Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
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UBCV
Slack, Penelope Jean
Master of Science - MSc
University of British Columbia
Many populations have low intakes of n-3 fatty acids, yet there is substantial evidence that the long chain n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3), found at high concentrations in the brain, is required for the proper development of the nervous system. However, less is known about requirements of long chain n-3 fatty acids for maintenance and function of the nervous system in later life. Several recent studies have reported that high amounts of long chain n-3 fatty acids reduce the extent of brain damage caused by cerebral ischemia in animals. However, whether or not a dietary deficiency of n-3 fatty acids increases the extent of injury when cerebral ischemia occurs has not been previously reported. The present studies, therefore, sought to determine if a diet deficient in n-3 fatty acids influences the extent of brain injury in the rat following cerebral ischemia. Male rats were fed an n-3 fatty acid adequate (control), an n-3 fatty acid deficient, or a high DHA diet for 5 weeks from weaning. Middle cerebral artery occlusion (MCAO) was induced and infarct volume was measured by 2,3,5,-triphenyltetrazolium chloride staining 24 hours after the procedure. Brain and platelet fatty acids were analyzed by gas liquid chromatography. DHA (22:6n-3) was 21-28% lower in brain phospholipids, and 17% lower in brain total fatty acids in the n-3 fatty acid deficient compared to control group, while 22:6n-3 was 12% higher in total brain fatty acids in the high DHA group than the control group. There was no significant difference in infarct volume (203, 220 and 218 mm³) among the control, n-3 fatty acid deficient, and high DHA groups, respectively. Platelet fatty acids and platelet aggregation were assessed to determine if these were influenced by the high DHA diet, and could possibly explain the observation of an apparent, but not statistically significant, higher number of rats with hemorrhages in the high DHA diet group. Platelet lipid arachidonic acid was not lower and platelet aggregation, assessed ex vivo using whole blood with a platelet function analyzer, was not longer in rats fed the high DHA compared to control or n-3 fatty acid deficient diets. In summary, dietary n-3 fatty acid deficiency did not increase the extent of brain injury following cerebral ischemia. The possibility that high dietary 22:6n-3 might increase susceptibility to cerebral hemorrhage will require further study.
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DIETARY N-3 FATTY ACIDS AND CEREBRAL ISCHEMIA/REPERFUSION by PENELOPE JEAN SLACK B.Sc. University of Guelph, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2009 © Penelope Jean Slack, 2009 ABSTRACT Many populations have low intakes of n-3 fatty acids, yet there is substantial evidence that the long chain n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3), found at high concentrations in the brain, is required for the proper development of the nervous system. However, less is known about requirements of long chain n-3 fatty acids for maintenance and function of the nervous system in later life. Several recent studies have reported that high amounts of long chain n-3 fatty acids reduce the extent of brain damage caused by cerebral ischemia in animals. However, whether or not a dietary deficiency of n-3 fatty acids increases the extent of injury when cerebral ischemia occurs has not been previously reported. The present studies, therefore, sought to determine if a diet deficient in n-3 fatty acids influences the extent of brain injury in the rat following cerebral ischemia. Male rats were fed an n-3 fatty acid adequate (control), an n-3 fatty acid deficient, or a high DHA diet for 5 weeks from weaning. Middle cerebral artery occlusion (MCAO) was induced and infarct volume was measured by 2,3,5,-triphenyltetrazolium chloride staining 24 hours after the procedure. Brain and platelet fatty acids were analyzed by gas liquid chromatography. DHA (22:6n-3) was 21-28% lower in brain phospholipids, and 17% lower in brain total fatty acids in the n-3 fatty acid deficient compared to control group, while 22:6n-3 was 12% higher in total brain fatty acids in the high DHA group than the control group. There was no significant difference in infarct volume (203, 220 and 218 mm3) among the control, n-3 fatty acid deficient, and high DHA groups, respectively. Platelet fatty acids and platelet aggregation were assessed to determine if these were influenced by the high DHA diet, and could possibly explain the observation of an apparent, but not statistically significant, higher number of rats with hemorrhages in the high DHA diet group. Platelet ii lipid arachidonic acid was not lower and platelet aggregation, assessed ex vivo using whole blood with a platelet function analyzer, was not longer in rats fed the high DHA compared to control or n-3 fatty acid deficient diets. In summary, dietary n-3 fatty acid deficiency did not increase the extent of brain injury following cerebral ischemia. The possibility that high dietary 22:6n-3 might increase susceptibility to cerebral hemorrhage will require further study. iii TABLE OF CONTENTS ABSTRACT ............................................................................................................................. ii TABLE OF CONTENTS ........................................................................................................ iv LIST OF TABLES.................................................................................................................. vii LIST OF FIGURES ............................................................................................................... viii LIST OF ABBREVIATIONS ................................................................................................. ix ACKNOWLEDGEMENTS.......................................................................................................x DEDICATION......................................................................................................................... xi CO-AUTHORSHIP STATEMENT ....................................................................................... xii CHAPTER 1: INTRODUCTION..............................................................................................1 1.1 Background................................................................................................................1 1.2 Introduction to Dietary Fatty Acids...........................................................................2 1.3 Fatty Acid Nomenclature...........................................................................................3 1.4 n-3 and n-6 Fatty Acids and their Metabolism ..........................................................4 1..5 n-3 Fatty Acid Intakes and Disease .........................................................................12 1.6 Long Chain n-3 Fatty Acids and Cerebral Ischemia in Animal Studies..................14 1.7 Primary Research Hypotheses .................................................................................21 1.8 Rationale ..................................................................................................................21 1.9 Thesis Objectives.....................................................................................................22 1.10 Subsidiary Objective................................................................................................22 1.11 Bibliography ............................................................................................................23 iv CHAPTER 2: AN N-3 FATTY ACID DEFICIENT DIET DOES NOT WORSEN INFARCT VOLUME AND A DIET HIGH IN DOCOSAHEXAENOIC ACID DOES NOT IMPROVE INFARCT VOLUME AFTER CEREBRAL ISCHEMIA IN RATS .....34 2.1 Introduction .............................................................................................................34 2.2 Materials and Methods ............................................................................................36 2.2.1 Animals and diets ................................................................................................36 2.2.2 Middle cerebral artery occlusion (MCAO) surgical procedure...........................37 2.2.3 Sensorimotor testing ............................................................................................38 2.2.4 Infarct measurement ............................................................................................38 2.2.5 Brain fatty acids...................................................................................................39 2.2.6 Platelet aggregation and lipid analysis ................................................................39 2.2.7 Statistical analyses ..................................................................................................40 2.3 Results .....................................................................................................................41 2.3.1 Characteristics of animals....................................................................................41 2.3.2 Effects of dietary fatty acids on brain fatty acids ................................................41 2.3.3 Sensorimotor scores.............................................................................................45 2.3.4 Infarct volume and edema volume ......................................................................45 2.3.5 Incidence of cerebral hemorrhage .......................................................................47 2.3.6 Platelet fatty acids and ex vivo aggregation ........................................................47 2.4 Discussion................................................................................................................50 CHAPTER 3: GENERAL DISCUSSION AND CONCLUSIONS....................................62 3.1 General Discussion and Conclusion: .......................................................................62 3.2 Influence of Diet on Brain Fatty Acid Composition ...............................................62 v 3.4 Cerebral Hemorrhages in Relation to Dietary n-3 Fatty Acid Intakes ....................65 3.6 Strengths and Limitations ........................................................................................69 3.7 Future Directions .....................................................................................................73 3.8 Bibliography ............................................................................................................74 APPENDICES .........................................................................................................................79 A ppendix 1: UBC Ethics Board Certificate...........................................................................79 Appendix 2: Supplemental Methods .......................................................................................81 Appendix 2.1 Diagram of MCAO ...................................................................................81 Appendix 2.2 Equipment .................................................................................................82 Appendix 2.3 Analysis of Brain Fatty Acids...................................................................82 Appendix 2.4: Platelet Fatty Acid Analysis ....................................................................85 Appendix 2.5 Platelet Aggregation Studies.....................................................................86 Appendix 2 Bibliography ................................................................................................87 vi LIST OF TABLES Table 1-1. Levels of oleic acid (18:1n-9), linoleic acid (18:2n-6), and alpha-linoleic acid (18:3n-3) in common dietary oils and fats1. ............................................................................. 5 Table 1-2. Arachidonic acid (20:4n-6), eicosapentaenoic acid (20:5n-3), and docosahexaenoic acid (22:6n-3) content of selected foods. ..................................................... 7 Table 1-3. Comparison of published studies assessing dietary n-3 fatty acids and infarct volume. ..................................................................................................................................... 1 Table 2-1. Major fatty acid components in brain of rats fed diets varying in n-3 and n-6 fatty acids for 5 weeks .................................................................................................................... 44 Table 2-2. Major fatty acid components in platelets of rats fed diets varying in n-3 and n-6 fatty acids for 5 weeks……………………………………………………………………….49 vii LIST OF FIGURES Figure 1-1: Schematic representation of saturated and unsaturated bonds.............................. 2 Figure 1-2: Schematic representations to show a saturated (stearic), monounsaturated (oleic), n-6 polyunsaturated (linoleic), and n-3 polyunsaturated (alpha-linolenic) fatty acid. ............. 3 Figure 1-3: Schematic representation of docosahexaenoic acid (22:6n-3) to show the positions of the unsaturated (double) bonds. ............................................................................ 3 Figure 1-4: Schematic of n-3 and n-6 fatty acid desaturation and elongation in animal cells.9 Figure 2-1: Major fatty acid components in brain cortex and subcortex of rats fed a control, n-3 fatty acid deficient, or high DHA diet for 5 weeks……………………………………...43 Figure 2-2: Representative TTC stained brain for infarct volume analysis…………………46 Figure 2-3: Infarct volume in animals, 24 hr after MCAO………………………………….46 viii LIST OF ABBREVIATIONS ADP: adenosine diphosphate ANOVA: analysis of variance CCA: common carotid artery DHA: docosahexaenoic acid, 22:6n-3 GLC: gas liquid chromatography HPLC: high performance liquid chromatography hr: hour kg: kilogram MCA: middle cerebral artery MCAO: middle cerebral artery occlusion mg: milligram min: minute PC: phosphatidylcholine PBS: phosphate buffered saline PE: phosphatidylethanolamine PFA: platelet function analyzer PI: phosphatidylinositol PS: phosphatidylserine TTC: 2,3,5-triphenyltetrazolium chloride SPSS: Statistical Package for Social Sciences ix ACKNOWLEDGEMENTS First of all, I would like to thank my supervisor, Dr. Sheila Innis, for giving me the opportunity to carry out this research, and for her continuous support, encouragement, knowledge, and wisdom throughout. I would also like to thank all the members of our lab for their support. I would like to thank Roger Dyer, Benny Chan, and Janette King for their wide range of help throughout. I am also very grateful to Nancy Gruber for her surgical expertise and support while setting up the model. Thanks to all my fellow students, Mohammad Abudullah, Russ Friesen, Sanjoy Ghosh, and Liz Novak, for your input. I would also like to thank my committee members, Dr. Wan Lam, Dr. Timothy Murphy, Dr. John O’Kusky, and Dr. Louis Wadsworth for their help throughout the project. I am very grateful to Dr. Timothy Murphy, as well as Andy Shih and Ping Li from his lab for showing me the MCAO model. I am also very appreciative of Dr. Wadsworth for his help with the platelet aggregation studies and Nellie Quiring for her expertise with the platelet function analyzer. I would also like to thank Penny Woo from the department of pathology for being so helpful and for always answering questions in such a timely fashion. Last, but not least, I would like to thank my family for always being supportive of my educational pursuits, even though this time it has meant being on the other side of the continent. x DEDICATION I dedicate this thesis to Kal. Words cannot convey how much your willingness to stand behind my pursuits has meant to me over the years. Thanks for always reminding me to stop and smell the roses, and listen to the music. xi CO-AUTHORSHIP STATEMENT A version of chapter 2 from this thesis will be submitted for publication: Slack, P.J., Wadsworth, L.D., and Innis, S.M. I completed the work described in this paper. Wadsworth, L.D. offered expertise in platelet aggregation studies. Innis, S.M. is the principal investigator and my supervisor, and was involved in all stages. All co-authors edited and reviewed this paper. xii CHAPTER 1: INTRODUCTION 1.1 Background High concentrations of the n-3 fatty acid docosahexaenoic acid (22:6n-3) in ethanolamine (PE) and serine (PS) containing glycerol phospholipids is a specific feature of mammalian brain gray matter membranes (1, 2). A substantial amount of research supports the role of 22:6n-3 in the proper development and maintenance of the nervous system, although many details of the roles of 22:6n-3 remain incompletely understood (3-5). Mammalian cells lack the enzymes necessary to form n-3 fatty acids and therefore rely on a dietary source of n-3 fatty acids for the 22:6n-3 needed for brain membranes (6). Several studies have suggested that high intakes of fish oil or 22:6n-3 can reduce the extent of brain damage in animal models of stroke (7-13), which is the 3rd leading cause of death in North America (14, 15). Despite a decrease in the incidence of stroke in the last 50 years, the severity has not changed; 30-50% of those who have suffered a stroke are dependent on others for care (16). Although high intakes of n-3 fatty acids appear to decrease the extent of damage in animal models of stroke, human diets in many nations are low in n-3 fatty acids (17) . The question of whether or not reduced levels of 22:6n-3 in the brain might worsen the extent of damage caused by cerebral ischemia, however, has not been addressed. 1.2 Introduction to Dietary Fatty Acids The diets of mammals include 3 macronutrient groups: carbohydrates, protein, and fat. A major component of dietary fat is triglyceride, which is composed of glycerol plus three fatty acids. This thesis is concerned with dietary fatty acids and in particular the n-3 polyunsaturated fatty acids. Fatty acids are hydrocarbon chains with a carboxyl group at one end, known as the delta (Δ) end, and a methyl group at the opposite end (methyl, n). The basic structure of a fatty acid is denoted below: (Methyl end) CH3 (CH)n COOH (Carboxyl end) (n) (Δ) Each carbon atom in a fatty acid chain is either saturated (all four bonds are linked to a separate carbon, hydrogen, or oxygen), or unsaturated (2 adjacent carbons share two bonds), shown in Figure 1-1. 2 HH H H H O OH H Figure 1-1: Schematic representation of saturated and unsaturated bonds. Fatty acids not containing any double bonds belong to the saturated family, whereas those with one carbon-carbon double bond belong to the monounsaturated family, and those with more than one carbon-carbon double bond belong to the polyunsaturated family (Figure 1-2). Two important families of polyunsaturated fatty acids are the n-3 and n-6 fatty acids. H CC Saturated Unsaturated CCC Figure 1-2: Schematic representations to show a saturated (stearic), monounsaturated (oleic), n- 6 polyunsaturated (linoleic), and n-3 polyunsaturated (alpha-linolenic) fatty acid. 1.3 Fatty Acid Nomenclature In common nomenclature, fatty acids are denoted by the length of their carbon chain, followed by a colon, then the number of double bonds, and finally by the position of the 1st double bond from the methyl (n) terminus. For example, the polyunsaturated fatty acid 22:6n-3, shown in Figure 1-3, has 22 carbons and 6 double bonds, with the 1st double bond occurring at the 3rd carbon from the methyl (n) terminus (2). 3 Figure 1-3: Schematic representation of docosahexaenoic acid (22:6n-3) to show the positions of the unsaturated (double) bonds. 1 3 4 COOH 22 6 7 9 10 12 13 15 16 18 19 CH3 3 4 COOH 6 7 9 10 12 13 15 16 18 CH3 15 183 4 COOH 6 7 9 10 12 13 16 CH3 3 4 COOH 6 7 9 10 12 13 15 16 18 CH3 Saturated (stearic acid) Monounsaturated (oleic acid) Polyunsaturated (n-6, linoleic acid) 3 4 COOH 6 7 9 10 12 13 15 16 18 CH3 Polyunsaturated (n-3, alpha-linolenic acid) n Δ Δ n 1.4 n-3 and n-6 Fatty Acids and their Metabolism In mammals, saturated fatty acids and monounsaturated fatty acids of the n-9 and n-7 series can be synthesized de novo from acetyl CoA. The n-6 and n-3 polyunsaturated fatty acids, however, cannot be synthesized by mammalian cells; this is due to the absence of the Δ12 and Δ15 desaturase enzymes required for insertion of a double bond at the 12th and 15th carbons from the carboxyl terminus, respectively, of 18 carbon chain fatty acids (2, 18). Linoleic acid (18:2n-6) and alpha linolenic acid (18:3n-3) are the precursors to longer chain, more highly unsaturated n-6 and n-3 fatty acids, respectively; these fatty acids, containing 20 or more carbons, are sometimes referred to as long chain polyunsaturated fatty acids (2). Because the n- 3 and n-6 polyunsaturated fatty acids are needed, but cannot be synthesized (19-22), they must be obtained from the diet and are termed essential dietary nutrients (23). It is important to note that n-6 fatty acids cannot be converted to n-3 fatty acids, and vice versa (24). This thesis focuses on the n-3 family of polyunsaturated fatty acids. However, because the n-3 and n-6 fatty acids interact in metabolism, and the n-6 fatty acids are also important in many physiological functions, the literature review also includes a discussion of these fatty acids. The n-3 fatty acid 18:3n-3 and the n-6 fatty acid 18:2n-6 are the essential dietary fatty acids for almost all animals (2). Plants are the major dietary sources of 18:3n-3 and 18:2n-6. Oils such as canola, soybean, and flaxseed, and certain nuts are rich dietary sources of 18:3n-3, while corn, soybean, sunflower, and safflower oils are rich dietary sources of 18:2n-6 for humans. The amounts of 18:3n-3 and 18:2n-6 in common oils and fats are given in Table 1-1. Oleic acid (18:1n-9) is a monounsaturated fatty acid commonly found in vegetable oils, and its content is also provided in Table 1-1. 4 Table 1-1. Levels of oleic acid (18:1n-9), linoleic acid (18:2n-6), and alpha-linolenic acid (18:3n-3) in common dietary oils and fats1. Oil/Fat 18:1n-9 18:2n-6 18:3n-3 % of fatty acids Flaxseed 18 16 57 Canola 61 21 11 Soybean 23 54 8 Safflower (high oleic) 77 14 1 Safflower2 13 78 < 0.1 Sunflower 16 71 1 Olive 75 9 1 Corn 29 57 1 Lard 47 9 1 Butter 28 3 1 Peanut 48 33 < 0.1 Cottonseed 19 54 < 0.1 Palm 39 10 < 0.1 Coconut 7 2 < 0.1 1 Adapted from (25). Oleic acid (18:1n-9), linoleic acid (18:2n-6), and alpha linolenic acid (18:3n-3) in some common dietary oils and fats. The remainder of the fatty acids in each oil are monounsaturated and/or saturated. 2 Fatty acid composition is from (23). 5 Longer chain n-3 fatty acids, such as eicosapentaenoic acid (20:5n-3) and 22:6n-3, and the longer chain n-6 fatty acid arachidonic acid (20:4n-6) are all present in the acyl moieties of cell membrane phospholipids. These fatty acids can be obtained from the diet, but are only found in animal tissue lipids. The richest dietary sources of 20:5n-3 and 22:6n-3 are fatty fish, whereas the predominant sources of 20:4n-6 in most human diets are poultry, eggs and other animal tissue lipids (23) . Arachidonic acid (20:4n-6) is also present in fish, although at lower levels than 20:5n-3 and 22:6n-3 (23). The content of 20:4n-6, 20:5n-3, and 22:6n-3 in some selected foods is shown in Table 1-2. 6 Table 1-2. Arachidonic acid (20:4n-6), eicosapentaenoic acid (20:5n-3), and docosahexaenoic acid (22:6n-3) content of selected foods. Food 20:4n-6 20:5n-3 22:6n-3 g/100 g food Seal blubber 0.40 4.30 5.40 Mackerel (salted) 0.26 1.62 2.97 Salmon, (Atlantic, wild) 0.34 0.41 1.43 Tuna (bluefin) 0.06 0.36 1.14 Herring (Atlantic) 0.08 0.91 1.11 Halibut (Atlantic or Pacific) 0.18 0.09 0.37 Shrimp (canned) 0.10 0.29 0.25 Tuna (canned) 0.03 0.05 0.22 Chicken 0.15 0.01 0.06 Egg 0.15 0.01 0.04 Pork 0.08 <0.001 0.01 Beef 0.07 <0.00 <0.00 Adapted from The Canadian Nutrient File [database on the internet]. Ottawa, ON: Health Canada; c2007 [cited June 24, 2008]. Available from: http://205.193.93.51/cnfonline/newSearch.do?applanguage=en_CA (26). 1Reported as 0. 7 Alternatively, the longer chain polyunsaturated fatty acids can be synthesized from their 18 carbon n-6 and n-3 fatty acid precursors as shown in Figure 1-4. Linoleic acid (18:2n-6) can be desaturated and elongated to several 20 and 22 carbon chain n-6 fatty acids, among which 20:4n-6 is particularly important. Alpha linolenic acid (18:3n-3) is desaturated and elongated to longer chain n-3 fatty acids including 20:5n-3 and 22:6n-3 in animals, including humans (Figure 1-4), (2, 18). Linoleic acid (18:2n-6) is metabolized to 20:4n-6, by Δ6 desaturase, elongation, and an additional desaturation by Δ5 desaturase on the endoplasmic reticulum (Figure 1-4). Alpha linolenic acid (18:3n-3) is similarly desaturated and elongated to 20:5n-3, using the same enzymes. For conversion to 22:6n-3, the currently accepted pathway includes two additional elongations from 20:5n-3 to 24:5n-3, followed by desaturation again using Δ6 desaturase to form 24:6n-3. Then, 24:6n-3 is transported to the peroxisome where chain shortening by two carbons (partial beta-oxidation) yields 22:6n-3 (27-29). The same process is believed to lead to the formation of docosapentaenoic acid (22:5n-6) (27, 28). Levels of 22:5n-6 in tissue lipids are usually very low, but increase in the liver, brain, and other organs of animals fed an n-3 fatty acid deficient diet (30-34). 8 18:3n-3 18:2n-6 9 18:4n-3 18:3n-6 20:4n-3 20:3n-6 20:5n-3 20:4n-6 22:5n-3 22:4n-6 24 :5n-3 24 :4n-6 24:6n-3 24 :5n-6 24:6n-3 24:5n-6 22:6n-3 22:5n-6 Elongase Δ6-desaturase Chain Shortening Endoplasmic Reticulum Δ6-desaturase Elongase Δ5-desaturase Elongase Peroxisome Figure 1-4: Schematic of n-3 and n-6 fatty acid desaturation and elongation in animal cells. Adapted from (18). The activity of the essential fatty acid desaturation pathway is generally believed to be more active in rodents than in humans (2). This interpretation is based on the higher levels of 22:6n-3 in plasma and liver lipids of rodents fed diets containing soybean or other oils containing 18:3n-3 but not 22:6n-3, when compared to the usual circulating plasma levels of 22:6n-3 in humans. However, some caution is warranted as humans do not usually consume diets containing a single oil source. In humans, about 90% of dietary 18:3n-3 appears to be directly β-oxidized (2), with < 1% of a trace dose of stable isotope labeled 18:3n-3 converted to 22:6n-3 (35-37). Interpretation of the proportion of dietary 18:3n-3 converted to 22:6n-3 is not necessarily an indication that the conversion is limited by the activity of the ∆6 and ∆5 desaturases, or that the conversion is too low. For example, the intake of 18:3n-3 may be greatly in excess of that needed for 22:6n-3 synthesis for incorporation into membranes. Alternatively, inhibition of 18:3n-3 conversion to 20:5n-3 and 22:6n-3 by high dietary intakes of 18:2n-6 could also explain low recovery of stable isotopically labeled 22:6n-3 in plasma following administration of the labeled 18:3n-3 as a precursor. As introduced above, it is important to note that the relative amounts of n-3 and n-6 fatty acids in the diet have important ramifications for the fatty acid elongation and desaturation pathways. In vitro studies have shown that the Δ6 desaturase enzyme has a preference for 18:3n-3 over 18:2n-6 (38-40) . However, numerous animal studies have shown that high dietary intakes of 18:2n-6 reduce the desaturation of 18:3n-3 and decrease 20:5n-3 and 22:6n-3 in tissue lipids (41, 42). Current mean intakes of 18:2n-6 in Canada and the U.S.A. are about 5-7% dietary energy, while 18:3n-3 represents about 0.5% energy; this results in an 18:2n-6 to 18:3n-3 of 10:1 or higher (42-44). The activities of the ∆6 and ∆5 desaturase enzymes are also influenced by the dietary intake of preformed 20:4n-6, 20:5n-3, and 22:6n-3; this is consistent with the expected product inhibition of the desaturation-elongation pathway. For example, high 10 intakes of fish (high in 20:5n-3 and 22:6n-3) are associated with lower blood levels of 20:4n-6 and decreased synthesis of eicosanoids formed from 20:4n-6 (2). The median intake of long chain n-3 fatty acids (20:5n-3 plus 22:6n-3) is estimated to be about 200-300 mg/day in the U.S.A. and in Canada (17, 44, 45). It is believed that up until 150 years ago humans consumed a diet with an n-6 to n-3 fatty acid ratio of about 1:1 (46). The high, approximate 10:1 ratio of n-6 compared to n-3 fatty acids, in modern diets, has been suggested to antagonize n-3 fatty acid metabolism, resulting in elevated n-6 fatty acids and a pro-inflammatory environment (46). The low intakes of n-3 fatty acids, but high intakes of n-6 fatty acids that characterize typical western diets have been suggested to be a cause for concern; this is based largely on the opposing biological roles of the metabolites of 20:5n-3 and 20:4n-6, and the important role of 22:6n-3 in the central nervous system (2). Phospholipids and their fatty acids are continuously turned over as part of cell signaling pathways, leading to release of unesterified 20:4n-6 and 20:5n-3 for further metabolism to eicosanoids (2), and unesterified 22:6n-3 for further metabolism to resolvins and docosanoids (47). Eicosanoids have multiple roles, among which are the initiation, potentiation, and termination of inflammatory pathways, as well as platelet aggregation and vessel wall constriction and permeability (48). In many of these pathways, metabolites derived from 20:4n- 6 are pro-inflammatory, while those derived from 20:5n-3 are either weakly-inflammatory or anti-inflammatory; however, the lipoxins derived from 20:4n-6 are important in the termination of inflammatory responses. Eicosapentaenoic acid (20:5n-3) is high in circulating blood cells, but is low in the brain, and is reported to be much lower than 20:4n-6 (49). Recently, a series of metabolites from 22:6n-3, termed resolvins and docosanoids, were identified and shown to have important anti-inflammatory actions in neural cells (47). The release of 20:4n-6, 20:5n-3, and 22:6n-3 from cell membrane phospholipids is important under conditions of injury or disease. 11 Under pathological states, the activities of phospholipase enzymes that regulate fatty acid cleavage from phospholipids are elevated (50). In addition, cyclooxygenase and lipoxygenase enzymes, induced in eicosanoid production, are elevated during injury (50). Due to their important roles in the body, and because the n-3 fatty acids are essential dietary nutrients, a substantial amount of research has recently been dedicated to understanding the relationship between the n-3 and n-6 fatty acid intakes, eicosanoids, inflammation and human health. 1 .5 n-3 Fatty Acid Intakes and Disease Epidemiological studies, beginning in the 1970’s, revealed interesting trends in disease incidence between populations that consumed high amounts of long chain n-3 fatty acids compared to those that consumed low amounts of these fatty acids. The population in Greenland, following a marine diet high in n-3 fatty acids from seal and other marine mammals and fish, were found to have substantially lower than expected rates of myocardial infarction and coronary heart disease (51, 52). However, the incidence of cerebral hemorrhage was substantially higher than expected in the Greenland population (52); estimates on the expected incidence of each type of disease were based on numbers from a predominantly Danish European population. In regards to coronary health, recent epidemiological studies have reported a lower coronary heart disease rate among men who ate some fish weekly, as well as a lower relative risk of death from coronary heart disease of 0.62, and a relative risk of non-sudden death from myocardial infarction of 0.33 among men who ate at least 35 g of fish per day (about 2 servings/week) when compared to men who ate no fish (53). The Nurses’ Health Study reported an inverse relationship between fish intake and between n-3 fatty acids and death from coronary heart disease among women (54) . Similarly, Zhang et al. (55) reported an association across 36 12 countries between higher fish consumption and lower rates of ischemic heart disease mortality. In clinical intervention studies involving fish or fish oil, the lower mortality from cardiovascular disease appears to be due to lower sudden death (56). Based on epidemiological and clinical studies to indicate that long chain n-3 fatty acids are associated with a decreased risk of cardiovascular disease mortality, particularly sudden death, many expert bodies have recommended 1-2 meals of fish per week as part of a healthy diet (53, 57, 58). The relationship between dietary n-3 fatty acids and stroke incidence and mortality is more complicated, owing to the presence of two subtypes of stroke; both occur from the cessation of blood flow in the brain, but ischemic stroke results from the occlusion of an artery and hemorrhagic stroke results from the rupture of a blood vessel. Epidemiological studies in this field suggest that long chain n-3 fatty acids have different effects on these 2 subsets of stroke, having a positive association with hemorrhagic stroke and an inverse association with ischemic stroke (52, 59). As introduced above, the early epidemiological study comparing the population of Greenland to a predominantly Danish European population found a higher than expected incidence of cerebral hemorrhage in Greenlanders. Interestingly, other studies have noted that aneurysms and hemorrhages were much more frequent than myocardial infarctions in native Alaskans (60), and a high bleeding tendency in Greenland natives was noted as far back as the 15th century (61). In contrast to the apparent susceptibility to hemorrhage in populations consuming high amounts of n-3 fatty acids, a meta-analysis of cohort studies recently reported that regular fish intake may be inversely associated with ischemic stroke incidence (62). A recent epidemiological study across 38 countries reported an association between lower morbidity and mortality rates from all causes of stroke among countries that had higher intakes of long chain n-3 fatty acids (17); the authors suggested that higher stroke morbidity and 13 mortality rates, seen among some countries, may be partially attributable to low intakes of long chain n-3 fatty acids. 1 .6 Long Chain n-3 Fatty Acids and Cerebral Ischemia in Animal Studies The observed beneficial effects of long chain n-3 fatty acids on cardiovascular disease has stimulated interest in the roles these fatty acids have in other aspects of disease. The ability of dietary n-3 fatty acids to modulate tissue fatty acid compositions (2), including their tendency to replace n-6 fatty acids, has brought interest to their possible roles in pathological conditions, where cell membrane destruction leads to a higher than normal level of fatty acids in their free form. In this regard, several studies have examined the effects of high dietary amounts of fish oil or 22:6n-3 alone on the extent of damage that occurs following cerebral ischemia in animals. A stroke causes an area of damage referred to as an infarct, which is an outcome measured in several of these studies. To date, most animal studies assessing the effects of dietary 20:5n-3 and 22:6n-3 in models of cerebral ischemia have reported evidence to show a reduced infarct volume in animals fed these fatty acids for 3-10 weeks prior to cerebral ischemia (7-11). Black et al. (7) reported that cats fed a diet of cat chow supplemented with an additional 8% of energy as menhaden fish oil for approximately 3 weeks had a significantly smaller average infarct volume after middle cerebral artery occlusion (MCAO) than cats fed standard chow without fish oil. It must be noted that cats have either no or very low Δ6 desaturase activity and depend on a dietary source of preformed 20:5n-3 and 22:6n-3, as well as 20:4n-6 (63, 64). The species is an obligate carnivore, and also differs from humans in requiring the terminal products of the transulphuration pathway, such as taurine. Relton and colleagues (8) reported a 36% lower infarct volume when a diet containing fish oil (0.9% 20:5n-3 and 0.6% 22:6n-3 by weight of the 14 diet) was fed to rats for 6 weeks prior to permanent MCAO compared to rats fed a diet with 6% olive oil. Similarly, rats fed a diet containing 14% by weight menhaden fish oil for 6 weeks had significantly smaller infarct volumes when measured after MCAO followed by 24 hrs of reperfusion than in rats fed standard chow (10). Rats fed chow supplemented with 300 mg/kg/day 22:6n-3 for 6 weeks had smaller infarct volumes when compared to rats fed chow only, in a model that involved middle cerebral artery thrombosis, using rose bengal and green light irradiation to induce cerebral ischemia (9). The latter study also administered tissue plasminogen activator 30 minutes after irradiation and found that regardless of whether or not the middle cerebral artery was reopened, infarct volume was smaller in rats treated with 22:6n-3. In other studies, gerbils treated with 200 mg/kg/day of the ethyl-ester of 22:6n-3 by gavage for 10 weeks, in addition to feeding pellets, had a 35% lower infarct volume after bilateral common carotid artery occlusion for 30 minutes, followed by reperfusion, than that observed in animals treated with vehicle alone (11). Beneficial outcomes that have been reported in conjunction with feeding a diet containing high amounts fish oil or following supplementation with ethyl esters of 22:6n-3 that led to a reduction in infarct volume included better gait score and righting reflex (7), and reduced cerebral edema and mortality (11). Marcheselli et al. (65) also reported that infusing 22:6n-3 or 10, 17 S-docosatriene (a metabolite of 22:6n-3), immediately following cerebral ischemia, reduced the infarct volume in mice. At the time that the studies described in this thesis were designed and commenced, only one publication contradicted the reduction in infarct volume associated with fish oil or 22:6n-3. This study involved rats fed a diet containing 20% energy as Norwegian cod liver oil for 7 weeks, with the finding that infarct volume, after transient occlusion of both common carotid arteries and the right middle cerebral artery, was higher than in rats fed chow alone (66). However, more recently, Skryzypki et al. (67) reported that rats fed 0.6% 22:6n-3 did not show improvement, and even had tendencies toward larger 15 infarct volumes after focal cerebral ischemia. A summary of studies published to date concerning dietary n-3 fatty acids and infarct volume following cerebral ischemia is provided in Table 1-3. 16 N ot es 1 an im al in th e ex pe rim en ta l gr ou p di ed o f hy po te ns iv e sh oc k In fa rc t V ol um e Sm al le r i n ex pe rim en ta l gr ou p, m ea su re d 3 da ys a fte r 36 % sm al le r in ex pe rim en ta l gr ou p, 2 4 hr af te r La rg er in ex pe rim en ta l gr ou p, 2 4 hr af te r C er eb ra l Is ch em ia M od el M C A O , pe rm an en t M C A O , pe rm an en t Fo ca l is ch em ia (te m po ra ry oc cl us io n of M C A a nd bo th C C A s fo r 4 5 m in ) Sp ec ie s C at R at , S pr ag ue D aw le y T ab le 1 -3 . C om pa ri so n of p ub lis he d st ud ie s a ss es si ng d ie ta ry n -3 fa tty a ci ds a nd in fa rc t v ol um e. R at , s tra in n ot sp ec ifi ed D ur at io n of F ee di ng 18 -2 4 da y, a du lt ca ts , ag e no t s pe ci fie d 6 w ee ks , f ro m w ea ni ng 7 w ee ks , f ro m w ea ni ng 17 D ie ts C on tro l: ca t c ho w Ex pe rim en ta l: ca t ch ow pl us 8 % e ne rg y m en ha de n fis h oi l C on tro l: se m is yn th et ic di et w ith 6 % fa t, by w ei gh t, fr om o liv e oi l Ex pe rim en ta l: se m is yn th et ic d ie t w ith 0. 9% 2 0: 5n -3 a nd 0 .6 % 22 :6 n- 3 fr om fi sh o il, b y w ei gh t C on tro l: ra t c ho w Ex pe rim en ta l: co d liv er oi l a dd ed a t 2 0% e ne rg y to ra t c ho w B la ck e t a l. 19 79 (7 ) R el to n et a l. 19 93 (8 ) La i e t a l.1 99 3 (6 6) R ef er en ce 18 N ot es In fa rc t V ol um e Sm al le r i n ex pe rim en ta l gr ou p, m ea su re d 24 hr a fte r Sm al le r i n ex pe rim en ta l gr ou p, 2 4 hr af te r 35 % sm al le r in ex pe rim en ta l gr ou p, 2 4 hr af te r C er eb ra l Is ch em ia M od el M id dl e ce re br al ar te ry th ro m bo si s m od el (r os e gr ee n lig ht M C A O , 2 h r G lo ba l is ch em ia , oc cl us io n of bo th C C A s Sp ec ie s R at , W is ta r R at , S pr ag ue - D aw le y G er bi l D ur at io n of F ee di ng 8 w ee ks , f ro m 6 w ee ks o f a ge 10 w ee ks , f ro m w ea ni ng 6 w ee ks , f ro m w ea ni ng D ie ts C on tro l: ra t c ho w Ex pe rim en ta l: 30 0 m g/ kg /d ay 2 2: 6n -3 a dd ed to ra t c ho w C on tro l: A IN 9 3G d ie t Ex pe rim en ta l: A IN 9 3G di et p lu s 1 4% m en ha de n fis h oi l C on tro l: ve hi cl e Ex pe rim en ta l: 20 0 m g/ kg /d ay e th yl e st er 22 :6 n- 3 by g av ag e R ef er en ce U m em ur a at a l. 19 95 (9 ) C ho i-K w on e t a l. 20 04 (1 0) C ao e t a l. 20 07 (1 1) The mechanism by which the long chain n-3 fatty acids, 20:5n-3 and 22:6n-3 or 22:6n-3 alone may reduce infarct volume during cerebral ischemia is not fully understood, but several mechanisms have been suggested. Because high dietary intakes of fish oil are often associated with decreased tissue 20:4n-6 (due to inhibition of 18:2n-6 desaturation and elongation to 20:4n- 6), one possible mechanism could involve lower amounts of 20:4n-6 in brain lipids, potentially leading to reduced production of 20:4n-6-derived metabolites that might contribute to tissue damage (68, 69). Eicosapentaenoic acid (20:5n-3) competes with 20:4n-6 for cyclooxygenase and lipoxygenase enzymes for the production of prostaglandin and leukotriene synthesis (70), although 20:5n-3 levels are low in the brain even in animals fed high 20:5n-3 fish oils. The positive results found in animals given ethyl esters of 22:6n-3, or after infusing 22:6n-3 or 10, 17 S-docosatriene into the brain, however, suggest that the protective effect on infarct size involves 22:6n-3, rather than 20:5n-3. Although 20:5n-3 levels are low in the brain, some studies have reported that 22:6n-3 is important in regulating brain 20:4n-6 and 20:4n-6-derived eicosanoid production (11, 65, 66). With respect to the potential benefits of 20:5n-3 plus 22:6n-3 or 22:6n-3 alone, it is important to note that the amounts added to the diet or given in supplements to animals in the studies described above were well above the intakes found in any known human populations including Greenland Inuit. Furthermore, populations with the highest known intakes of long chain n-3 fatty acids that have a lower incidence of some diseases, such as myocardial infarction, also have higher incidences of other conditions, such as cerebral hemorrhage (52). In many, if not most nations, dietary intakes of n-3 fatty acids are low, not high, and this occurs for 18:3n-3, and 20:5n-3 and 22:6n-3; furthermore, the low intakes of n-3 fatty acids often occurs in combination with high intakes of n-6 fatty acids (71). It is known that dietary deficiency of n-3 fatty acids decreases the amount of 22:6n-3 in the brain, with a compensatory increase in n-6 19 fatty acids, particularly 22:5n-6, and that this is associated with impaired neurogenesis, altered neurotransmitter metabolism, and altered gene expression during early development (43). The question of whether low dietary n-3 fatty acid intakes lower brain 22:6n-3, which increases the brain n-6:n-3 fatty acid ratio, possibly worsening the extent of brain damage during cerebral ischemia, has not been addressed. 20 1 .7 Primary Research Hypotheses 1) Feeding a diet deficient in n-3 fatty acids for 5 weeks to young adult rats will reduce the levels of 22:6n-3 in brain phospholipids by at least 20%. 2) Feeding a diet deficient in n-3 fatty acids will increase brain damage, when assessed as infarct volume, when compared to a diet providing adequate n-3 fatty acids, or containing high amounts of 22:6n-3. 1.8 Rationale One objective was to alter the levels of 22:6n-3 in brain solely by diet to an extent that is considered comparable to the effects of dietary fatty acids on the human brain. Decreasing dietary 22:6n-3 by 20% in rats was considered an acceptable strategy because autopsy studies of brain tissue from infants fed formula containing high 18:2n-6 and low 18:3n-3 with no 22:6n-3 have reported approximately 30% lower brain 22:6n-3 than in the brains of infants fed human milk (72). Previous studies have reported that increasing 22:6n-3 protects the brain from cerebral ischemia damage. Therefore, in addition to an n-3 fatty acid deficient diet, a diet containing supplemental 22:6n-3 was included, therefore, further increasing the range of brain 22:6n-3 levels in which to seek an association between 22:6n-3 and outcome following cerebral ischemia. 21 1. 9 Thesis Objectives This thesis had 2 objectives A) To establish a model of cerebral ischemia/reperfusion in the rat at the Child and Family Research Institute. 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Arch Dis Child. 1995 Mar;72(3):198-203. 33 CHAPTER 2: AN N-3 FATTY ACID DEFICIENT DIET DOES NOT WORSEN INFARCT VOLUME AND A DIET HIGH IN DOCOSAHEXAENOIC ACID DOES NOT IMPROVE INFARCT VOLUME AFTER CEREBRAL ISCHEMIA IN RATS1 2.1 Introduction High concentrations of the n-3 fatty acid 22:6n-3 in phosphoglycerides, particularly PE and PS, is a specific feature of mammalian brain gray matter membranes (1, 2). Mammalian cells lack the enzymes necessary to form n-3 fatty acids and therefore rely on a dietary source of n-3 fatty acids for the 22:6n-3 needed for brain membranes (3). The 22:6n-3 for incorporation into brain phosphoglycerides can be derived from endogenous synthesis by desaturation and elongation of 18:3n-3 provided in the diet, or from a dietary source of preformed 22:6n-3 (3). The n-3 fatty acid 18:3n-3 is abundant in some vegetable oils, such as canola, soybean and flaxseeds, but is very low, <1 g 18:3n-3/100 g fatty acids, in most common vegetable and hydrogenated oils (4). DHA (22:6n-3) is present only in animal tissue lipids, with the richest dietary source being fatty fish, which is also high in 20:5n-3. The n-6 fatty acid 18:2n-6, which is present in high amounts in many vegetable oils, and represents 90% of the polyunsaturated fatty acids in modern western diets (5-7), competes with 18:3n-3 for the desaturase enzymes, and itself is further metabolized to 20:4n-6 (3, 8). Epidemiological studies have shown an association between higher intakes of 20:5n-3 and 22:6n-3 from fish and other marine foods and decreased mortality from myocardial infarction (9) and all causes of stroke (10). In this regard, several studies have reported that dietary 34 1 A version of this chapter will be submitted for publication. Slack, P.J., Wadsworth, L.D., and Innis, S.M. An n-3 fatty acid deficient diet does not worsen infarct volume and a diet high in docosahexaenoic acid does not improve infarct volume after cerebral ischemia in rats. supplementation with fish oil or 22:6n-3 at doses of 200-300 mg/kg/day 22:6n-3 can reduce the extent of brain damage in animal models of cerebral ischemia (11-17). However, rather than consuming diets high in n-3 fatty acids, most western diets are high in 18:2n-6 and low in 18:3n-3 with recent estimates indicating that the median intake of 18:2n-6 and 18:3n-3 are about 17 g/day and 1.6 g/day ; this represents 5.5% and 0.6% of energy, respectively (5). In addition, the intakes of preformed 20:5n-3 and 22:6n-3 are also low, with average intakes of 100-200 mg/day 20:5n-3 plus 22:6n-3, equivalent to less than 0.05% of total energy (5). Thus, dietary intakes in humans are in the same range as that given per kg to animals in studies linking 20:5n-3 and 22:6n-3 to protection against brain damage in cerebral ischemia. On the other hand, the important question of whether or not low n-3 fatty acid intakes, sufficient to lower brain 22:6n-3, increases the extent of brain damage during cerebral ischemia has not been addressed. The present studies, therefore, sought to determine if short-term dietary deficiency of n-3 fatty acids decreases brain 22:6n-3 and increases infarct volume after cerebral ischemia/reperfusion, and if supplementation of a diet high in 18:2n-6, deficient in 18:3n-3, but with 22:6n-3 confers protection. To achieve these objectives, rats were fed a diet high in 18:3n- 3 (control), deficient in n-3 fatty acids, or the same diet deficient in n-3 fatty acids with supplemental 22:6n-3 for 5 weeks from weaning, then ischemia outcome was addressed after transient MCAO. 35 2.2 Materials and Methods 2.2.1 Animals and diets Male Sprague Dawley rats (Charles River Laboratories, Saint-Constant, QC, Canada), 21 days of age, were housed in a humidity and temperature controlled animal facility with a 12 hr:12hr light:dark cycle. The animals were randomly assigned to one of three semi-synthetic diets providing 16.5% of energy from fat, and identical in all nutrients except for fatty acids (18). The diets were provided fresh daily, and were fed ad libitum for approximately 5 weeks. The diets contained per kg, 70 g oil, 200 g vitamin-free casein, 200 g sucrose, 400 g cornstarch, 50 g non-nutritive cellulose, 10 g multi-vitamin mix (Teklad, AIN 93-VX), and 35 g multimineral mix (Teklad, AIN 93G-MX), with an additional 150 μg SeO2, 1 g choline chloride, and 3 g L-methionine (18). Because the goal was to determine if changes in brain n-3 and n-6 fatty acids influence cerebral ischemia/reperfusion infarct volume, the diets prepared included a control diet with a low 18:2n-6/18:3n-3 ratio to provide as a percent of energy, 3.2% 18:2n-6 and 1.1% 18:3n-3 using canola oil as the fat source; an n-3 fatty acid deficient diet with 12.4% 18:2n-6 and 0.05% 18:3n-3 from safflower oil; and a 22:6n-3 diet prepared by inclusion of 22:6n-3 from single cell triglycerides (Martek Biosciences, Columbia, MD, U.S.A) in the n-3 fatty acid deficient diet, which resulted in a diet providing 0.5% 22:6n-3, 11.5% 18:2n-6, and 0.05% 18:3n-3. Because the total fat content was constant, the three diets also differed in 18:1n- 9, with 10.2, 2.4, and 2.4% energy from 18:1n-9 in the control, n-3 fatty acid deficient, and high DHA diets, respectively. All procedures involving animals were approved and carried out in accordance with the animal care committee of the University of British Columbia and conformed to the guidelines of the Canadian Council on Animal Care Guidelines. A copy of the animal care certificate for this study is located in appendix 1. 36 2.2.2 Middle cerebral artery occlusion (MCAO) surgical procedure After feeding the assigned diet for approximately 5 weeks, and at a body weight of 280- 340 g, MCAO was performed following the procedures of Longa et al. (19), and Shih et al. (20), with slight modification. Briefly, after an overnight fast, the rats were anesthetized with 5% isoflurane carried by O2 (1 lb/min), and when adequate anesthesia was reached, the isoflurane flow was decreased to 2.5% and O2 decreased to 0.5 lb/min. A ventral neck incision was made, and the left external carotid artery ligated and cauterized above the occipital branch using a heat coagulator (Fine Science Tools, North Vancouver, BC, Canada). Branches from the external and internal carotid arteries were cauterized just above the bifurcation, and microvascular aneurysm clips placed on the left common and internal carotid arteries. A small incision was made in the external carotid artery stump and a 3 cm long, 3-0 monosof nylon filament (Assoc. Veterinary Purchasing, Abbotsford, BC, Canada) with a heat blunted end then advanced through the incision into the external carotid artery and advanced through the internal carotid artery to occlude the origin of the middle cerebral artery. A diagram of the occluded artery and surrounding arteries in this procedure can be found in appendix 2.1. Body temperature was maintained between 36.5 and 37.5°C using a feedback regulated temperature controlled heating pad (FHC Inc., Bowdoin, ME, U.S.A.). The incision was then sutured, and the animal returned to room air. After 90 min (occlusion), the rat was re-anesthetized and the filament removed to allow reperfusion. Buprenorphine (0.01 mg/kg) was administered as an analgesic immediately after closing the incision. Sensorimotor testing was done on all animals, immediately before removing the filament to confirm proper placement and occlusion of the artery. Animals who became hypothermic at any time, defined as a temperature < 36°C determined by rectal probe, had a sensorimotor score <10, cerebral hemorrhage evident on brain dissection, or no visible 37 infarct were not included in analysis of infarct volume, edema, or sensorimotor score. Detailed information on equipment used in this project can be found in appendix 2.2. 2.2.3 Sensorimotor testing The sensorimotor testing followed published procedures (20-22) and involved 6 tests, scored from 0 to 2, with 0 indicating no deficit, 1 moderate deficit, and 2 a severe deficit in performance, for a maximum potential score of 12. The tests used measured the function of the contralateral (right) side of the body and were: contralateral thorax twisting, forelimb flexion, forelimb bracing against a table edge, resistance to lateral push, table edge sensation, and forelimb placing on a table edge. Sensorimotor testing was repeated twice for each animal, immediately prior to reperfusion and again 24 hr after reperfusion. A score of 10 or higher prior to reperfusion was taken as the index of occlusion, and animals with a score <10 were removed from the study. 2.2.4 Infarct measurement The rats were euthanized 24 hr after MCAO under isoflurane anesthesia, exsanguinated, and the brains rapidly removed using a rongeur to remove the skull, and sliced into 2 mm coronal sections using a brain matrix (Plastics One, Roanoke, VA). The first, third, fifth, and seventh sections were stained with 2% triphenyl-tetrazolium chloride (TTC) in PBS for 30 min, then fixed in 4% paraformaldehyde for 24 hr (23, 24). Images of the front and back surfaces of each brain section were recorded using a digital camera, and the infarct areas measured using NIH Image J version 1.36b. Infarct volumes in mm3 were calculated as the sum of the products of the infarct area of the section and the distance between sections. The edema volume was calculated as the difference between the volume of the contralateral hemisphere and the ipsilateral hemisphere. 38 2.2.5 Brain fatty acids For analysis of brain phospholipid fatty acids, brains from rats fed the diets not subjected to MCAO, but fed for an equivalent duration, were rapidly dissected into frontal cortex and subcortex (including caudate putamen, corpus callosum, and frontal portion of hippocampus). These analyses provided the ability to establish the extent of alteration in fatty acids in different brain phospholipids caused by feeding the diets in the present studies for 5 weeks from weaning. Previous studies in the field relating diet to ischemia/reperfusion injury have reported brain total lipid fatty acids in animals fed diets with fish oil or supplemented with 22:6n-3 (14, 16, 25). To enable comparison of results from the present study to other published studies, brain total lipid fatty acids were also analyzed. For analyses of total fatty acid, the undamaged hemisphere, collected 24 h after MCAO, was used. All tissue samples were flash frozen in liquid nitrogen, and then stored at -70°C until analysis. For analysis, total lipids were extracted, and the fatty acids directly methylated, or PS, PE, phosphatidylcholine (PC), and phosphatidylinositol (PI) separated, quantified by high performance liquid chromatography (HPLC), and recovered, and their fatty acids then converted to their respective methyl esters, and separated and quantified by gas liquid chromatography (GLC) (18, 26-28). Detailed methods are included in appendix 2.3. 2.2.6 Platelet aggregation and lipid analysis 39 To address the possibility that diet-induced alterations in platelet fatty acids might contribute to cerebral hemorrhage, analysis of platelet fatty acids and tests of ex vivo platelet aggregation were conducted. These tests were done on animals that did not undergo MCAO surgery. Blood was collected by cardiac puncture into tubes containing 3.8% sodium citrate (1.5 v:v) under isoflurane anesthesia, centrifuged 5 min at 4°C and 300 x g, and the platelets recovered by centrifugation at 500 x g for 15 min, after addition of 20% sodium citrate solution. Platelets were then frozen at -70°C until analysis. For analysis of fatty acids, the platelets were thawed in cold water, and the fatty acids converted to methyl esters for separation and quantification by GLC (18, 26, 27, 29, 30). Platelet number was determined on aliquots of whole blood using an automated blood counter (Toa-Sysmex, Los Alamitos, CA) in the Division of Hematopathology, B.C. Children’s Hospital. Platelet aggregation, ex vivo, was determined using a PFA-100 platelet function analyzer with collagen/ADP test cartridges (Dade Behring, Mississauga, ON, Canada), following the manufacturer’s instructions and as described previously (29, 30). Detailed methods are included in appendix 2.4-2.6. 2.2.7 Statistical analyses Data were analyzed using the Statistical Package for Social Sciences (SPSS) for Windows, version 15.0. Results are given as means ± SEM, unless otherwise stated. Means of the three groups were compared using one way ANOVA with Fisher’s protected least significant difference for post-hoc analysis. Chi-square was used to determine whether the number of rats that had cerebral hemorrhages was different among diet groups. Differences with P < 0.05 were considered statistically significant. 40 2.3 Results 2.3.1 Characteristics of animals In order to ensure a rigorous approach to the MCAO procedure, male rats were fed their assigned diet until a body weight of 280-340 g was achieved. Body weight at the time of MCAO did not differ among the groups; 313 ± 3.6 g (n=14), 314 ± 4.8 g (n=13), and 319 ± 3.0 g (n=14), (P > 0.05) for the control, n-3 fatty acid deficient, and high DHA groups, respectively. The duration of feeding the diets was 35.4 ± 0.9, 34.4 ± 0.8, and 36.1 ± 0.6 days for the control, n-3 fatty acid deficient and high DHA groups, respectively, (P > 0.05). A number (4/14, 3/13, and 2/14 in the control, n-3 fatty acid deficient, and high DHA groups, respectively) of rats subjected to MCAO had either no visible infarct or a sensorimotor score <10, this is likely explained by the nylon filament not fully blocking the middle cerebral artery; these animals were excluded from the studies of infarct and edema volume. Cerebral (subarachnoid) hemorrhages were present in 3/14, 3/13, and 7/14 rats in the control, n-3 fatty acid deficient, and high DHA groups, respectively. To address the effects of the diets on the fatty acid composition of the brain phospholipids, without possible influences of the MCAO procedure, a further 6 rats for each diet group were fed for the same duration. 2.3.2 Effects of dietary fatty acids on brain fatty acids The subcortical region represents the core of the infarct in MCAO and is the initial site of cell death where damage is irreversible, whereas the cortex represents a large part of the penumbra, the area of moderate ischemic damage surrounding the area of severe ischemia, which is considered to be potentially salvageable (31). Phospholipid fatty acids have been reported to differ among different regions of the brain (32). Therefore, phospholipid fatty acids were analyzed in both the cortex and subcortex of the rats fed the diet for 5 weeks with the 41 results shown in Fig. 2-1. Rats fed the n-3 fatty acid deficient diet had levels of 22:6n-3 in cortex and subcortex PE, PS, PC and PI that were 21-28% lower than in rats fed the control diet (P < 0.05). Conversely, levels of 22:5n-6, in both the cortex and subcortex, were 445-842% higher in rats fed the n-3 fatty acid deficient diet than those fed the control diet, (P < 0.05). These results also show significantly higher levels of 20:4n-6 in cortex PE and PI, and subcortex PE and PS in n-3 fatty acid deficient compared to the control diet group (P < 0.05). In contrast, the levels of 22:6n-3 were higher in PE and PC of the subcortex, but not cortex, of rats fed the high DHA compared to control diet. Arachidonic acid (20:4n-6) was higher in PI in the cortex and subcortex, and in PC in subcortex, and 22:5n-6 was higher in PC in the cortex and subcortex in rats fed the high DHA diet when compared to those fed the control diet (P < 0.05). The results of brain total lipid fatty acid analysis are shown in Table 2-1. When compared to rats fed the control diet, rats fed the n-3 fatty acid deficient diet had lower levels of 22:6n-3, and higher levels of 18:2n-6, 20:4n-6, 22:4n-6 and 22:5n-6 in brain total fatty acids (P < 0.05). The brain lipid 20:4n-6/22:6n-3 ratio was 0.71 ± 0.01 and 0.94 ± 0.01 (P < 0.05) in the control and n-3 fatty acid deficient group, respectively. The levels of saturated fatty acids were not different, but the levels of 16:1(n-9) and 18:1(n-9) were both lower in brain total fatty acids of rats fed the n-3 fatty acid deficient than in those fed the control diet (P < 0.05). Rats fed the high DHA diet had higher levels of 18:3n-3 and 22:6n-3, 18:2n-6, 20:4n-6 and 22:4n-6, but lower levels of 20:5n-3, 16:1n-9 and 18:1n-9 in brain total fatty acids than in rats fed the control diet. The brain lipid 20:4n-6/22:6n-3 ratio, however, was not different between rats fed the control and those fed the high DHA diet 0.71 ± 0.01 and 0.68 ± 0.01 respectively, (P > 0.05). Rats fed the high DHA diet, in comparison to those fed the n-3 fatty acid deficient diet, had higher 18:3n-3, 22:6n-3, 18:2n-6, and 20:3n-6, but lower 20:5n-3, 22:4n-6, and 22:5n-6. 42 43 Fig. 2-1 Major fatty acid components in brain cortex and subcortex of rats fed a control, n-3 fatty acid deficient, or high DHA diet for 5 weeks from weaning. Bars are means +SEM, n=5 group. Means with a different subscript are different, P < 0.05; a,b,c. Subcortex PS PI Cortex 0 10 20 30 40 50 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y ac id s g/ 10 0 g fa tty a ci ds ortex PI 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac id s g/ 10 0 g fa tty a ci ds ab b a b a a b b Cortex PI PC Cortex 0 1 2 3 4 5 6 7 8 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y ac id s g/ 10 0 g fa tty a ci ds Cortex PC 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac id s g/ 10 0 g fa tty a ci ds a b b c a a Cortex PC PI Subcortex 0 10 20 30 40 50 1 2 3 g/ 10 0g fa tt y ac i DHA, ARA, DPA g/ 10 0 g fa tty a ci ds Subcortex PI 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds b b a b a a a a,b b Subcort x PI PC Subcortex 0 1 2 3 4 5 6 7 8 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds S cortex PC 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds a b c a b a b ubcortex PC a,b c PS Cortex 0 5 10 15 20 25 30 35 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds Cortex PS 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds a b b b a a Cortex PS Subcortex PE 0 5 10 15 20 25 30 35 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds Subcortex PE 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds b c High ALA n-3 Deficient High DHA Control a a b a a a b Cortex PE 0 5 10 15 20 25 30 35 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds Cortex PE 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y ac i g/ 10 0 g fa tty a ci ds Subcortex PE ortex PE b b n-3 fatty acid deficient a i A ba a b aa P Su cortex 0 5 10 15 20 25 30 35 1 2 3 DHA, ARA, DPA g/ 10 0g fa tt y a Subcorte S c g/ 10 0 g fa tty a ci ds 22:6(n-3) 20:4(n-6) 22:5(n-6) g/ 10 0g fa tt y a Subcortex PS c g/ 10 0 g fa tty a ci ds b b a a bb a aa Table 2-1. Major fatty acid components in brain of rats fed diets varying in n-3 and n-6 fatty acids for 5 weeks1 Control n-3 Deficient High DHA Fatty Acid g/100g fatty acid 18:3n-3 0.01 ± 0.01 a 0.01 ± 0.01 a 0.05 ± 0.00b 20:5n-3 0.12 ± 0.02a 0.09 ± 0.02a 0.03 ± 0.00b 22:6n-3 14.4 ± 0.47b 16.1 ± 0.09c 12.0 ± 0.16a 0.57 ± 0.02a 0.83 ± 0.03b 0.97 ± 0.04c18:2n-6 0.05 ± 0.00b 0.04 ± 0.00a 0.04 ± 0.00a18:3n-6 0.51 ± 0.02a 0.48 ± 0.01a 0.58 ± 0.02b20:3n-6 10.1 ± 0.27a 11.3 ± 0.13b 10.9 ± 0.08b20:4n-6 3.20 ± 0.08a 4.10 ± 0.07c 3.60 ± 0.04b22:4n-6 0.52 ± 0.01a 3.40 ± 0.21b 0.59 ± 0.03a22:5n-6 16:0 19.7 ± 0.29 19.6 ± 0.13 19.3 ± 0.14 18:0 21.4 ± 0.30 21.3 ± 0.09 21.1 ± 0.03 0.28 ± 0.01b 0.24 ± 0.00a 0.24 ± 0.00a16:1(n-9) 19.8 ± 0.44b 17.8 ± 0.15a18:1(n-9) 17.7 ± 0.14a 1 Values are means ± SEM, with n = 9, 8, and 8 for the control, n-3 fatty acid deficient, and high DHA diets groups, respectively. Means in the same row with different superscripts are significantly different, P < 0.05, by ANOVA. 44 2.3.3 Sensorimotor scores Sensorimotor function, when tested immediately prior to or 24 hr after reperfusion, was not different among rats fed the 3 diets, with scores of 10.4 ± 0.29, 10.4 ± 0.20, 10.4 ± 0.24 prior to, and 7.4 ± 1.3, 8.0 ± 0.44, 9.4 ± 0.6 at 24 hr after reperfusion in the control, n-3 fatty acid deficient and high DHA groups, respectively (P > 0.05). 2.3.4 Infarct volume and edema volume The infarct volumes when assessed by TTC staining 24 hr after MCAO were 203 ± 35.5 (n=7), 221 ± 34.8, (n=7), and 218 ± 38.9 mm3 (n=5) for the control, n-3 fatty acid deficient and high DHA diet groups, respectively, (P > 0.05), (Fig 2-3). Note that infarct volume was not assessed on rats showing cerebral hemorrhages. Although the mean edema volume appeared higher in the high DHA group, variability was also high in this group of animals, and no statistically significant differences were present among the groups, 43.4 ± 8.1, 50.6 ± 9.8, 67.0 ± 27.3 mm3 for the control, n-3 fatty acid deficient, and high DHA groups, respectively. 45 Fig 2-2 Representative TTC stained brain for infarct volume analysis. The brains were sliced into 2 mm coronal sections using a brain matrix, and the 1st, 3rd, 5th, and 7th sections stained with 2% TTC for 30 minutes. Brain slices were photographed, and then infarct areas were measured with NIH image J version 1.36b. Infarct volumes were calculated as the sum of the products of the infarct area of the section and the distance between sections. 46 0 50 100 150 200 250 300 In fa rc t V ol um e (m m3 ) High ALA (n-3) Deficient High DHA In fa rc t v ol um e (m m 3 Control n-3 fatty acid deficient High DHA ) Fig 2-3 Infarct volume (mm3) in animals, 24 hr after MCAO, which were fed either a control, n=7, n-3 fatty acid deficient, n=7, or high DHA diet, n=5, from weaning to 8 weeks of age. 2.3.5 Incidence of cerebral hemorrhage A higher number of rats with cerebral hemorrhages was observed in the high DHA diet group compared with the other 2 diet groups. However, using chi-square for analysis of the data, the incidence of cerebral hemorrhage did not differ significantly by diet group, χ2 (2, N = 41) = 0.193. It is important to note some of the diet groups had less than 5 animals with a hemorrhage, indicating that this study methodology was underpowered to detect a significant difference in hemorrhage among the 3 diet groups. There is no statistical significance using chi- square regarding the observation that more rats in the high DHA group having cerebral hemorrhages compared with the other 2 groups. However, an increased incidence of cerebral hemorrhage does occur in populations with high intakes of fish (9), therefore this observation warrants further exploration. 2.3.6 Platelet fatty acids and ex vivo aggregation The platelet levels of the n-3 fatty acids, 18:3n-3, 20:5n-3 and 22:6n-3 were all lower, and 18:2n-6, 22:4n-6 and 22:5n-6 were higher in rats fed the n-3 fatty acid deficient diet when compared to the control diet (P < 0.05), (Table 2-2). Rats fed the high DHA diet, on the other hand, had higher levels of 22:6n-3, 18:2n-6 and 20:4n-6, but lower 18:3n-3 and 20:5n-3 in platelet lipids than in the control group (P < 0.05). The platelet lipid 20:4n-6/20:5n-3 ratios in rats fed the n-3 fatty acid deficient or high DHA diet were higher than in rats fed the control diet, 151 ± 21.2, 172 ± 53.6, and 25.3 ± 4.4, respectively, (P < 0.05). The platelet lipid 20:4n- 6/22:6n-3 ratio, on the other hand, was lower in rats fed the high DHA diet, but higher in rats fed the n-3 fatty acid deficient diet than in the control group (5.0 ± 0.67, 272 ± 2.4, 7.9 ± 0.63, respectively, P < 0.05). The 20:4n-6/20:5n-3+22:6n-3 ratio was significantly higher in the n-3 47 fatty acid deficient than control and high DHA diet groups (24.6 ± 1.9, 5.9 ± 0.6, 4.8 ± 0.7, respectively, P < 0.05). There were no significant differences in platelet number among rats fed the control, n-3 fatty acid deficient, and high DHA diets (data not shown). In an ex vivo assay that measures the time required for blood to clot while flowing through a collagen/ADP aperture, the closure times in seconds were 112 ± 11.9 (n=5), 120 ± 10.5 (n=5) and 78.8 ± 5.5 (n=5) for rats fed the control, n- 3 fatty acid deficient, and high DHA diets, respectively, with the closure time for the high DHA group shorter, not longer than for the control group (P < 0.05). 48 Table 2-2. Major fatty acid components in platelets of rats fed diets varying in n-3 and n-6 fatty acids for 5 weeks1. Control n-3 Deficient High DHA Fatty Acid g/100g fatty acid 0.41 ± 0.06b 0.09 ± 0.03a 0.10 ± 0.05a18:3n-3 0.84 ± 0.10b 0.08 ± 0.03a 0.19 ± 0.05a20:5n-3 2.50 ± 0.15b 0.81 ± 0.06a 4.90 ± 0.62c22:6n-3 7.10 ± 0.59a 12.7 ± 2.10b 14.3 ± 1.60b18:2n-6 19.1 ± 0.82a 21.5 ± 1.10a,b 22.8 ± 0.50b20:4n-6 1.80 ± 0.15a 3.30 ± 0.35b 2.10 ± 0.53a22:4n-6 0.17 ± 0.04a 1.60 ± 0.27b 0.29 ± 0.03a 22:5n-6 1 Values given are means ± SEM, with n=6, 6, and 5 for the control, n-3 fatty acid deficient, and high DHA diet, respectively. Means in the same row with different superscripts are significantly different, P < 0.05, by ANOVA. 49 2.4 Discussion In the present study, rats were fed diets differing in n-3 and n-6 fatty acids, at the extremes of levels expected in human diets for 5 weeks from weaning, and had altered brain levels of 20:4n-6, 20:5n-3, 22:5n-6 and 22:6n-3. However, using MCAO as a model of cerebral ischemia-induced injury, there was no evidence that the diet-induced differences in brain fatty acids were associated with worsening of the infarct size or extent of edema in animals fed an n-3 fatty acid deficient diet, nor was there evidence of benefit in animals fed the 22:6n-3 supplemented diet. Whereas the immature brain is generally considered most vulnerable to the effects of unbalanced or inadequate dietary intakes of n-6 and n-3 fatty acids (33, 34), epidemiological studies have drawn attention to a relationship between a decreased intake of fish-derived n-3 fatty acids and increased risk of all causes of stroke (10), as well as aging-related cognitive decline in adults (35-37). The present study demonstrates a decrease by 17% in 22:6n-3 with a substantially higher (550%) 22:5n-6 in brain fatty acids of rats fed an n-3 fatty acid deficient diet compared to control diet for only 5 weeks from weaning. Stable isotope tracer studies have estimated the half-life of 22:6n-3 from adult rat brain phospholipids is 33 days, which increased to 90 days in rats fed an n-3 fatty acid deficient diet for 15 weeks (38). These results show that the more mature brain is plastic to changes in fatty acid composition when a diet high in 18:2n-6 and deficient in n-3 fatty acids is fed. On the other hand, feeding 0.5% energy 22:6n-3 led to an increase of 25% in 22:6n-3, with no increase in 22:5n-6 as found in rats fed the same diet without added 22:6n-3, this being the n-3 fatty acid deficient diet. The results show that rats fed the high DHA diet had lower levels of 22:4n-6 and 22:5n-6 in brain total fatty acids than rats fed the n-3 fatty acid deficient diet. This suggests inhibition of elongation after 20:4n-6 and/or inhibition of Δ6 desaturation at the level of 24:4n-6 to 24:5n-6 by 22:6n-3. The n-3 fatty acid 50 deficient and high DHA diets provided 12.4 and 11.5% dietary energy from 18:2n-6, respectively, with both diets providing about 0.05% energy from 18:3n-3. The analyses of the brain total fatty acids show higher 18:2n-6, 20:4n-6, and 22:4n-6 in the high DHA group than in rats fed the control diet with 3.2% and 1.1% energy from 18:2n-6 and 18:3n-3. This suggests, however, that feeding a diet high in 18:2n-6 and low in 18:3n-3 results in an increased accumulation of some n-6 fatty acids in the brain, regardless of the addition of 22:6n-3. Epidemiological associations have shown a relationship between habitual diets low in 20:5n-3 and 22:6n-3 from fish, and increased risk of stroke and neurological problems (10, 35-37). The present study provides experimental results to suggest that associated differences in disease could involve differences in tissue n-6 as well as n-3 fatty acids. This was the first study to assess the effects of n-3 and n-6 fatty acids, over a range of dietary intakes, on the extent of damage after cerebral ischemia. The present study shows that diets ranging in n-3 and n-6 fatty acids, when fed for 5 weeks, results in brain total fatty acid levels of 22:6n-3 that differed between 11 and 25%, and levels of 20:4n-6 that differed between 3 and 10 %. Epidemiological studies indicate that the incidence of morbidity and mortality from stroke is higher among populations consuming diets that are low rather than high in n-3 fatty acids (10); this has, in part, stimulated interest in the possible protective role of n-3 fatty acids in animal models of stroke. At the outset of this study, a number of studies had reported that high dietary long chain n-3 fatty acids reduced the extent of damage caused by cerebral ischemia in animal models; only one study reported larger infarct volumes in rats fed a high fish oil diet. Despite the differences in brain fatty acid levels induced by diets in this study, rats fed these diets did not have statistically significant differences in infarct volumes after cerebral ischemia. Rather, the findings of the current study, and two other publications that have found long chain n-3 fatty acids to have detrimental effects, raise the question of whether or not high levels of n-3 51 fatty acids present during cerebral ischemia are safe. Lai et al. (40) reported a 25% larger infarct volume, 24 hr after MCAO, in rats fed chow supplemented with 20% energy from fish oil when compared to rats not given fish oil. In another model, involving intraperitoneal injection of 22:6n-3, 60 min after transient focal cerebral ischemia, there was a dose-dependent increase in cerebral infarct volume in rats (41). However, there are a number of reports that fish oil reduces the extent of damage caused by cerebral ischemia in animals. Relton et al. (12) reported that rats fed a semisynthetic diet with 0.9% 20:5n-3 and 0.6% 22:6n-3/ kg diet from fish oil for 6 weeks had smaller infarct volumes than rats fed a control diet with 6% olive oil/kg diet. Differing from the diets in this study, olive oil is a monounsaturated oil with low amounts of 18:2n-6. In cats, the mean infarct volume was lower 3 days after MCAO in animals fed cat chow with 8% energy fish oil rather than no fish oil (11). Also different from the current study, the activity of Δ6 desaturase activity is very low in cats (42, 43), and cat chow provides 20:4n-6. Similarly, Choi-Kwon et al. (14) reported smaller infarct volumes 24 hr after 2 hr of MCAO in rats fed a diet with 14% fish oil rather than chow for 6 weeks from weaning. Recently, one laboratory reported that rats given 400mg/kg/day Marincap fish oil for 14 days by gavage had significantly fewer apoptotic neurons in both the hippocampus and prefrontal cortex after cerebral ischemia injury, induced by 45 min of occlusion of both common carotid arteries and hypotension followed by 30 min of reperfusion, compared to controls given saline (44, 45). In addition to dietary supplementation with fish oils, the potential neuroprotective effect of 22:6n-3 has been addressed through administration of ethyl esters of 22:6n-3 by gavage or infusion into the brain. Using an ethyl ester of 22:6n-3 at 300 mg/kg/day in the chow diet for 8 weeks, cerebral lesions in rats receiving 22:6n-3 were lower than in those not receiving it, after 24 hr (13). DHA (22:6n-3), at 200 mg/kg/day given perorally for 3 weeks was also associated with less histological damage in the hippocampus, 8 days after transient forebrain ischemia than in 52 rats not given 22:6n-3 (16). Similarly, Cao et al. (15) reported that 200 mg/kg/day ethyl esters of 22:6n-3 given by gavage for 10 weeks was associated with a 35% lower infarct volume in gerbils when measured 24 hr after transient global ischemia. Together, the latter studies suggest that 22:6n-3, rather than, or in addition to 20:5n-3, has neuroprotective effects in decreasing damage in animal models of cerebral ischemia. Consistent with this, 10,17 S-docosatriene, a metabolite of 22:6n-3, given infused into the brain for 48 hr immediately following cerebral ischemia was associated with a lower mean infarct volume in mice (46). However, the current study and that by Lai et al. (40) and Yang et al. (41), outlined above, suggest that 22:6n-3 does not necessarily have any beneficial effects and can also have detrimental effects on cerebral ischemia outcome. Understanding the reason for the discrepancy amongst those studies reporting positive and negative effects of fish oil or ethyl esters of 22:6n-3 in animal models of cerebral ischemia is complicated by the use of different species, diets, doses and methods of dietary administration, as well as surgical procedures. Comparison of the reported effects of fish oil or 22:6n-3 supplementation on brain levels of 22:6n-3, 20:4n-6, or the 20:4n-6/22:6n-3 ratio also offers no clear reason why n-3 fatty acids offer protection in some studies (11-14, 16, 46), adverse effects in others (40, 41), and in the present study had no significant effect on infarct size or edema volume following cerebral ischemia. Although it is tempting to speculate that the high DHA diet might have made the rats fed this diet more susceptible to cerebral hemorrhage in this study, chi-square analysis did not reveal any statistically significant difference in the incidence of cerebral hemorrhage among the diet groups. However, this study was neither designed nor powered to test the hypothesis of an increased susceptibility to hemorrhage among rats fed different diets. Design of an experiment to properly test this hypothesis may be warranted, due to the epidemiological data to suggest 53 that populations with high intakes of fish have a higher than expected rate of cerebral hemorrhage (9). Because some studies have reported that platelet fatty acids and aggregation are altered with high DHA intakes (47-50), experiments were conducted to explore the possibility that platelet fatty acids or aggregation might be altered in the high DHA diet group, which could possibly provide supporting evidence to the observation of more animals with hemorrhage in this diet group. Analyses of platelet fatty acids from rats fed the diets showed the anticipated diet- dependent increase in platelet 22:6n-3 in rats fed the 22:6n-3 supplemented diet, although the 20:4n-6 levels were higher, not lower than in rats fed the control diet. Further, the ex vivo platelet aggregation tests found no evidence of decreased platelet aggregation in rats fed the high DHA compared to control or n-3 fatty acid deficient diet. In summary, although low intakes of n-3 fatty acids have been associated with an increased risk of all causes of stroke (10), in the current study there was no evidence that a diet deficient in n-3 fatty acids increased the infarct volume after cerebral ischemia in the rat, although brain levels of 22:6n-3 were decreased. In addition, feeding a diet supplemented with 22:6n-3 was not associated with a reduction in infarct volume following cerebral ischemia. 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Blood. 1981 Nov;58(5):880-5. 61 CHAPTER 3: GENERAL DISCUSSION AND CONCLUSIONS 3.1 General Discussion and Conclusion: The present studies addresses whether dietary n-3 fatty acid deficiency, or supplementation of an n-3 fatty acid deficient diet with 22:6n-3 alters brain n-3, and n-6 fatty acids, and whether this is associated with changes in infarct volume when measured 24 hrs after MCAO. 3.2 Influence of Diet on Brain Fatty Acid Composition The first hypothesis, that an n-3 fatty acid deficient diet when fed to rats for 5 weeks from weaning would lower the level of 22:6n-3 in brain phospholipids by at least 20% when compared to rats fed an n-3 fatty acid adequate (control) diet was proven. In previous studies using similar diets, dams fed an n-3 fatty acid deficient diet throughout gestation produced offspring that, at embryonic day 19, had 55-65% lower 22:6n-3 in brain phospholipids (1). When fed from weaning, n-3 fatty acid deprivation for 15 weeks resulted in 27-43% lower levels of 22:6n-3 in brain phospholipids in rats (2). Very little information is available to relate dietary n-3 fatty acid restriction to brain fatty acids in humans. However, fatty acid analysis of autopsy samples from infants fed formula with high 18:2n-6 and low 18:3n-3 and no 22:6n-3 or 20:5n-3 reported levels of 22:6n-3 that were approximately 30% lower than in the brains of infants who were breastfed before death (3, 4). Current human diets, particularly those in many Western countries are believed to be low in all n-3 fatty acids, and to contain an abundance of n- 6 fatty acids (5). The replacement of 0.5% energy from safflower oil with 22:6n-3 from single cell triglycerides resulted in significantly higher levels of 22:6n-3 in brain phospholipids of rats fed the high DHA compared to the n-3 fatty acid deficient diet. The presence of 22:6n-3 in the diet also prevented the accumulation of high levels 22:5n-6 that were present in brain phospholipids of rats fed the n-3 fatty acid deficient diet, and which are a characteristic feature 62 of n-3 fatty acid deficiency. Several recent studies have linked increased risk of aging-related cognitive decline and incidence of dementia to low dietary intakes of n-3 fatty acids (6-8), raising the question of whether or not low dietary intakes of n-3 fatty acids alter brain fatty acids, with consequences for neurological function after infancy. 3.3 Influence of Dietary n-3 Fatty Acids on Cerebral Ischemia/Reperfusion Outcome The second hypothesis, that rats fed an n-3 fatty acid deficient diet would have a larger infarct volume 24 hrs after MCAO when compared to rats fed the control diet or diet high in 22:6n-3 was rejected. The edema volume was also not significantly different among the groups, and no differences in the sensorimotor deficits were detected 24 hrs after the MCAO procedure. The absence of a beneficial effect of the high DHA diet on infarct volume, compared to the control diet is consistent with some other studies that have found 22:6n-3, either added separately or through fish oil, does not benefit stroke outcome. Lai et al. (9) reported larger infarct volumes after temporary focal ischemia in rats fed a diet containing 20% of energy as fish oil for 7 weeks from weaning when compared to rats fed chow. Similarly, Skrzypski et al. (10) found a tendency toward larger infarct volumes in rats fed a diet containing 0.6% energy as 22:6n-3 when focal cerebral ischemia was induced. In another study, Yang et al. (11) reported that intraperitoneal injection of 22:6n-3 given 60 min after transient focal ischemia was associated with a dose-dependent increase in cerebral infarct volume in rats. However, other reports described in Chapter 2 have reported smaller infarct volumes and beneficial outcomes in animals given dietary fish oil or 22:6n-3 (12-18). Comparison of currently published studies, to address potential reasons for the discrepancy, revealed no clear explanation when considering factors such as the species of animal used, the 63 model of cerebral ischemia, the duration of feeding, or the amount of fish oil or 22:6n-3 given. The findings in the present study, together with those from other laboratories show no benefit or larger infarct volumes after cerebral ischemia in animals given 22:6n-3 or fish oil. This emphasizes the need for further research to consider the possibility of adverse effects, particularly using models of diets and supplementation paradigms relevant for human populations. Recent studies have suggested that a limiting amount of n-3 fatty acids may be detrimental to the brain’s ability to create new synapses during pathological states, such as stroke and Alzheimer’s disease (19, 20). Whether or not the decrease of 20% in brain membrane phospholipid 22:6n-3 in rats fed the n-3 fatty acid deficient diet might have any relevance to later recovery following cerebral ischemia was not addressed in this research, but may be important. DHA (22:6n-3) can be further metabolized to 10-17 S-docosatriene, which is produced during ischemia/reperfusion injury (21). When infused into the brain, 10-17 S- docosatriene may reduce the extent of injury through inhibition of inflammatory and apoptotic processes (21). Whether the brain, following injury, can produce 10-17 S-docosatriene endogenously and in amounts sufficient to afford neuroprotection is not known (21). However, the brain pool of 22:6n-3 is very large, while available evidence indicates 10-17 S-docosatriene production is quantitatively very low (21). Whether dietary deficiency of n-3 fatty acids, leading to decreased brain 22:6n-3, can alter 10-17 S-docosatriene production remains to be elucidated. 64 3.4 Cerebral Hemorrhages in Relation to Dietary n-3 Fatty Acid Intakes The model of MCAO used in the current study is known to sometimes result in cerebral (subarachnoid) hemorrhage. The advancement of a nylon suture through the internal carotid artery to block the middle cerebral artery can result in damage to these arteries which might cause them to rupture. In reviewing published studies using this procedure, many have not reported an incidence of cerebral hemorrhage, however, Shimamura et al. (22) and Yang et al. (23) reported incidences of 16.7% and 20%, respectively. In the current study, the incidences of cerebral hemorrhage were 23.1% and 21.4 in the n-3 fatty acid deficient and control groups, respectively, and 50% in the high DHA diet group. Notably, the assessment of hemorrhage was not an intended outcome of the current study. There was an apparent, but not statistically significant increase using the chi-square, in the incidence of cerebral hemorrhage in the high DHA diet group compared to the other 2 groups. Possible explanations for this observation include the possibility that the DHA feeding increased susceptibility to hemorrhage or that there was an incidental difference in technique during surgery. There are several risk factors and causes for cerebral hemorrhage in human populations. Causes of cerebral hemorrhage include chronic hypertension, vascular malformations, anticoagulant or antiplatelet therapy, and loss of vascular integrity, among others (24). Platelet fatty acids and aggregation were assessed to determine whether or not rats in the high DHA diet group had characteristics, such as an increased 20:5n-3+22:6n-3/20:4n-6 ratio in platelet fatty acids or decreased platelet aggregation which could increase their susceptibility to bleeding. None of these characteristics were found in rats fed the high DHA diet. Although not measured in this study, it seems unlikely that elevated blood pressure could be involved because there is no evidence that 22:6n-3 increases blood pressure; rather 22:6n-3 is associated with lower blood 65 pressure in both humans and rats (25, 26). Very little information is available to address whether or not 22:6n-3 influences vascular integrity. One study reported that treatment of neutrophils with 22:6n-3 in vitro, increased endothelial detachment (27), and this was suggested to reflect potential increased susceptibility of endothelial cells to injury. At this time, there is a paucity of information on the effects of high DHA intakes on vascular tissues. Alternatively, rather than a cause and affect association between dietary DHA and cerebral hemorrhage, it is possible that the apparent increased incidence of hemorrhage in the high DHA group occurred by chance. As is clear from published data, other laboratories performing this model of MCAO report that about 16-20% of animals experience cerebral hemorrhage (22), (23). Although it seems unlikely that the apparent higher number of hemorrhages in the high DHA group occurred by chance observation, the low number of animals does make a chance observation possible. Two approaches are possible for further exploring the question of whether or not supplementation with high amounts of 22:6n-3 increases the risk of cerebral hemorrhage; first, the present study could be repeated with sufficient power to detect a two-fold increase in the incidence of hemorrhage, based on the present findings. Alternatively, it may be more appropriate, if an increased incidence of cerebral hemorrhage is of interest, to design a study using an appropriate animal model to enable this to be rigorously tested and mechanisms explored. A possible example to assess vessel fragility is a variation is the Rumple-Leede test used in rats (28). To summarize, further studies will be required to determine whether or not rats fed high 22:6n-3 diets are more susceptible to cerebral hemorrhage than rats fed control diets without 22:6n-3. Consideration of this possibility is important because of the epidemiological reports of higher incidences of cerebral hemorrhage in populations with high intakes of n-3 fatty acids from fish and marine animals than among populations with habitual low intakes of the same 66 foods (29), and the current trends to use high dose fish oil supplementation for primary and secondary prevention of cardiovascular disease (30), as well as widespread addition of 22:6n-3 and fish oils to many foods. 67 3.5 Conclusion In conclusion, although lower stroke mortality rates have been reported in populations who consume higher amounts of long chain n-3 fatty acids, the present study found no evidence that a diet deficient in n-3 fatty acids leads to a greater extent of brain damage, or that a diet high in 22:6n-3 leads to a smaller amount of damage after cerebral ischemia/reperfusion in the rat, when measured 24 hrs after the injury. However, the possibility that important differences in recovery may emerge with a longer-term study have not been addressed. The potential higher rate of cerebral hemorrhage in rats fed a high DHA diet requires further research to confirm this observation, elucidate possible mechanisms, and better understand the relevance to long chain n- 3 fatty acid supplementation in human populations, particularly because increased bleeding among individuals consuming high amounts of fish is acknowledged as a concern. 68 3.6 Strengths and Limitations The current study addressed the effects of diets differing in n-3 fatty acids on infarct volume following ischemic stroke. A limitation in this field of research is the variability in the approaches to provide n-3 fatty acids to animals, including addition to the diet, gavage, infusion into the brain, and intraperitoneal injection. Although addition to the diet may seem simplest, fatty acids also provide energy, thus addition of fat to a complete diet increases the energy content per kg and thus decreases the protein, vitamin, and mineral content per kg. Many studies have compared diets that have different amounts of fat; a common approach was adding fish oil to a stock diet and comparing it to a control diet not containing fish oil. A major strength of the current study was that a rigid dietary design was used in which the energy densities, protein, and vitamin and mineral content were constant, and only the composition of fatty acids was altered. Three diets were included to enable comparison of the effects of n-3 fatty acid deficiency, n-3 fatty acid adequacy, and high amounts of 22:6n-3 on ischemic stroke outcome. An unavoidable limitation is that in order to keep total fat constant, as the amount of one fatty acid is changed, another must also change. Thus, the levels of 18:1n-9 and 18:2n-6 also differed between the 3 diets, since fat was supplied by canola oil in the control group and safflower oil in the n-3 fatty acid deficient and high DHA groups. It would have been possible to limit the difference between the levels of n-6 fatty acids in the control and n-3 fatty acid deficient diets by using high oleic acid safflower oil (77% 18:1n-9, 14% 18:2n-6) rather than high linoleic acid safflower oil (13% 18:1n-9, 78% 18:2n-6). However, since no difference in the size of infarct volume was found at 24 hrs after the procedure, the difference in 18:1n-9 and 18:2n-6 between the control and n-3 fatty acid deficient diets would not likely be an important modifier of the outcome. 69 The difference in fatty acid composition of the diets fed, and the 5 week feeding duration was successful in altering the levels of 22:6n-3 in brain phospholipids by 20% between the control and n-3 fatty acid deficient diet groups. This relatively large difference in brain phospholipid 22:6n-3, induced solely by feeding diets of different fatty acid composition, adds strength to a conclusion that within the context of usual human diets, diet-dependent differences in brain fatty acids are not likely to influence the extent of injury in the early period following stroke. However, some caution is warranted. First, the analyses included total brain lipid fatty acids and the phospholipid fatty acid composition of the four major species, PC, PE, PI, and PS. The analyses, however, did not include specific cells such as neurons, astrocytes, or capillary endothelial cells. It is possible that the effects of the diet differed across cell types and after 5 weeks, insufficient incorporation or depletion of 22:6n-3 from cell types that might influence the extent of the infarct volume size was not achieved. Several different animal models of ischemic stroke have been described in the literature. The animal model of cerebral ischemia used in the present research is considered to be among the most relevant to human ischemic stroke (31), and is similar to models used in several other studies assessing the impact of n-3 fatty acids on cerebral ischemia (9, 12, 13, 16). The use of an accepted model of ischemic stroke that has also been used by others investigating the effect of n-3 fatty acids on stroke outcome was a strength of the present study, and it also enabled comparison between the results of the current study and published studies. Regardless of the consistency among studies in terms of the animal model used, both positive and negative effects of n-3 fatty acids on cerebral ischemia outcome have been reported when similar protocols have been used (9, 10, 12, 13, 16). A limitation of the present study was that outcome was assessed 24 hrs after MCAO, but recovery at later time points was not part of the objective. TTC staining was performed at 24 70 hrs. This is considered optimal since the size of the infarct does not change significantly after this point, for at least 14 days after MCAO (32). However, recovery at later time points, including improvements in behavioural outcomes may or may not necessarily correlate with the extent of damage measured soon after occlusion. Assessment of longer-term recovery including behavioural testing was considered, but was not included because of the large number of animals that would be required for the procedure, and the absence of any adverse effect of n-3 fatty acid deficiency which was the major focus of this research. In the present study, the number of animals assigned to each dietary group was relatively large. However, although the careful exclusion of animals that did not have an ischemic stroke was a strength, it also resulted in a smaller number of animals that could be assessed for infarct volume. This raises the possibility that a low sample size might have led to the inability to detect important effects of the diets in modifying infarct size. However, this seems unlikely because the results showed no evidence of any trend towards altered infarct volume. Furthermore, rats expected to have negligible infarct volumes due to insufficient occlusion of the middle cerebral artery were identified by a sensorimotor score of less than 10. If n-3 fatty acids protected the brain by reducing the infarct volume size, then more animals in the control and 22:6n-3 groups might have had a sensorimotor score less than 10 than among those in the n- 3 fatty acid deficient group; this was not the case. Rats that had a cerebral hemorrhage were identified and also excluded from analysis. Inclusion of measures of the infarct volume from these groups could have influenced the results, but the interpretation of such results is questionable. A further strength of this study is that the model used and experimental approach enabled the observation that cerebral hemorrhage may be increased in rats fed a diet high in 22:6n-3. Although the mechanism is currently unknown, human populations habitually 71 consuming diets high in long chain n-3 fatty acids do have an increased incidence of cerebral hemorrhage (29). The current study successfully tested the effect of whether or not differences in 22:6n-3 in brain lipids, achieved by feeding an n-3 fatty acid deficient diet compared to an adequate or enriched diet, influences infarct volume in rats subjected to cerebral ischemia/reperfusion. Although there are many aspects of damage and recovery to consider in cerebral ischemia, the results show that using infarct volume as the outcome measure, there is no worsening of infarct volume when brain 22:6n-3 is altered by 20% in brain phospholipids by dietary means. 72 3.7 Future Directions 1 Assess histological damage beyond 24 hrs to determine whether the diets varying in n-3 fatty acids impact the extent of damage or recovery at later time points. 2 Assess behavioural outcomes, post cerebral ischemia, using tests of sensorimotor function such as the horizontal ladder, or staircase to determine whether the diets affect such outcomes. 3 Explore the possibility of an increased incidence of cerebral hemorrhage with high DHA feeding, using either sufficient power with the current model, or using an animal model which would assess vessel fragility. 73 3.8 Bibliography 1. Coti Bertrand P, O'Kusky JR, Innis SM. Maternal dietary (n-3) fatty acid deficiency alters neurogenesis in the embryonic rat brain. J Nutr. 2006 Jun;136(6):1570-5. 2. DeMar JC,Jr, Ma K, Bell JM, Rapoport SI. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids. J Neurochem. 2004 Dec;91(5):1125-37. 3. Farquharson J, Jamieson EC, Abbasi KA, Patrick WJ, Logan RW, Cockburn F. Effect of diet on the fatty acid composition of the major phospholipids of infant cerebral cortex. Arch Dis Child. 1995 Mar;72(3):198-203. 4. Makrides M, Neumann MA, Byard RW, Simmer K, Gibson RA. Fatty acid composition of brain, retina, and erythrocytes in breast- and formula-fed infants. Am J Clin Nutr. 1994 Aug;60(2):189-94. 5. Simopoulos AP. Human requirement for N-3 polyunsaturated fatty acids. Poult Sci. 2000 Jul;79(7):961-70. 6. Beydoun MA, Kaufman JS, Sloane PD, Heiss G, Ibrahim J. n-3 fatty acids, hypertension and risk of cognitive decline among older adults in the atherosclerosis risk in communities (ARIC) study. Public Health Nutr. 2008 Jan;11(1):17-29. 74 7. Van Gelder BM, Tijhuis M, Kalmijn S, Kromhout D. Fish consumption, n-3 fatty acids, and subsequent 5-y cognitive decline in elderly men: The zutphen elderly study. Am J Clin Nutr. 2007 Apr;85(4):1142-7. 8. Schaefer EJ, Bongard V, Beiser AS, Lamon-Fava S, Robins SJ, Au R, Tucker KL, Kyle DJ, Wilson PW, Wolf PA. Plasma phosphatidylcholine docosahexaenoic acid content and risk of dementia and alzheimer disease: The Framingham heart study. Arch Neurol. 2006 Nov;63(11):1545-50. 9. Lai ML, Hsu CY, Liu TH, He YY, Xu J, Navidi M, Sun G, Hogan EL. Effects of fish oil supplementation on acute ischemic brain injury in the rat. Neurology. 1993 Jun;43(6):1227-32. 10. Skrzypski J, Tessier C, Bertrand N, Mossiat C, Chardigny JM, Narce M. In: 8th meeting of the international society for the study of fatty acids and lipids. May 17-22, 2008; Kansas City, Missouri. ; 2008. 11. Yang DY, Pan HC, Yen YJ, Wang CC, Chuang YH, Chen SY, Lin SY, Liao SL, Raung SL, et al. Detrimental effects of post-treatment with fatty acids on brain injury in ischemic rats. Neurotoxicology. 2007 Nov;28(6):1220-9. 12. Black KL, Culp B, Madison D, Randall OS, Lands WE. The protective effects of dietary fish oil on focal cerebral infarction. Prostaglandins Med. 1979 Nov;3(5):257-68. 13. Relton JK, Strijbos PJ, Cooper AL, Rothwell NJ. Dietary N-3 fatty acids inhibit ischaemic and excitotoxic brain damage in the rat. Brain Res Bull. 1993;32(3):223-6. 75 14. Umemura K, Toshima Y, Asai F, Nakashima M. Effect of dietary docosahexaenoic acid in the rat middle cerebral artery thrombosis model. Thromb Res. 1995 Jun 1;78(5):379-87. 15. Okada M, Amamoto T, Tomonaga M, Kawachi A, Yazawa K, Mine K, Fujiwara M. The chronic administration of docosahexaenoic acid reduces the spatial cognitive deficit following transient forebrain ischemia in rats. Neuroscience. 1996 Mar;71(1):17-25. 16. Choi-Kwon S, Park KA, Lee HJ, Park MS, Lee JH, Jeon SE, Choe MA, Park KC. Temporal changes in cerebral antioxidant enzyme activities after ischemia and reperfusion in a rat focal brain ischemia model: Effect of dietary fish oil. Brain Res Dev Brain Res. 2004 Aug 18;152(1):11-8. 17. Cao D, Yang B, Hou L, Xu J, Xue R, Sun L, Zhou C, Liu Z. Chronic daily administration of ethyl docosahexaenoate protects against gerbil brain ischemic damage through reduction of arachidonic acid liberation and accumulation. J Nutr Biochem. 2007 May;18(5):297-304. 18. Bas O, Songur A, Sahin O, Mollaoglu H, Ozen OA, Yaman M, Eser O, Fidan H, Yagmurca M. The protective effect of fish n-3 fatty acids on cerebral ischemia in rat hippocampus. Neurochem Int. 2007 Feb;50(3):548-54. 19. Cansev M, Wurtman RJ, Sakamoto T, Ulus IH. Oral administration of circulating precursors for membrane phosphatides can promote the synthesis of new brain synapses. Alzheimers Dement. 2008 Jan;4(1 Suppl 1):S153-68. 76 20. Sakamoto T, Cansev M, Wurtman RJ. Oral supplementation with docosahexaenoic acid and uridine-5'-monophosphate increases dendritic spine density in adult gerbil hippocampus. Brain Res. 2007 Nov 28;1182:50-9. 21. Marcheselli VL, Hong S, Lukiw WJ, Tian XH, Gronert K, Musto A, Hardy M, Gimenez JM, Chiang N, et al. Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression. J Biol Chem. 2003 Oct 31;278(44):43807- 17. 22. Shimamura N, Matchett G, Tsubokawa T, Ohkuma H, Zhang J. Comparison of silicon- coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model. J Neurosci Methods. 2006 Sep 30;156(1-2):161-5. 23. Yang Y, Yang T, Li Q, Wang CX, Shuaib A. A new reproducible focal cerebral ischemia model by introduction of polyvinylsiloxane into the middle cerebral artery: A comparison study. J Neurosci Methods. 2002 Aug 30;118(2):199-206. 24. Norrving B. Cerebral hemorrhage. In: Ginsberg MD, Bogousslavsky J, editors. Cerebrovascular Disease: Pathophysiology, diagnosis, and management. U.S.A.: Blackwell Science; 1998. p. 1447. 25. Mori TA, Bao DQ, Burke V, Puddey IB, Beilin LJ. Docosahexaenoic acid but not eicosapentaenoic acid lowers ambulatory blood pressure and heart rate in humans. Hypertension. 1999 Aug;34(2):253-60. 77 26. Kimura S, Minami M, Saito H, Kobayashi T, Okuyama H. Dietary docosahexaenoic acid (22: 6n-3) prevents the development of hypertension in SHRSP. Clin Exp Pharmacol Physiol Suppl. 1995 Dec;22(1):S308-9. 27. Bates EJ, Ferrante A, Harvey DP, Nandoskar M, Poulos A. Docosahexanoic acid (22:6, n-3) but not eicosapentaenoic acid (20:5, n-3) can induce neutrophil-mediated injury of cultured endothelial cells: Involvement of neutrophil elastase. J Leukoc Biol. 1993 Dec;54(6):590-8. 28. Kozam G. Capillary fragility in the skin of the albino rat and its relation to the hair cycle. Am J Physiol. 1952 Jun;169(3):529-36. 29. Kromann N, Green A. Epidemiological studies in the Upernavik district, Greenland. incidence of some chronic diseases 1950-1974. Acta Med Scand. 1980;208(5):401-6. 30. Kris-Etherton PM, Harris WS, Appel LJ, American Heart Association. Nutrition Committee. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation. 2002 Nov 19;106(21):2747-57. 31. Macrae IM. New models of focal cerebral ischemia. Br J clin, Pharmac. 1992;34:302-308. 32. Xu XH, Zhang SM, Yan WM, Li XR, Zhang HY, Zheng XX. Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats. Life Sci. 2006 Jan 11;78(7):704-12. 78 APPENDICES A ppendix 1: UBC Ethics Board Certificate THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A06-0450 Investigator or Course Director: Sheila M. Innis Department: Paediatrics Animals: Rats Sprague Dawley 106 Start Date: September 26, 2006 Approval Date: November 9, 2006 Funding Sources: Funding Agency: Bristol-Myers Squibb, US (Princeton) Funding Title: Unrestricted Nutrition Research Grant Unfunded itle: N/A t The Animal Care Committee has examined and approved the use of animals for the above experimental project. 79 This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 80 Appendix 2: Supplemental Methods Appendix 2.1 Diagram of MCAO CCA ECA ICA MCA ACA ACOA MCA BA VA ICA PCA PCOA Diagram of filament placement during MCAO in the rat. ACA, anterior cerebral artery; ACOA, anterior communicating artery; BA, basilar artery; CCA, common carotid artery; ECA, external carotid artery; MCA, middle cerebral artery; PCA, posterior cerebral artery; PCOA, posterior communicating artery; VA, vertebral artery. Adapted from (1). 81 Appendix 2.2 Equipment Centrifugation was performed using a Sorvall Biofuge Stratus centrifuge with swinging basket rotor (Mandel Scientific, Guelph, ON, Canada). Fatty acid methyl esters were separated and quantified by GLC with a 6850 gas chromatograph from Agilent Technologies (Mississauga, ON, Canada), equipped with flame ionization detection and an IBC computer system with GC chemostation software (Agilent Technologies). Glass capillary columns (30.3 m x 250 um) were from Supelco Canada Ltd. (Oakville, ON). Individual phospholipids (PE, PS, PC, PI) were separated using HPLC with a Waters 2695 Alliance HPLC, (Milford MA), equipped with an auto-sampler and column heater. The column was a Waters YMC-Pack Diol 120NP, 25 cm x 4.6 mm id, 5 μm particle size and 12 nm pore size. The column eluant was split using an Alltech (model 2000) evaporative light scattering detector (ELSD) from Mandel Scientific and a Gilson FC204 fraction collector (Mandel Scientific). Surgery was performed using a Nikon SMZ645 dissecting microscope (Mississauga, ON, Canada), and a Fiber-Lite MI- 150 illuminator (Lawrence, MA, U.S.A.). Sterilization of surgical instruments was by autoclave or using a Steri 350 bead sterilizer (Inotech, Rockville, MD, U.S.A.). Appendix 2.3 Analysis of Brain Fatty Acids Lipid Extraction Total lipids were extracted according to the Folch method (2), with minor modifications. Brain tissue (100-150 mg) was added to 17 volumes of saline, and sonicated with a sonic dismembrator, model 500 (Fisher Scientific). Fatty acids were extracted with solvent proportions of chloroform:methanol:saline, 6:3:2.25 (v/v/v) . The samples were centrifuged and the resulting bottom organic layer was filtered into a test tube, and the aqueous layer transferred to a new tube and 6 ml chloroform added. The aqueous layer and chloroform were centrifuged, 82 and the resulting aqueous layer discarded, and the 2nd organic layer filtered into the test tube with the original organic layer. Samples were dried under nitrogen gas and for phospholipid separation by HPLC, and reconstituted in methanol:chloroform:hexane:acetone, 3:2:0.5:0.5 (v/v/v/v). For fatty acids analyzed in their total fraction, (17:0, heptadecanoic acid) was added prior to methylation as an internal standard. Betulin (50 ul of 1 mg/ml) was added as an internal standard prior to quantification by HPLC for phospholipid fatty acid analysis. Separation of phospholipid classes by high performance liquid chromatography (HPLC) Individual phospholipids (PE, PS, PC, PI) were separated using a HPLC. The sample chamber was kept at 18°C and the column heater at 35°C. A quaternary solvent system of hexane-petroleum ether, 97:3 (v,v); methanol-triethylamine-acetic acid, 765:15:13 (v/v/v); acetone-triethylamine-acetic acid, 765:15:13 (v/v/v);isopropanol-acetic acid, 800:40 (v/v) in a linear gradient with a flow rate of 2 ml/min was used. The column eluant was split 10:90 to an ELSD and a fraction collector. ELSD detection and quantitation of the separated lipid classes was performed with a nitrogen flow rate of 1.8 ml/min, a drift tube temperature of 60°C, and the impactor OFF. Calibration curves to determine the linear range of the analysis were established using authentic standards for each lipid class, and samples were quantified using the external standard method. Methylation of samples Methylation of fatty acids is carried out to hydrolyze complex lipids to free fatty acids and convert the carboxylic groups to methyl esters in preparation for quantification of individual fatty acids by GLC. This procedure neutralizes functional groups on fatty acids that would otherwise react with the GLC columns and creates compounds that are more volatile and more 83 easily analyzed by GLC. Phospholipid fractions were methylated with 1 ml of 14 % boron trifluoride in methanol, and total fatty acids were methylated with 0.7 ml 14 % boron trifluoride in methanol (Pierce Biotechnology, Rockford, IL, U.S.A.) at 110°C for 30 minutes. Methyl esters were recovered by two extractions with 3 ml saline and 6 ml pentane, and 3 ml saline, respectively. The pooled pentane layers were dried under nitrogen gas and resuspended in hexane for GLC analysis. Analysis of fatty acids by gas-liquid chromatography Phospholipid fatty acids and total fatty acids were separated and analyzed as their respective methyl esters by GLC. Separation was carried out with 30.3 m x 250 μm ID, 0.20 μm nominal, fused silica polar SP2380 columns (Supelco, Belefronte, PA, U.S.A.). Helium was used as the carrier gas, at a flow rate of 25 ml/min, inlet pressure of 18.6 pounds per square inch, and inlet splitter set at 10 to 1. Samples were injected at 80°C. The temperature program was as follows: samples remained at 80°C for 2 minutes, then increased to 165°C at 5°C/min, were held for 14 min, then increased to 180°C at 1.5°C/min, were held for 2.5 min, then increased to 240°C at 20°C/min and held for 20 min. The temperatures of the injectors and detectors were 240°C and 260°C, respectively. Retention times of fatty acid methyl esters were compared with those of authentic standards for identification. Fatty acids identified were 18:3, 20:5, 22:5, 22:6 from the n-3 series; 18:2, 18:3, 20:3, 20:4, 22:4, 22:5 from the n-6 series; 16:1, 18:1 from the n-7 series; 14:1, 16:1, 18:1, 20:1, 22:1, 24:1 from the n-9 series; 22:1 from n-11 series; and 8:0, 10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0, 24:0 of the saturated fatty acids. For computation of fatty acid peak areas, GC Chemstation software (Agilent Technologies) was used. 84 Appendix 2.4: Platelet Fatty Acid Analysis Methylation of platelet total fatty acids Platelet samples were thawed in cold water. A 0.1 ml aliquot of platelets and 10 ul of C17:0 internal standard was methylated with 2 ml benzene:methanol, 1:4 (v/v) following the method of Lepage and Roy (1), with slight modifications. Samples were vortexed, and 200 ul of acetyl chloride added before capping tubes and heating at 100°C for 60 minutes. During the incubation, tubes were vortexed every 15 minutes. Samples were then cooled at 4°C to prevent loss of medium chain fatty acid methyl esters, which are very volatile and subject to evaporation. Five ml of 6 % (weight/v) potassium carbonate solution was then added to the samples for neutralization, and the samples were briefly shaken. Two extractions with 6 ml pentane were carried out to extract methyl esters. The pooled pentane layers were dried under nitrogen gas and then resuspended in 200 μl hexane for analysis by GLC. Analysis of fatty acids by gas-liquid chromatography Fatty acids were separated and analyzed as their respective methyl esters by gas liquid chromatography, as described above for brain phospholipid and total fatty acid samples. 85 Appendix 2.5 Platelet Aggregation Studies Original method of blood collection for complete blood counts and platelet function tests Rats were anesthetized with isoflurane. Samples for CBC were taken by cardiac puncture (500 ul), using a syringe rinsed with EDTA, and put into an EDTA collection tube (supplied by hospital). Blood samples were taken to BC Children’s hospital (Vancouver, BC, Canada) for analysis. For platelet aggregation studies, blood was collected by cardiac puncture, using a 3 ml syringe, with 1” 20 G needle, both rinsed with 3.2% sodium citrate. Blood was put immediately into 2 ml 3.2% sodium citrate vaccutainer tubes. An aliquot of 800 ul of blood was added to each test cartridge, and tested in the PFA-100. The results from this initial attempt resulted in closure times of greater than 300 seconds (maximum time) with both the collagen/EPI and collagen/ADP cartridges for all rats tested. Revised method of blood collection for complete blood counts and platelet function tests Rats were anesthetized with isoflurane anesthesia. For complete blood counts, a 500 ul blood sample was taken by cardiac puncture, with 20 G needle and 1 ml syringe, rinsed with EDTA. The sample was collected into an EDTA tube and taken to hematology at BC Children’s Hospital (Vancouver, BC, Canada) for CBC analysis. For platelet aggregation studies, the method of Dunleavy et al. (3) was followed. Rats were injected intraperitoneally (IP) with heparin (1000 IU/kg). Blood was collected by cardiac puncture, using a 3 ml syringe, with a 1” 20 G needle, both rinsed with heparin (5000 IU/100 ml), and was immediately put into a 3ml heparin/lithium vacutainer tube. Platelet aggregation was assessed with 800 ul of blood, using a platelet function analyzer (PFA-100, Dade sciences), and collagen/ADP and collagen/EPI cartridges. 86 Appendix 2 Bibliography 1. Macrae IM. New models of focal cerebral ischemia. Br J clin, Pharmac. 1992;34:302-308. 2. Lepage G, Roy CC. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J Lipid Res. 1984 Dec 1;25(12):1391-6. 3. Dunleavy M, Dooley M, Cox D, Bradford A. Chronic intermittent asphyxia increases platelet reactivity in rats. Exp Physiol. 2005 May;90(3):411-6. 87
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Dietary n-3 fatty acids and cerebral ischemia/reperfusion
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