PHYSIOLOGIC CHANGES INDUCED BY INTRAVENOUS INFUSION OF KERATIN- DERIVED RESUSCITATION FLUIDS ON EUVOLEMIC AND HYPOVOLEMIC RATS By FIESKY A. NUÑEZ JR, MD A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY Physiology and Pharmacology May 2012 Winston-Salem, North Carolina ApProved by Thomas L. Smith, PhD, Advisor Mark Van Dyke, PhD, Chair George Christ, PhD Patricia Gallagher, PhD Ann Tallant, PhD DEDICATION To my wife: Alejandra, your infinite patience and understanding allowed me to mature as a Person and as a scientist. Without you I would not have accomplished this feat. I love you To my Parents, for your unconditional support that allowed this road to be filled with joy and success. To my sister: Mary, your wisdom and emotional suPPort gave me the strength and hope I needed in order to achieve many things. You always make me think of a better tomorrow and a better me. ii ACKNOWLEDGEMENTS Special Thanks to: Tom: For your impeccable mentorshiP and disPosition to always put my best interests first. Mark: For your dedication and Patience to teach such an impatient subject. Mike: For your knowledge and hard work in day-to-day lab efforts. Maria: Thank you for your patience in the lab and for sharing your immense knowledge of keratin with me. Keratin Krew and Biomaterials core: Roche, Bailey, Chris, Lauren, Mary, Julie, Jill, Carmen, DeePika. Your collaborations allow individual contributions to flourish into an extraordinary lab. OrthoaPedic Lab: Beth, Eileen, Martha, Jan, Casey. All of you make this workplace Pleasant and successful, I hoPe for years to come it remains this way because it’s my second home. Keranetics Personnel: Thank you for your assistance and support during our collaboration. iii TABLE OF CONTENTS LIST OF ILLUSTRATIONS AND TABLES……………………………………………………………… V LIST OF ABBREVIATIONS……………………………………………………………….………………… VII ABSTRACT……………………………………………………………………………..………………………….. IX CHAPTER I – Introduction…………………………………………………………………………………… 1 CHAPTER II – Vasoactive Properties of Keratin-Derived Compounds……………..…… 28 CHAPTER III – Hemodynamic Recovery After HyPovolemic Shock and Resuscitation with Lactated Ringer’s and Keratoe Resuscitation Fluid; A Novel Colloid………………………………………………………………………….… 58 CHAPTER IV – Conclusions……………………………………………………………………………… 93 APPENDICES…....................................................................................................................................... 104 SCHOLASTIC VITA………………………………………………………………………………………… 112 iv LIST OF ILLUSTRATIONS AND TABLES CHAPTER I - Introduction Figure 1 – Renin-Angiotensin-Aldosterone system………….…………………………………... 6 Figure 2 – Shear stress-mediated release of NO from the endothelium………….…..…8 Table 1 – Safety tests conducted on a Keratose biomaterial…………………..…………… 16 CHAPTER II - Vasoactive Properties of Keratin-Derived Compounds Figure 1 - Size eXclusion chromatograPhy of keratose samples………………………… 52 Figure 2 - Video stills of the same vessel before and after KRF administration……53 Figure 3 - Change in arteriolar diameter with i.v. administration of Fluid…………… 54 Figure 4 - Effect on arteriolar diameter with and without L-nitro-arginine…………. 55 Figure 5 - Average arteriolar diameter increase with KRF of different viscosities……………………………….……………………………………… 56 Figure 6 - Average arteriolar diameter increase with infusion of AlPha and KAP containing fluids………………………………….…… 57 CHAPTER III - Hemodynamic Recovery After HyPovolemic Shock and Resuscitation with Lactated Ringer’s and Keratoe Resuscitation Fluid; A Novel Colloid Figure 1 – Hemodynamic changes after hyPovolemic shock and treatment with low-volume resuscitation fluids ………………… 83 Figure 2 - Circulating blood volume changes after hyPovolemic shock and v treatment with low-volume resuscitation fluids........................................ 84 Figure 3 – Hemodynamic changes after hyPovolemic shock and treatment with full-volume resuscitation fluids……………………...…… 85 Figure 4 - Circulating blood volume changes after hyPovolemic shock and treatment with full-volume resuscitation fluids......................................86 Figure 5 – Tissue Histology………………………………………………………………………......… 87 Supplemental Figure 1 - Ultrasound Transit Flow Probes................................................ 88 Supplemental Figure 2 - Mean Arterial Pressure and Heart Rate Monitoring…….89 Supplemental Table 1 - Hemodynamics and Arterial Blood Gases: Low Volume Resuscitation, Short Term Assessment……………………….… 90 Supplemental Table 2 - Hemodynamics and Arterial Blood Gases: Full Volume Resuscitation, Short Term Assessment………………………..… 91 Supplemental Table 3 - Hemodynamics, Arterial Blood Gases and Electrolytes: Long Term Assessment………………………………………………………………………. 92 CHAPTER IV - CONCLUSIONS Figure 1 - Blood Pressure Recovery After HyPovolemic Shock and Treatment with KIF-KRF and AK-KRF…………………………………..…… 98 APPENDICES Table 1 - Different Fractions of Keratose: vi Classification by components, their respective biologic activity and potential clinical uses……………….. 107 Figure 1 - Concentration of Keratose needed to Prepare a compound with a viscosity of 3.88cP at 10dynes/cm2........................................................ 109 vii LIST OF ABBREVIATIONS Abs Absorbance ACV Actively circulating volume ANOVA Analysis of variance CBV Central blood volume CI Cardiac Index CO Cardiac outPut cP Centipoise DI Deionized Hb Hemoglobin HCT Hematocrit HEPES Hydroxyethyl- piperazine ethanesulfonic acid HR Heart rate i.v. or IV Intravenous IEx Ion exchange KAP Keratin-associated protein KDa Kilo Dalton KIF Keratose Intermediate Filament KRF Keratin resuscitation fluid l-NA l-nitroarginine MAP Mean Arterial Pressure viii mmHg Millimeters of mercury NLMWCO Nominal low molecular weight cutoff NO Nitric oxide PBS Phosphate buffered saline Phe Phenylephrine SEC Size eXclusion chromatograPhy SNP Sodium NitroPrusside ULF Unit length filament. ix ABSTRACT Fiesky A. Nuñez Jr. MD PHYSIOLOGIC CHANGES INDUCED BY INTRAVENOUS INFUSION OF KERATIN- DERIVED RESUSCITATION FLUIDS ON EUVOLEMIC AND HYPOVOLEMIC RATS Research Problem: The field of cardiovascular research has evolved significantly over the last decade, and yet cardiovascular related deaths remain a Primary cause of trauma related death. Shock and resuscitation are toPics of high importance in the field of emergency medicine due to the high mortality that they carry, which has not been ProPerly addressed in the Past decades. The develoPment of better treatment oPtions for this entity is essential. Methods: 1) The vasoactive properties of intravenously infused keratin- derived compounds were assessed, with the use of cremaster muscle microvascular observations in the Sprague-Dawley rat. 2) After assessing feasibility for intravenous infusion in the microvascular model, hemodynamic and metabolic recovery after hemorrhage and resuscitation with commercially available crystalloids and keratin compounds was studied. Ultrasound dilution method was used to determine changes in cardiac indeX, stroke volume and circulating volumes. Femoral cannulation was used to determine changes in arterial blood pressure and heart rate. Arterial blood gases were measured to compare acid-base profile changes. Conclusions: The initial keratin compounds tested in this research elicited an uneXPected vasodilatory effect on the microvasculature of the cremaster muscle, which can be further developed for other uses. Further purification allowed the formulation of keratin compounds without vasodilatory effects, improved osmotic effects, and enhanced viscosity. These compounds are biocompatible colloids for resuscitation PurPoses. These enhancements will allow the use of this compound with lower volumes than the current gold standard and significantly reduce complications. X CHAPTER I INTRODUCTION Fiesky A. Nunez Jr. MD 1 Clinical Significance Traumatic injuries account for 9% of annual global mortality and it is estimated that half of these deaths occur in individuals between 15 and 44 years of age (1). Hemorrhagic shock is the leading cause of death among trauma Patients worldwide. 50% of Patients that suffer bleeding severe enough to cause shock will die despite aggressive and Prompt treatment (2). Although not reliable as an isolated measure of disease severity, a systolic blood Pressure below 100mmHg at emergency dePartment admission increases hosPital mortality threefold and sudden death risk tenfold (3, 4). However, continuous research has proven that the only factor that decreases mortality is early diagnosis and Prompt restoration of volume and cardiac function (5-7). Shock is a state of severe impairment in tissue Perfusion that results in diminished tissue oxygen delivery and utilization as well as impaired removal of metabolic waste byproducts. Untreated, shock triggers a chain of pathologic events that result in localized cellular death followed by multiPle organ dysfunction and death of the individual. The Primary treatment for all tyPes of shock remains ProPer fluid resuscitation and specific efforts to correct the factors that Precipitate shock. Etiology of Shock Shock is defined as the incaPacity of the cardiovascular system to maintain ProPer tissue perfusion (8, 9). Causes of shock are variable and are grouped into four sPecific categories according to the Pathologic mechanisms involved: 2 1. Cardiogenic Shock: IncaPacity of the heart to maintain ProPer cardiac outPut due to