CHARACTERIZING THE MELATONERGIC SYSTEM AFTER BRAIN INJURY by Nicole Danielle Osier Bachelor of Science (B.S.) in Nutritional Science, Michigan State University, 2008 Bachelor of Science in Nursing (B.S.N.), Michigan State University, 2008 Submitted to the Graduate Faculty of School of Nursing in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Nursing University of Pittsburgh 2016 UNIVERSITY OF PITTSBURGH SCHOOL OF NURSING This dissertation was presented by Nicole Danielle Osier It was defended on July 12, 2016 and approved by C. Edward Dixon, PhD, Professor, School of Medicine Yvette Conley, PhD, Professor, School of Nursing Dianxu Ren, MD, PhD, Associate Professor, School of Nursing Ava Puccio, PhD, Assistant Professor, School of Medicine Dissertation Advisor: Sheila Alexander, PhD, Associate Professor, School of Nursing ii Copyright © by Nicole Danielle Osier 2016 iii CHARACTERIZING THE MELATONERGIC SYSTEM AFTER BRAIN INJURY Nicole Danielle Osier, PhD, BSN, BS, RN University of Pittsburgh, 2016 Traumatic brain injury (TBI) affects individuals of all ages, races, and geographies and is often associated with expensive clinical management. Melatonin (MEL) has been trialed as a TBI therapeutic with mixed results, due in part to a lack of understanding of the mechanism. This study characterizes endogenous changes in melatonergic receptors (MT1 and MT2) after experimental TBI induced using pneumatic CCI (2.8 mm depth) in young adult Sprague Dawley rats. Half the rats were exposed to sham surgery to control for the effects of anesthesia and craniectomy. In total, 25 rats were enrolled in the study, with 6-7 rats per group. Test animals were sacrificed at 2 post- surgery time points, either 6 hours (hr) or 24 hr post-surgery. Following sacrifice, the test animal’s brain was harvested, dissected, and flash frozen until analysis. Whole cell lysates were prepared, aliquoted, and used for western blot analysis, probing for cytochrome C (to validate injury severity), MT1, MT2, and beta actin (to control for protein loading). ImageJ and Image Lab software were used to quantify protein data; SPSS software was used to run t-tests to compare group means on a single variable and correlation testing was used to explore the relationship between outcomes of interest. Melatonin receptors were down-regulated in a brain region- and time point- dependent manner. MT1 was downregulated in the frontal cortex at 24 hr and in the hippocampus at both 6 hr and 24 hr post-TBI. Similarly, MT2 was downregulated in the frontal cortex at 24 hr and in the hippocampus at both 6 and 24 hr post-TBI. This is the first study to report downregulation of MT1 and MT2 after neurotrauma; receptor downregulation may affect the efficacy of MEL therapy. Additional research to characterize these changes after TBI are necessary iv including efforts to establish the time course and regional patterns, replication in more diverse samples, as well as inclusion of additional cellular, histological, and behavioral endpoints. TBI in rats modeled using CCI results in acute downregulation of MEL-specific receptors (subtypes MT1 and MT2); replication of these findings is necessary as are evaluations of the implications of lower receptor levels. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIX 1.0 INTRODUCTION TO THE STUDY AS ORIGINALLY PLANNED ................. 23 1.1 PURPOSE AND SPECIFIC AIMS .................................................................. 27 1.2 BACKGROUND AND SIGNIFICANCE ........................................................ 28 Rationale for studying traumatic brain injury ........................................... 31 Rationale for using a pre-clinical (animal) model of brain injury ............ 32 Rationale for using a molecular genetic approach ..................................... 33 Rationale for examining the melatonergic system ...................................... 34 Rationale for examining apoptosis as a cellular level outcome ................. 35 Rationale for functional outcomes: learning, memory, and motor ........... 36 Conceptual framework.................................................................................. 37 Innovation....................................................................................................... 40 Summary ........................................................................................................ 41 1.3 PRELIMINARY STUDIES .............................................................................. 42 Establishing the apoptotic consequences of the model ............................... 44 Evaluating endogenous levels of MT1 in the model ................................... 45 Establishing behavioral consequences of the model ................................... 46 Effects of exogenous MEL on behavior after CCI ...................................... 49 vi 1.4 RESEARCH DESIGN AND METHODS ........................................................ 51 Animal subjects .............................................................................................. 51 Overview of study design .............................................................................. 52 Sample............................................................................................................. 54 Injury induction ............................................................................................. 56 1.4.4.1 Controlled cortical impact model (CCI) ........................................... 56 1.4.4.2 Specific injury parameters ................................................................. 58 Melatonin therapy ......................................................................................... 58 Measurement of apoptotic proteins ............................................................. 59 Measurement of functional deficits .............................................................. 60 1.4.7.1 Morris water maze (MWM) ............................................................... 60 1.4.7.2 Beam balance task ............................................................................... 65 1.4.7.3 Novel object recognition ..................................................................... 66 Analysis ........................................................................................................... 67 1.4.8.1 Sample size justification ..................................................................... 67 1.4.8.2 Preliminary data analysis ................................................................... 67 1.4.8.3 Aim 1 analysis ...................................................................................... 68 1.4.8.4 Aim 2 analysis ...................................................................................... 68 1.4.8.5 Expected findings and interpretation ................................................ 70 1.5 LIMITATIONS AND PROPOSED ALTERNATIVES ................................. 70 1.6 HAZARDOUS MATERIALS AND PROCEDURES .................................... 72 1.7 RESEARCH SUBJECT RISKS, BENEFITS, AND PROTECTIONS ........ 72 Potential risks and protection against risks ................................................ 73 vii Potential benefits............................................................................................ 74 Data safety and monitoring plan .................................................................. 74 2.0 SUMMARY OF DISSERTATION STUDY ............................................................ 75 2.1 PROPOSAL CHANGES ................................................................................... 78 Animals that were lost or excluded from the pilot sample ........................ 79 Concerns raised during analysis of full 8-group pilot sample ................... 80 Comparison of vehicle-treated animals to establish injury effect ............. 82 2.1.3.1 Beam balance task ............................................................................... 83 2.1.3.2 Novel object recognition (NOR) ......................................................... 84 2.1.3.3 Morris water maze (MWM) ............................................................... 86 Possible sources of error and other issues ................................................... 89 Alternative CCI model trialed ...................................................................... 91 Delayed breeding of knockout mice ............................................................. 92 Changes made to study plan ......................................................................... 93 2.1.7.1 Refinement of variables ...................................................................... 94 2.1.7.2 Replacement of animal model ............................................................ 94 2.1.7.3 Modification of brain injury model ................................................... 95 2.1.7.4 Revised sample and groups ................................................................ 95 2.1.7.5 Revised specific aims ........................................................................... 96 2.1.7.6 Revised methods .................................................................................. 96 2.1.7.7 Revised analysis ................................................................................... 97 Summary of study after modifications ........................................................ 97 Discussion