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Development of a Z-Stack Projection Imaging Protocol for a Nerve Allograft

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Development of a Z-Stack Projection Imaging Protocol for a Nerve Allograft DEVELOPMENT OF A Z-STACK PROJECTION IMAGING PROTOCOL FOR A NERVE ALLOGRAFT by SELVAANISH SELVAM Submitted in partial fulfillment of the requirements for the degree of Master of Science Dissertation Advisor: Dr. George F. Muschler Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY August 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Selvaanish Selvam candidate for the degree of (Master of Science) *. Committee Chair Dr. George F. Muschler Committee Member Dr. Eben Alsberg Committee Member Dr. Robert Kirsch Committee Member Cynthia Boehm Date of Defense May 4th 2018 *we also certify that written approval has been obtained For any proprietary material contained therein 2 Table of Contents List of Tables……………………………………………………………………..4 Figures List……………………………………………………………………….5 Acknowledgments………………………………………………………………..6 List of Abbreviations………………………………………………………….....7 Abstract…………………………………………………………………………...8 Introduction……………………………………………………………………..10 Methods………………………………………………………………………….21 Data and Analysis………………………………………………………………31 Conclusions and Future Directions……………………………………………51 References……………………………………………………………………….53 3 List of Tables Table 1: Overall summary of ratios and retention rates……………………..31 Table 2: Selection Ratio for grafts (3X – 10-minute rinse) ………………….35 Table 3: Selection Ratio for grafts (15-minute soak) ………………………...35 4 List of Figures: Figure 1: 2D 10X DAPI stained image using current techniques…….……….9 Figure 2: Peripheral Nerve Anatomy………………………………………….10 Figure 3: Nerve grade injury…………………………………………………..11 Figure 4: Different types of peripheral nerve repair…………….…………...13 Figure 5: Z-stack of images creating a Z-stack projection image……...……18 Figure 6: Jablonski diagram for Fluorescence molecules……………………19 Figure 7: Nerve allograft…..…………………………………………………...22 Figure 8: Nerve graft after rinse……………………………………………….22 Figure 9: Nerve graft with media at Day 0 of cell culture……………………23 Figure 10: Cell Culture at day 0………………..…………………………...…25 Figure 11: Cell Culture at day 3………………………………..……..……….25 Figure 12: C2 Graft……………………………………………..……...………39 Figure 13: E1 Graft………………………………………………………….….40 Figure 14: E2 Graft…………………………………………….………….……41 Figure 15: F2 Graft………………………………….…………….……………43 Figure 16: F3 Graft……………………………………………………………..44 Figure 17: Week 3 (G1, G2, and G3 graft)…………………………………....45 Figure 18: Z-stack projection image of D1 Graft…………………………….47 Figure 19: Z-stack projection image of D2 Graft…………………………….49 5 Acknowledgement I would like to thank Dr. George Muschler, Cynthia Boehm, Dr. Nicolas Piuzzi, Wes Bova, and Viviane Luangphakdy and Ratnam Mantripragada from the Cleveland clinic in helping me formulate, solve, and present this project. I also would like to thank Dr. Robert Kirsch, Dr. Eben Alsberg, Amrish Selvam, and the rest of Case Western Reserve University in helping me complete my project. 6 List of Abbreviations 2D: Two Dimensional BMA: Bone Marrow Aspirate BMC: Bone Marrow Aspirate Concentrate CTP: Connective Tissue Progenitor CTPs: Connective Tissue Progenitor cells DAPI: 4',6-diamidino-2-phenylindole stain FDA: Food and Drug Administration HBSS: Hank’s Balanced Salt Solution NGF: Nerve Growth Factor PBS: Phosphate-Buffered saline 7 Development of a Z-Stack Projection Imaging Protocol for a Nerve Allograft Abstract By SELVAANISH SELVAM Peripheral nerve injuries have traditionally been treated with a variety of different surgical procedures, but the use of allografts for these injuries remain to be a largely unexplored concept. As a result, there has yet to be a successful protocol created for imaging cells on the surface of nerve allografts. In this thesis, I developed a z-stack projection imaging protocol for nerve allografts and demonstrated the ability to take clear and focused images of cell retention and proliferation on the surface of the allograft. I also optimized the preparation of the nerve graft to improve cell and connective progenitor cell (CTP) retention rates. 8 Problem Statement and Purpose Bone Marrow Aspirate has been used in many graft applications and has shown significant clinical improvement. 1 However, when using a fluorescence light microscope to image the graft, limited focusing capabilities and the uneven topography of the graft make it difficult to obtain a clear image of the cells on the surface of the graft. Figure 1 shows an example of a graft using current 2D image techniques. The figure indicates cell attachment, but the image acquired by the present technique produces a blurry and inconclusive image of the graft. With such an unfocused image little to no analysis can be done to understand the number of cells retained and proliferating on the nerve graft. Figure 1. A 2D 10X image of a DAPI stained nerve graft. The few small white spots in the middle appear to be cells but cannot be confirmed due to the quality of the image. Large portions of the image remain distorted and increases the need for a better image acquisition protocol for grafts. The purpose of this experiment will be to resolve this imaging issue by developing and introducing a workable z-stack imaging protocol to have a clear view of cells on the top surface of the nerve graft. To test this imaging protocol, nerve allografts will be imaged. The preparation of this graft will also be optimized to improve overall cell and connective tissue progenitor (CTP) retention rates. 9 Introduction Nerves are a bundle of fibers that relay information from the brain and spinal cord to muscles and skin. They use electrical and chemical signals to send information quickly to different parts of the body to help establish motor and sensory functions. 2,3 Various injuries to the structure of nerves can cause detrimental damage to the patient and their quality of life. Before looking into the different classifications of these nerve injuries it is paramount to understand nerve anatomy. Each nerve fiber is composed of connecting axons and are protected by a layer of connective tissue known as the endoneurium. When multiple nerve fibers are combined together the bundle is known as a fascicle. The fascicle is encircled by a sheath of connective tissue called a perineurium and is known as the smallest structure capable of accepting sutures.4 Multiple fascicles and blood vessels combined together make up the peripheral nerve architecture. The peripheral nerve is surrounded by a loose outer sheath of blood vessels known as the epineurium. Figure 2 is a descriptive image that shows the previously described structures under one diagram . Figure 2. Schematic illustration of peripheral nerve anatomy and vital structures.5 10 Categorization of Nerve Injury When specific structures of the nerve anatomy are injured, various levels of nerve pathophysiology are affected. The most minimal form of peripheral nerve injury is neurapraxia which is defined as focal demyelination with no damage to the connective tissues or the axons. This can lead to muscle weakness and can be resolved within a few days after injury. The Figure 3. Image representing functioning nerve with no second level of nerve damage, Grade IV damaged nerve, and Grade V damaged nerve.6 injury is known as axonotmesis and can be classified under Grade II, Grade III, and Grade IV of the Sunderland nerve injury scale. 6 Axonotmesis is categorized as damage to axons along with focal demyelination. Grade II axonotmesis is classified with no damage to surrounding connective tissue and the endoneurium is kept intact. Grade III is observed when the endoneurium is damaged, but the perineurium is still kept intact. Grade IV classification extends damage to the perineurium, but the epineurium is unbroken. The most severe peripheral nerve injury category is Grade V or neurotmesis and involved complete transection of the nerve. In this level of injury, the perineurium is damaged and axons are fully disconnected.6–8 Grade IV and Grade V injuries require surgical techniques for recovery.4 Figure 3 shows a diagram of a proper nerve along with Grade 11 IV and Grade V nerve injuries. Large portions of the nerve are seen transected and muscle function is compromised. Natural Nerve Reinnervation Process Following a peripheral nerve injury several natural mechanisms try to reinnervate the proximal end of the injured nerve to the distal end. If injuries affect greater than 90% of the axons then axonal regeneration is the primary method for reinnervation.9 The three primary steps to achieve full recovery are: Wallerian degeneration, axonal regeneration, and end-organ reinnervation. 7 A phenomenon known as Wallerian degeneration occurs within the first week of injury and causes the breakdown of axonal cytoskeleton. In the distal stump, granular disintegration occurs after an influx of extracellular ions and results in a process resembling apoptosis. On the other hand, the proximal stump is also broken down but much more limited and breakdown typically only progresses to the first node of Ranvier on the proximal end. Once Wallerian degeneration is fully complete the environment is now conducive to nerve regeneration since myelin debris and existing damaged nerves have mostly been removed. Regeneration begins when a growth cone is formed on the distal tip of the proximal stump. Neurotropic factors and guidance molecules like Collapsin-1 help guide the growth cone towards the distal end of the injured nerve. Schwann cells are also critical in promoting suitable axonal regeneration as they display nerve growth factors (NGFs)
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