extraordinary pool of prostaglandins, cytokines, growth factors, chemokines, tumour necrosis factors, and nitric oxide (Galli et al, 2005; Pulendran & Ono, 2008). The cells are remarkably mobile (Persinger, 1977). Within 1 hr of the infusion of purified peritoneum, dye-tagged, mast cells they are evident within the velum interpositum within the dorsal thalamic- hippocampal interface (Silverman, et al., 2000). 87 Intrathalamic injection of Compound 48/80 into the dorsal thalamus induced MC degranulation (Persinger, 1983) in a manner similar to most tissues (Olsson, 1966) . The chemospecificity of the response was demonstrated by the paucity of degranulation in brains that were mechanically distorted before fixation (Persinger, 1983). Incubation of arteries in 10 pM carbachol elicited degranulation that could be blocked by 10~7 M Verapamil and was similar to the degranulation evoked by incubation with C 48/80 or by calcium-dependent mechanisms in response to cholinomimetic drugs (Aubineau & Dimitriadou, 1989). Baloyannis and Theoharides (1982) found that brain sections exposed to medium enriched with MCs that were degranulated by C 48/80 demonstrated extensive demyelination with pronounced MC accumulation around the affected areas. Thymopoietin, or thymic hormone, which influences the activity of T- lymphocytes, induces histamine and granule release from MCs (Erjavec and Ferjan, 1991). Lewis et al (1989) reported that injection of Compound 48/80 into the cerebroventricles of rats induced head and body shakes, paw tremor, excessive grooming, unusual posture, and gate, mild diarrhoea, piloerection, extreme agitation, irritability to the touch and a later stage of sedation. This syndrome is very similar to the behaviours 88 displayed by rats injected with lithium and pilocarpine before the onset of rearing and forelimb clonus associated with the limbic seizures. The release of MC degranulating peptide by C 48/80 has been shown to produce convulsions (Ide, et al., 1989), enhance long-term potentiation (LTP) in hippocampal slices (Ben Ari et al, 1989) through delayed release of proteins into extracellular space rather than enhanced release of glutamate or aspartate, and create ion channels (particularly K+) de novo in synthetic bilayer lipid membranes (Ide et al., 1989). That a cytological or correlative chemical vulnerability to later seizure-induced damage might be determined by the early chemical environment occupied by MCs may explain the individual differences in seizure-induced damage in these areas. The numbers of MCs in the preweaned thalamus is reduced by more than half in handled rats (Persinger, 1977). Sexual activity in mice increases MCs within the velum interpositus over the thalamus (Yang et al., 1999) and light deprivation increases their numbers by a factor of 20 in the leptomeninges over the lateral geniculate bodies (Mares & Bruckner, 1977). Because most MCs are situated around blood vessels and contain a plethora of compounds that influence membrane constituents and even calcium-sequestering protein, we compared the areas of 89 delayed damage within the thalamus of the rat with the area known in other studies to be associated with MCs. Here we present evidence of a conspicuous overlap of these regions. 4.2 Method Brains from rats in which lithium and pilocarpine seizures had been induced (Persinger, et al., 1993) and treated with acepromazine immediately upon the onset of forelimb clonus had been fixed following decapitation in ethanol formalin acid on various days (1 to 50) after the induction of the seizures. The forebrains had been processed, infiltrated with paraffin, and sectioned at 10 pm with a microtome. Toluidine blue O stains of some sections verified the classic neuronal dropout, gliosis, and formation of cystic lesions (Persinger, et al, 1988). In the present study, unstained sections were processed using the mineralization stain Alizarin Red using the method of McGee-Russell (1958) . The sample consisted of two rat sacrificed at one day post-seizure, two rats sacrificed at six days post-seizure, two rats sacrificed at ten days post seizure, one rat sacrificed at twenty-five days post-seizure and three rats sacrificed at fifty days post-seizure. 90 4.3 Results As indicated in Figure 4.1, the emergence of the Alizarin red diffuse stains began about six days after the induction of the seizures, was evident after day 10 and was conspicuous by 25 days. However, by 50 days post-seizure the diffuse and topographically significant nature of the reaction had diminished. The primary residual were small, highly focused, punctuate stains with marked granulation. This was similar to those reported by Lafreniere et al (1992) . 91 Days post Examples seziure 6 10 25 50 Figure 4.1. Examples of the right posterior thalamus of rats killed between 1 and 50 days post seizure._ 92 4.4 Discussion Mast cells outside of the brain are known for their involvement in immunological profiles, including inflammation, antimicrobial responses, cytokines, hypersensitivity, angiogenesis, and tumor growth (Beaven, 2009). MCs appear to participate in Type IV hypersensitivity reactions including multiple sclerosis and experimental allergic encephalomyelitis. Stimulated MCs rapidly form inflammatory lipids from arachidonic acid from membrane phosopholipids after activation of phospholipase A2. This requires substantial pools of calcium. Between postnatal days 15 and 25, rat MCs move from the leptomeninges (interpositus) velum covering the dorsal thalamus into the parenchyma (Persinger, 1981). Although they appear to only follow the spatial patterns occupied by the microvasculature, by the time the full complement occupies thalamic space, it is likely that proximity to most of the extracellular matrix and the neuronal and glial cells occurs. That some cells degranulate and dissipate completely as indicated by the reduction by about one-half of the numbers of MCs that were within the leptomeninges compared to the numbers later appearing within the parenchyma (Persinger, 1981). 93 During this period, which precedes the development of histidine decarboxylase, the enzyme required for neuronal histamine synthesis, most of the histamine is contained within MCs (Schwartz, 1975) . Both the occupation of this space and the loss of about 50% of MCs between the disappearance from the leptomeninges and appearance in the parenchyma might be associated with the release of their multiple products into this area. This non-specific diffusion could potentially sensitise the response of the tissue and its extracellular matrix to the consequences of subsequent intense cholinergic stimulation which is known to degranulate MCs and cause histamine secretion in other tissues (Massini et al., 1988). Physiological distress, which is associated with the acute state of the seizures, releases endogenous opiates from p and 3 opoid receptor stimulation that in turn produces a dose-dependent increase in serum corticosterone levels (Turon et al., 1991) . In rats pretreated with C 48/80 24 hours earlier, this response to the stimulating effects of morphine, leu-enkephalin and beta-endorphin is markedly attenuated. Even the small number of MCs in the hypothalamic median eminence can affect metabolic activity. C 48/80 injected into this region produced elevated thyroid 94 stimulating hormone for as long as 90 minutes after the injection (Nunes & Britto, 1989). A similar thalamic pattern of wide diffuse calcium deposition has been reported appearing 4 days and disappearing 24 days following administration of 5mg/kg dimethyl mercury (Mori et al., 2000). This congruence of spatial and temporal pattern of damage suggests that the effects reported are likely indicative of a general physiological distress resulting in neuronal damage rather than one specific to seizure-based damage. The results of this study indicate that the thalamic region which displays the greatest emergence of calcium-like staining following neuronal dropout in adults overlaps conspicuously with the area initially occupied by mast cells during late infancy. Whether or not this is correlative or causal is not clear. However, the hypothesis could be tested by experimentally modifying the numbers of thalamic mast cells. Because rats that are handled daily before weaning show a 50% reduction in MC numbers (Persinger, 1977) one would predict that the extent of diffuse thalamic Alizarin Red stain during the weeks that follow seizure- induction during adulthood would be significantly less. Similarly, Sugimotor, et al. (1998) reported that the degranulating effects of Compound 48/80-induced scratching 95 behavior could be blocked by Hi receptor antagonists terfenadine, epinastine and astemizole while cromolyn sodium and tranilast, antiallergic drugs without Hi receptor antagonistic activity were much less effective. Several review papers (Persinger, 1977; Theoharides, 1990; Silver et al., 1996) have implicated brain mast cells as gateways to brain-blood immunological reactions that may have long term or residual impacts upon the space they occupied during earlier ontogeny. Treatment with these compounds that affect these reactions during the peak in MC numbers within the thalamus might also be employed to test the hypothesis. 4.5 References Aubineau, P., and Dimitriadou, V. (1989). Mast Cells in the Cerebral Circulation. Characterization and Possible Function.In Neurotransmission and cerebrovascular function II, J. Seylaz and R. Sercombe, ed. (Elsevier Science Publishers B.V. Biomedical Division), pp. 293- 307 . Baloyannis, S.J, and Theoharides, T.C. (1982). Mast Cells Many Induce Demyelination of Rat Brain Slices In Vitro Clinical Research. 30(2): 407A. 96 Beaven, M.A. (2009). Our perception of the mast cell from Paul Ehrlich to now. European Journal of Immunology. 39: 11-25. Ben Air, Y., Aniksztejn, L., Roisin, M.P., Charriaut- Marlangue, C. (1989). Mechanism of Induction of Long Term Potentiation by the Mast Cell Degranulating Peptide. Pharmacopsychiatry. 22: 107-110. Dube,C., Boyet, S., Marescaux, C., and Nehlig, A. (2000). Progressive Metabolic Changes Underlying the Chronic Reorganization of Brain Circuits during the Silent Phase of the Lithium-Pilocarpine Model of Epilepsy in the Immature and Adult Rat. Experimental Neurology. 162: 146-157. Erjavec, F., and Ferjan, I. (1991). Characteristics of histamine and serotonin release from rat mast cells induved by thymic peptides. Agents and Actions. 33: BO- 32. Galli, S.J., Nakae, S., and Tsai, M. (2005). Mast cells in the development of adaptive immune responses. Nature Immunology. 6(2): 135-142. Handforth, A., and Treiman, D.M. (1995). Functional Mapping of the Early Stages of Status Epilepticus: A 14C-2- deoxyglucose Study in the Lithium-Pilocarpine Model in Rat. Neuroscience. 64(4), 1057-1073. Ide, T., Taguchi, T., Morita, T., Sato, M., Ikenaka, K., Aimoto, S., Kondo, T., Hojo, H., Kasai, M., and Mikoshiba, K. (1989). Mast Cell Degranulating Peptide Forms Voltage Gated and Cation-Selective Channels in Lipid Bilayers. Biochemical and Biophysical Research Communications. 163(1): 155-160. Lafreniere, G.F., Peredery, 0., and Persinger, M.A. (1992).Progressive accumulation of large aggregates of calcium-containing polysaccharides and basophilic debris within specific thalamic nuclei after lithium/pilocarpine-induced seizures. Brain Research Bulletin. 28(5): 825-830. Lewis, S.J., Quinn, M.J., Fennessy, M.R., and Jarrott, B. (1989). Acute Intracerebroventricular Injections of the Mast Cell Degranulator Compound 48/8 0 and Behavior in Rats. Pharmacology, Biochemistry, and Behavior. 33: 75- 79. Mares, V., and Bruckner, G. (1977). Increased Number of Mast Cells in the Brain of Light Deprived Rats. Physiologia Bohemoslovaca. 26: 458-459. McGee-Russell, S.M. (1958). Histochemical methods for calcium. Journal of Histochemistry and Cytochemistry. 6 : 22. 98 Mori, F., Tanji, K., and Wakabayashi, K. (2000). Widespread calcium deposits, as detected using the alizarin red S technique, in the nervous system of rats treated with dimethyl mercury. Neuropathology. 20: 210-215. Nunes, M.T., and Britto, L.R.C. (1989). Mast Cell Degranulation Acutelly Increases Thyrotropin in Serum Levels in the Rat. Neuroendocrinology Letters. 11(3), 131-137. Olsson, Y. (1966). The Effect of the Histamine Liberator, Compound 48/80 on Mast Cells in Normal Peripheral Nerves. Acta Pathologica et Microbiologica Scandinavica. 68: 563-574. Paxinos, G., and Watson, C. (1986). The Rat Brain in Sterotaxic Coordinates. 2nd Ed (San Diego: Academic Press Inc.). Peredery, 0., Persinger, M.A., Parker, G., and Mastrosov, L. (2000). Temporal changes in neuronal dropout following inductions of lithium/pilocarpine seizures in the rat. Brain Research. 881:9-17. Persinger, M. A. (1977). Mast cells in the brain: Possibilities for physiological psychology. Physiological Psychology. 5(2): 166-176. 99 Persinger, M.A. (1977). Preweaning body marking reduces brain mast cell numbers in rats. Behavioral Biology. 21(3): 426-431. Persinger, M.A. (1981). Developmental alterations in mast cell numbers and distributions within the thalamus of the albino rat. Developmental Neuroscience. 4(3): 220- 224 . Persinger, M.A. (1983). Degranulation of Brain Mast Cells in Young Albino Rats. Behavioral and Neural Biology. 39: 299-306. Persinger, M.A., Bureau, Y.R.J., Kostakos, M., Peredery, O., and Falter, H. (1993). Behavior of Rats With Insidious, Multifocal Brain Damage Induced by Seizures Following Single Peripheral Injections of Lithium and Pilocarpine. Physiology and Behavior. 53: 849-866. Persinger, M.A., Makarec, K., and Bradley, J.C. (1988). Characteristics of limbic seizures evoked by peripheral injections of lithium and pilocarpine. Physiology and Behavior. 44(1): 27-37. Pulendran, B., and Ono, S.J. (2008). A shot in the arm for mast cells. Nature Medicine. 14(5): 489-490. Rucci, L., Masini, E., Cirri Borghi, M.B., Giannella, E., and Mannaioni, P.F. (1988). Histamine release from nasal mucosal mast cells in patient with chronic 100 hypertrophic non-allergic rhinitis after parasympathetic nerve stimulation. Agents and Actions. 25: 314-320. Schwartz, J.C. (1975). Histamine as a Transmitter in Brain. Life Science. 17: 503-518. Silver, R., Silverman, A.J., Vitkovic, L., and Lederhendler, 1.1. (1996). Mast cells in the brain: evidence and functional significance. Trends in Neuroscience. 19(1): 25-31. Silverman, A.J., Sutherland, A.K., Wilhelm, M., and Silver, R. (2000). Mast cells migrate from blood to brain. The Journal of Neuroscience. 20(1): 401-408. Sugimoto, Y., Umakoshi, K., Nojiri, N., and Kamei, C. (1998).Effects of histamine HI receptor antagonists on compound 48/80-induced scratching behavior in mice. European Journal of Pharmacology. 351: 1-5. Theoharides, T.C. (1990). Mast Cells: The Immune Gate to the Brain. Life Science. 46: 607-617. Turon, M., Chlap, Z., Gadek-Michalska, A., Bugajski, J., and Polczynska-Konior, G. (1991). Effect of brain mast cells degranulation on the corticosterone response to stimulation of central opioid receptors in rats. Archives Internationales de Pharmacodynamie et de Therapie. 313: 151-160. 101 Vasconcelos, A.P., Ferrandon, A., and Nehlig, A. Local Cerebral Blood Flow During Lithium-Pilocarpine Seizures in the Developing and Adult Rat: Role of Coupling Between Blood Flow and Metabolism in the Genesis of Neuronal Damage. Journal of Cerebral Blood Flow and Metabolism. 22: 196-205. Walton, N.Y., Gunawan, S., and Treiman, D.M. (1990). Brain Amino Acid Concentration Changes during Status Epilepticus Induced by Lithium and Pilocarpine. Experimental Neurology. 108: 61-70. Yang, M.F., Chien, C.L., and Lu, K.S. (1999). Morphological, immunohistochemical and quantitative studies of murine brain mast cells after mating. Brain Research. 846: 30- 39. 102 Chapter 5 - Mast Cells in Mouse Melanomas Following Patterned Magnetic Field Exposures That Suppress B16 Cell Proliferation in Vitro Abstract Previous experiments indicated that the suppression effect upon melanomas from mice exposed to a particular spatial-temporal configuration of 1 pT magnetic fields was associated with leukocytic infiltration and vascularization. To the role of mast cells in this process the numbers of mast cells and their state of degranulation within the tumours were measured. A strong interaction between magnetic field presence and time (day vs night) of the exposure indicated the magnetic fields normalized the circadian changes in intrinsic numbers. Whether or not this effect contributed to the marked field-induced reduction in tumor size remains to be elucidated. 103 5.1 Introduction Hu et al. (2010) reported that repeated 3 hr nocturnal exposures of C57 mice to weak, frequency-modulated extremely low frequency magnetic fields remarkably reduced the size or prevented the growth of ectopic B16-BL6 melanoma tumours whose initial cells had been injected subcutaneously more than two weeks before the measurement. The visually obvious reduction in tumor size occurred only in those mice exposed nocturnally to a temporally patterned magnetic field that was presented sequentially over repeated 2 s periods in each of the three spatial planes. In melanoma cell cultures application of the same patterned magnetic field attenuated B16-BL6 proliferation and affected T-type calcium channels (Buckner, 2011). Histochemical analyses of the tumours of the mice exposed to this particularly patterned magnetic field indicated alterations in the infiltration of leukocytes or vascularization (Hu et al., 2010). The discernible presence of mast cells in the extracted tumours suggested a potential marker of tumour activity and a means by which the mechanisms of their reduction in mass might be revealed. In this paper we explored this possibility. Mast cells (MCs) are derived from hematopoietic progenitor cells but circulate as premature cells that 104 differentiate to histomorphologically-typical adult MCs depending upon environment (Kalesnikoff and Galli, 2008; Beaven, 2009; Galli et al, 2008). The cells display a marked longevity, similar to macrophages and monocytes, and can re-enter cycles of proliferation and infiltration (migraton) following specific stimulation (Persinger et al, 1983). There are an extraordinary number of substances released by mast cells (Galli, et al, 2005) that include lipid-derived prostaglandins and related inflammatory products of archadonic acid, cytokines, growth factors, chemokines and free radicals as well as the classic heparin, chrondroiton sulfates and substance P. Their involvement with allergic reactions, local and systemic, is well known. Most of these compounds, such as cyclo-oxygenase and prostaglandin E2 (Schmauder-Chock, & Chock, 1989) are located in the secretory granules whose metachromasia with appropriate staining define the MC. Degranulation occurs in response to a variety of mechanical, chemical, and electromagnetic stimuli (Padawer, 1974), including Pavlovian conditioning (MacQueen et al, 1989). Because MCs are found near vasculature and nerve endings, they could modulate the tissue environment through humeral and neurogenic stimulation. That the number of MCs increase by about a 105 factor of 10 in surrounding tissue following dennervation is well known (Ollson & Sjostrand, 1969; Sanchez-Mejorada and Alonso deFlorida, 1992). Many of the peptides localized to unmyelinated nerves, such as substance P, calcitonin gene- related peptide, somatostatin, and vasoactive intestinal peptide, have been implicated in neurogenic inflammation (Coderre, et al, 1989). The increased numbers of MCs surrounding tumors was first reported by Erhlich (see Beaven, 2009) and has been attributed to the similarity between tumor growth and inflammatory processes. In the viscera, MCs are closely apposed to nerves (Stead et al, 1989) with greatest concentrations within the appendix. Schadendorf et al (1995) found a range of a factor of 10 in numbers of MCs/mm2 in tumors regardless of subtype. There were significantly more at tumour borders of naevi of the malignant tumours compared to benign tumours. Increased numbers of MCs accompany increased collagen synthesis in fibroblasts (Takeda, et al, 1989) . We report here the presence and degranulations of mast cells within melanoma tumors following tumour-reducing magnetic field exposures. 106 5.2 Method Fixed mouse melanoma tissue from a total of 26 male C57 mice that had been exposed to the experimental paradigm described by Hu, et al. (2010) were selected. The mice had been exposed for 3 hr per day from the time of subcutaneous injection with 0.2 million B16-BL6 melanoma cells to a frequency-modulated magnetic field (shown in Martin, et al, 2004) that was "rotated" through the three pairs of solenoids oriented in each of the three spatial planes. Magnetic field exposures occurred at either 0900h - 1200h or 1200h - 15h00h local time for the diurnally treated animals and at 2100h - OOOOh and OOOOh - 0300h local time for the nocturnally treated animals. Groups were exposed to the sham field condition (an identical plastic box) during the diurnal and nocturnal exposure times. Half of the numbers of mice in each condition were exposed to the field or sham treatment and killed during the day and the remainder was exposed and killed during the scotophase (midnight to 0:300 hr) . Approximately 20 days after the injection (and the daily or nightly exposures to the fields or sham field conditions) of the cells, the mice were decapitated. At that time the tumour sizes within the sham group were conspicuous 107 and near the threshold for termination according to the approved animal care protocol. The melanomas were removed with scissors and placed within 5 minutes into ethanol- formalin-acetic acid fixative. After processing and embedding in paraffin, sections were cut at 6 pm using a microtome. A sample section from the middle of the tumor was stained with thionin (pH = 2.3) and an adjacent sample was stained with safranain. Total numbers of MCs were counted, as defined by their metachromasia, at 400 X and classified according to 5 types that reflected increasing degranulation. They were: 1 (no extracellular granules, 2) 1 to 5 extracellular granules, 3) 6 to 10 discrete granules, 4) up to H of granules extracellular, and 5) complete or near complete degranulation. The basic experimental design was a three way analysis of variance with two between subject (time of day of exposure; field condition) and one within subject (type of MC) level. Post hoc analyses involved combinations of Tukey's and paired t-tests. All analyses involved SPSS PC 16 software. 108 5.3 Results Three-way analyses of variance for one within level (five classes of mast cell degranulation) and two between levels (day/night exposures; sham/magnetic field exposure) demonstrated a statistically significant interaction between day/night exposures and field condition for the number of thionin-positive MCs [F(2,20) =4.51, p <.05; eta2 = 31%]. The results are shown in Figure 5.1. Post hoc analysis indicated that the primary source of the interaction was due to the lower MC counts (M = 6.0) for melanomas of mice exposed to the sham-fields during the nocturnal phase compared to those exposed during the the diurnal phase (M = 28.6). The differences in MC numbers for tumours of mice exposed to the magnetic field as a function of day vs. night periods contributed a small but statistically significant contribution to the interaction. There was a significant difference between types of MCs [F(4,80) = 41.70, p <.001]. A significant result for the proportion of MCs was the interaction between type of MC and day/night exposures [F(4,80)=2.75, p <.03; partial eta2=.12] regardless of treatment and is shown in Figure 5.2. This was due in large part to the greater percentage of degranulation MCs in mice exposed during the night (M,SEM=15%, 3%) vs the 109 day (6%, 1%) for type 5 MCs (maximum degranulation) but fewer grade 4 cells (11%, 2%; vs 19%,4%) whereas the other three types did not differ. Two-way analysis of variance did not demonstrate any significant differences between the lengths of the minor and major axis of the tumour sections. However there was an interaction between time of exposure and field condition for the numbers of mast cells whose axes were perpendicular [F (2, 20) =5.06, p < 0.01; eta2=20%] but not parallel [F= 1.93, P > 0.05] to the nearest tumour surface. The perpendicular effect was due to the fewer MCs in this orientation in the magnetic field exposed tumors compared to controls for the day time exposures but no differences between treatments for the nocturnal exposures. The mean area of the tumor sections was 83 mm2 and the average MC density was 0.26 cells/mm2. Although significantly fewer mast cells were observed with safranin as compared with thionin staining [t (25) = 4.92, p < 0.001], the overall mast cell counts were correlated [r(26) = 0.77, p < 0.001). The means and standard error of the means for overall MC counts between thionin and safranin stains were 18.46 ± 2.56 and 10.50 ± 2.03 respectively. 110 40 ■o 35 0) s 30 « 25 <3 S 20 ■Night 1 15 Day I ■a io 5 o Sham 09h00/21h00 12h00/00h00 Experimental Group Figure 5.1. Mean number of observed mast cells by exposure condition and photophase. 0.4 0.35 s 0 3 1 0.25 * & 0.2 S □ Day * 0.15 ■ Night "Si * 0.1 0.05 0 State of Degranulation Figure 5.2. Proportion of mast cells in each of the five specified degranulation conditions for all observed samples. Ill 5.4 Discussion The presence of MCs around tumours has been known since Erhlich first described these cells (Beaven, 2009). Given the proximity of MCs around tumours, particularly within the stroma and connective tissues and their multipotent role in inflammation, vascular passage of compounds, and even growth factors, we examined whether or not these cells would respond to the same patterned magnetic fields that reduce melanoma growth in mice and in culture. The results indicated that the magnetic fields did not, themselves, affect MC numbers. The treatment by time of day of exposure effect was due to the markedly different numbers of mast cells in the tumors exposed and night vs. day to the sham fields. The magnetic field exposure appears to have "normalized" these diurnal differences. The number of MCs in the tumours of sham field-exposed mice during the day was almost 5 times the value measured in mice exposed at night. Diurnal variations in MC numbers have been noted in other tissue. For example, there was almost twice the number of thyroidal perifollicular MCs in the evening compared to the morning (Modlinger, 1989) and their degranulation was more intense. Whether or not the diminished numbers of MCs in the nocturnally exposed tumours 112 reflect actual emigration of MCs from the area was not confirmed. We suggest that the magnetic field effects of maintaining the MC numbers during the night, that was similar to values for the daytime, may have modulated the vagotonic influence similar to those in the rat colon (Stead et al, 1989). Degranulation of mast cells in the heart is associated with reperfusion tissue injury (Keller et al, 1988). Granules in MCs can stimulate collagenase and beta- hexosaminidase production by fibroblasts which in turn can cause destruction in connective tissue (Takeda, 1989). The secreted granules of MCs are a major site of the archidonic cascade and eicosanoid production (Schmauder-Chock and Chock, 1989) . Phospholipase A2 , the enzyme necessary for arachidonic acid release from phosholipids of membranes, requires millimolar concentrations of Ca++ for its activity. Rapidly dividing cells, such as melanomas, contain such an environment. The enhanced proportion of MCs maximally degranulated during night-time compared to day-time manipulations indicated enhanced sensitivity to or division of melanomas within the nocturnal context. It is relevant that in histiocytes, lactic acid dehydrogenase (LDH) activity peaks 113 during the night (around 03 hr local time). Such diurnal variation in granule secretion may suggest that nocturnal exposures to appropriately patterned magnetic fields may have significantly greater effects on reducing melanoma proliferation and viability. 5.5 References Beaven, M.A. (2009) . Our perception of the mast cell from Paul Ehrlich to now. European Journal of Immunology. 39: 11-25. Buckner, C. (2011). Effects of electromagnetic fields on biological processes are spatial and temporal- dependent. Laurentian University Ph.D. Dissertation (Biomolecular Sciences): Sudbury, ON. Coderre, T.J., Basbaum, A.I., & Levine, J.D. (1989). Neural control of vascular permeability: interactions between primary afferents, mast cells, and sympathetic efferents. Journal of Neurophysiology. 62(1): 48-58. Galli, S.J., Grimbaldeston, M., Tsai, M. (2008). Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nature Reviews: Immunology. 8(6): 478-486. 114 Galli, S.J., Nakae, S., and Tsai, M. (2005). Mast cells in the development of adaptive immune responses. Nature Immunology. 6(2): 135-142. Hu, J.H., St-Pierre, L.S., Buckner, C.A., et al. (2010). Growth of injected melanoma cells is suppressed by whole body exposure to specific spatial-temporal configurations of weak intensity magnetic fields. International Journal of Radiation Biology. 86(2): 79- 88. Kalesnikoff, J., & Galli, S.J. (2008). New developments in mast cell biology. Nature Immunology. 9(11): 1215-1222. Keller, A.M., Clancy, R.M., Barr, M.L., et al. (1988). Acute reoxygenation injury in the isolated rat heatL role of resident cardiac mast cells. Circulation Research. 63(6): 1044-1052. MacQueen, G., Marshall. J., Perdue, M., et al. (1989). Pavlovian conditioning of rat mucosal mast cells to secrete rat mast cell protease II. Science. 243(4887): 83-85. Martin, L.J., Koren, S.A., & Persinger, M.A. (2004). Thermal analgesic effects from weak, complex magnetic fields 115 and pharmacological interactions. Pharmacology Biochemistry and Behavior. 78(2): 217-227. Modlinger, G. (1989). Circadian rhythm of the thyroid mast cells in untreated and methylthiouracil-treated rats. Folia Biologica. 37(3-4): 155-164. Olsson, Y., & Stostrand, J. (1969). Proliferation of mast cells in peripheral nerves during Wallerian degeneration.A radioautographic study.Acta Neuropathologica. 13(2): 111-121. Padawer, J. (1974). Editorial: The ins and outs of mast cell function. The American Journal of Anatomy. 141(3): 299- 302. Persinger, M.A. (1983). Degranulation of Brain Mast Cells in Young Albino Rats. Behavioral Neural Biology. 39: 299- 306. Sanchez-Mejorada, G., & Alonso-deFlorida, F. (1992). Changes in mast-cell distribution in skeletal muscle after denervation. Muscle & Nerve. 15(6): 716-719. Schadendorf, D., Kohlmus, C., Gawlik, C., et al. (1995).Mast cells in melanocytic tumours. .Archives of Dermatological Research. 287(5): 452-456. 116 Schmauder-Chock, E.A., & Chock, S.P. (1989).Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell. Journal of Histochemistry and Cytochemistry. 37(9): 1319-1328. Stead, R.H., Dixon, M.F., Bramwell, N.H., et al. (1989). Mast cells are closely apposed to nerve in the human gastrointestinal mucosa. Gastroenterology. 97(3): 575- 585. Takeda, K., Hatamochi, A., & Ueki, H. (1989). Increased number of mast cells accompany enhanced collagen synthesis in linear localized scleroderma. Archives of Dermatological Research. 281(4): 288-290. 117 Chapter 6 - General Results, Discussion, & Conclusion 6.1 Brain Mast Cells Between Studies Chapters two and three considered brain mast cells under a pathological condition and over the life span of rats respectively. Therefore, it is not surprising that these two studies demonstrated significantly different mast cell densities. In order to allow for direct comparisons between the mast cell numbers between these two studies overall, the formula described in 3.2.2 where the mean number of mast cells observed per slide for each animal were multiplied by 330, the approximate number of potential 10 pm sections where diencephalic mast cells reside was used. The rats in the EAE study where all rats were induced with EAE expressed an overall three-fold increase in diencephalic mast cells than those examined in the ageing study (F70 = 74.00, p < 0.01, r|2 = 0.52). These data alone are not sufficient to demonstrate an increase in brain mast cells in EAE compared to normal rats since these were two independent studies using different strains of rats. However, the magnitude and direction of this increase reflects those reported by Dimitriadou and colleagues (2000). 118 The distribution of mast cells across the five predefined degranulation states defined in figure 2.1 are displayed in figure 6.2. This implies that the aforementioned greater incidence of thalamic mast cells in Lewis rats inoculated with EAE as compared with a sample of non-experimental Wistar rats covering a full life span appears to be driven by stable, i.e. non-degranulated mast cells. 5000 4500 « 4000 x t 1 3500 z u 3000 | 2500 v 2000 | 1500 2 1000 500 0 EAE Ageing Figure 6.1.The mean estimated total number +SEM of brain mast cells by study. 119 0.8 0.60.6 □ EAE ■ Ageing Figure 6.2.The mean proportion ± SEM of mast cells in each of five predefined states of degranulation between experiments. 6.2 Mast Cell Locations Brain mast cells tend to be found in the posterior thalamus of rat brains bilaterally and the rostral-caudal distribution was displayed in figure 3.1. However, where differences between the total number of mast cells are found, the question remains whether these changes are proportionally distributed across this region. Goldschmidt and colleagues demonstrated a strong [r(9) = 0.99] relationship between the mast cells observed on a single coronal section taken at 2.00 mm caudal of Bregma and the total number of mast cells found throughout. the entire thalamus (1985). Using a similar approach we found a peak correlation [r(ll) = 0.90, p < 0.05] occurred at 3.30 mm 120 caudal of Bregma. This coronal level approaches the peak mast cell density reported in figure 3.1 and is within the affected area of the wide and diffuse alizarin red staining region reported in chapter 4. This suggests that the posterior thalamus is the most sensitive to changes in mast cell numbers since the overall thalamic mast cell count can be best predicted by a single coronal section in this area. Mast cells in the brain tend to localise around blood vessels, particularly blood vessel junctions (Silver, 1996). We found that this held true for the mast cells observed. A detailed quantification of this effect was taken for a single rat brain. The mast cells apposed to blood vessels observed where the major axis of the mast and direction of the blood vessel were evident. Of these, the mast cell's major axis was parallel to the direction of the blood vessel 90% of the time, with the remaining 10% being oriented at about 45° and none were perpendicular. The mast cells in melanoma have not been described in the literature with nearly as much detail as those in the brain so more details were taken during counting and assessment. Mast cells were generally exclusively located in the outer connective tissue capsule of the tumour. The mean distance and standard deviation from the nearest 121 surface in cell lengths is 7.42 ± 4.74. Orientation with respect to the mast cell's major axis was also assessed relative to this nearest surface. About half of all mast cells found were oriented parallel and about one eighth were perpendicular with the remainder not having a clear major axis in the section they were observed in (see figure 6.3). ■ Parallel ■ Perpendicular No M ajo r Axis Figure 6.3.The proportions of mast cell orientation to the surface of melanoma samples. 6.3 Magnetic Field Effects on Mast Cells The two experiments (chapters 2 and 5) looking at the effects of magnetic field exposures both found significant results in terms of both total mast cell number and state of degranulation. In chapter two we used the properties of a coaxial cable as a model of how such weak fields may 122 interface with the structure of mast cells. However, much remains to be elucidated in terms of these interactions. In chapter two all field exposures were performed during the scotophase while in chapter five exposures were performed during both photophase and scotophase. Interestingly, despite the different field parameters, exposure apparatuses, tissue-types, and species we found an increase in mast cells in both scotophase exposures of about 60% and 240% respectively. However, there was no true sham condition in chapter 2. If we assume that a truly unexposed group's brain mast cell count would reflect those reported in chapter 3 the mast cell increase in field-exposed animals would be closer to 280%. For animals exposed to magnetic fields during their photophase, the opposite effect, i.e., a decrease in mast cell counts as observed (see figure 5.1). The magnitude of this decrease was approximately two-thirds. However, all magnetic field exposed mice in chapter 5 did not have significantly different mast cell counts between them. This suggests that these fields strongly attenuate the magnitude of natural circadian periodicities in mast cell number. These circadian changes in mast cell number have been implicated in circadian periodicities in asthma attacks 123 (Seery, et al., 1998), and acupuncture effects (Chen, & He, 1989) . Surprisingly, the findings of Chen & He that mast cell density peaks during the scotophase (1989) are opposite from ours in chapter 5. Both magnetic field experiments also found differences in the relative proportion of type 3 mast cells. This class of mast cells represents the median classification on a five point scale and was defined by 6 to 10 discrete extra- cytoplasmic granules. The possibility that magnetic fields can activate mast cells would likely be accompanied with a characteristic pattern of activation. These categories are solely defined by light microscopic appearance, and thus, further work to define the molecular, and physiological correlates of these states need to occur before further interpretations can be made. However, following such an approach may be fruitful in describing a mechanism in which weak magnetic fields mediate mast cell activation. 124 0.4 □ Sham 1 2 3 4 5 Figure 6.4.The mean proportion ± SEM of mast cells in each of five predefined states of degranulation from melanoma samples which were either exposed to real or sham magnetic fields regardless of photoperiod. 6.4 Consequences of Brain Mast Cell Degranulation The notion that brain mast cells are necessary for the initiation of experimental allergic encephalomyelitis has been inferred from inability to induce this condition in mast cell deficient mice (Secor, et al., 2000) . This has been attributed to the ability of brain mast cells to open the blood-brain barrier (Zhuang, et al., 1996), likely through the ability of histamine to separate endothelial cells (Gilfillan & Beaven, 2011). While this connection is most salient in multiple sclerosis and related autoimmune demyelination, dysfunction in the blood-brain barrier has 125 been implicated in Alzheimer's dementia, HIV-related dementia, and bacterial meningitis (de Vries et al., 1997). Histamine is also a neurotransmitter. It is synthesised by the neurons of the tuberomammilary nucleus that send diffuse wide ranging projections to the cerebral cortex, thalamus, and basal ganglia. Histamine function is associated with the regulation of arousal, wakefulness (Gilfillan & Beaven, 2011), and actylcholine release from the basal forebrain. Since the brain mast cell can contain the majority of the brain's histamine (Goldschmidt et al., 1985), mast cell activation can conceivably result in psychophysiological effects. With further refinement, magnetic field exposures that can increase the number of degranulating mast cells can be used to test this hypothesis in vivo. The mechanism of brain mast cell degranulation differs from that of peripheral mast cells since immunoglobulin E is generally restricted from the brain parenchyma due to the blood-brain barrier (Theoharides, 2002). Thus, it is not surprising that brain mast cells were more stable than melanoma-associated mast cells in the preceding chapters with more than a two-fold increase in the proportion of un degranulated (state one) mast cells. 126 6. 5 Neuroinf lamination Mast cells comprise a small part of the population of the central nervous system, when compared to the astroglia and microglia which also contribute to the brain's innate immunity. Furthermore, they tend to be localised in the diencephanlon of rats. However, despite their low density and specific localisation they are critical to the development and regulation of neuroinflammation. They act as "gate-keepers" as a function of their ability to activate other inflammatory cells and their strategic location along the drainage sites of the thalamus (Orr, 1988). The interconnectivity of the microglia and mast cells in neuroinflammation has been recently reviewed by Skaper and collegues (2012). 6.6 Conclusion In chapter two we reported that an intensity threshold of 10 nT regardless of EMF frequency, resulting in an increase in brain mast cell number in rats with EAE. We attempted a biophysical model explaining this effect assuming the elongated shape of a mast cell would give it properties similar to a coaxial cable. Upon comparison with 127 'normal' brains sampled cross-sectionally from the life course of rats, we noted a significant increase of brain mast cells in EAE rat brains. We did not, however, demonstrate significant differences in different age groups of rats in terms of the number of brain mast cells. This was, in part, due to the large variances within age group cohorts. This large individual variation provides some support for the hypothesis that mast cells are markers of individual experience (Persinger, 1977). In chapter four, we found that large diffuse alizarin red staining patterns, resembling those found by Mori and collegues (2000) in space, time, and appearance, following seizure induction were isolated to regions where mast cell tend to aggreagate during early development. From these findings, particularly the specificity of the location, we hypothesised that mast cells may be involved in this region's unique response to injury. Leaving the brain in chapter five, we found that mast cells display a circadian periodicity in the density surrounding mouse melanomas. We also found that exposures to weak, patterned magnetic fields homogenise these peaks and troughs. Thus, we did demonstrate the weak magnetic 128 field exposure in the nanoTesla range can influence mast cell number in tissue. 6.7 References Chen, X.J., & He, Z.Y. (1989). Quantitative Study of Circadian Variations in Mat Cell Number in Different Regions of the Mouse. Acta Anatomica. 136: 222-225. de Vries, H.E., Kuiper, J., de Boer, A.G., et al. (1997) . The Blood-Brain Barrier in Neuroinflammatory Diseases. Pharmacological Reviews. 49(2): 143-156. Dimitriadou, V., Pang, X., Theohardies, T.C. (2000). Hydroxyzine inhibits experimental allergic encephalomyelitis (EAE) and associated brain mast cell activation. International Journal of Immunopharmacology. 22: 673-684. Gilfillan, A.M., & Beaven, M.A. (2011). Regulation of mast cell responses in health and disease. Critical Reviews in Immunology. 31(6): 475-529. Goldschmidt, R.C., Hough, L.B., & Glick, S.D. (1985). Rat Brain Mast Cells: Contribution to Brain Histamine Levels. Journal of Neurochemistry. 44(6): 1943-1947. 129 Mori, F., Tanji, K., and Wakabayashi, K. (2000). Widespread calcium deposits, as detected using the alizarin red S technique, in the nervous system of rats treated with dimethyl mercury. Neuropathology. 20: 210-215. Orr, E.L. (1988). Nervous-system-associated mast cells: Gatekeepers of neural and immune interactions. Drug Development Research. 15(2-3): 195-205. Persinger, M.A. (1977). Mast cells in the brain: Possibilities for physiological psychology. Physiological Psychology. 5(2): 166-176. Seery, J.P., Janes, S.M., Ind, P.W., et al. (1998). Circadian rhythm of cutaneous hypersensitivity reactions in nocturnal asthma. Annals of Allergy, Asthma, & Immunology. 80(4): 329-332. Silver, R., Silverman, A.J., Vitkovic, L., et.al. (1996). Mast cells in the brain: evidence and functional significance. Trends in Neuroscience. 19(1): 25-31. Skaper, S.D., Giusti, P., & Facci, L. (2012). Microglia and mast cells: two tracks on the road to neuroinflammation. Journal of the Federation of American Societies for Experimental Biology. In Press, doi: 10.1096/fj.11-197194. 130 Theoharides, T.C. (2002). Mast Cells and Stress - A Psychoneuroimmunological Perspective. Journal of Clinical Psychopharmacology. 22(2): 103-108. Zuang, X., Silverman, A.J., & Silver, R. (1996). Brain mast cells degranulation regulates blood-brain barrier. Journal of Neurobiology. 31: 393-403. 131