Browsing by Subject "Polyethylene glycol"
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Item cAMP and oxidative mechanisms of plasmalemmal sealing and the effects on rapid and long lasting repair of severed axons in vivo by polyethylene Glycol(2011-05) Spaeth, Christopher Scott; Bittner, George D.; Zakon, Harold; Ben-Yakar, Adela; Morgan, Jennifer; Dalby, KevinTraumatic neuronal injury inevitably causes plasmalemmal damage, and sometimes leads to axonal severance. For any eukaryotic cell to survive following traumatic injury, the plasmalemma must be repaired (sealed). Plasmalemmal sealing occurs via a Ca²⁺-dependent accumulation of vesicles or other membranous structures that form a plug at the damage site. Using uniquely identified and damaged rat hippocampal B104 cells that extend neurites with axonal properties, or rat sciatic nerves, plasmalemmal sealing is assessed by exclusion of an extracellular dye from each damaged B104 cell, or sciatic nerves ex vivo. B104 cells with neurites transected nearer (<50 [micrometres]) to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther (>50 [micrometres]) from the soma. Sealing in B104 cells is enhanced by 1) increased [cAMP], 2) increased PKA activity, 3) increased Epac activity, 4) H₂O₂ and 5) Poly-ethylene glycol (PEG). Sealing is decreased by 1) PKA inhibition, 2), Botulinum toxins A, B, E, 3) Tetanus toxin 4), NEM, 5) Brefeldin A, 6) nPKC inhibition, 7) DTT, 8) Melatonin and 9) Methylene Blue. Substances (NEM, Bref A, PKI, db-cAMP, PEG) that affect plasmalemmal sealing in B104 cells in vitro have similar effects on plasmalemmal sealing in rat sciatic nerves ex vivo. Based on data from co-application of enhancers and inhibitors of sealing, I propose a plasmalemmal sealing model having four partly redundant, parallel pathways mediated by 1) PKA, 2) Epac, 3) cytosolic oxidation and 4) nPKCs. The identification and confirmation of these pathways may provide novel clinical targets for repairing and/or recovery from traumatic injury. The fusogenic compound PEG rapidly repairs axonal continuity of severed axons, potentially by rejoining severed proximal and distal axons. PEG-fusion is influenced by plasmalemmal sealing, since unsealed axons are easier to PEG fuse. I demonstrate that PEG restores morphological continuity, and improves behavioral recovery following crush-severance to sciatic nerves in rats in vivo. Co-application of Mel or MB prior to PEG application further improves PEG fusion (as measured by electrophysiology) and behavioral recovery following crush-severance in vivo. These PEG data may provide novel clinical techniques for rapidly repairing axonal severance.Item Immunosuppressive effects of PEG-fusion in peripheral nerve allografts(2021-03-08) Smith, Tyler Aaron; Bittner, George D.; Tucker, Haley O.; Sakiyama-Elbert, Shelly E.; Zakon, Harold; Poenie, MartinSurgical repair of ablation-type peripheral nerve injuries by using peripheral nerve allografts (PNAs) has been hindered for decades due to slow and ineffective axon regeneration from proximal nerve ends as well as immunological rejection of PNAs. We have developed a polyethylene glycol (PEG)-fusion repair protocol for sciatic PNAs in rats that results in maintenance of myelinated axons that do not degenerate, neuromuscular junction innervation, and significantly improved behavioral recovery, as compared to current nerve repair methods. These phenotypes are maintained for weeks postoperatively without using tissue-matching, decellularization, or immunosuppressive drugs. That is, PEG-fused PNAs are functionally tolerated by the host immune system. This dissertation presents work performed to characterize and investigate mechanisms underlying immunological responses to PNAs treated with PEG-fusion with the aim of understanding how and why PEG-fused allografts are not rejected by the host immune system in rat sciatic nerve injury models. Chapter 1 provides a review of the clinical significance of peripheral nerve injury, including: Biological processes of axonal degeneration and regeneration, current methods of nerve repair, immunological rejection of PNAs, concepts underlying PEG-fusion repair and current experimental results of PEG-fusion in rat PNAs, as well as the hypotheses and aims explored. Chapter 2 characterizes innate and adaptive immune responses to PEG-fused PNAs using primarily immunohistochemistry, electron microscopy, and quantitative reverse transcription PCR (RT-qPCR). Our results suggest that PEG-fused PNAs achieve immunotolerance via attenuated innate and adaptive immune responses. Chapter 3 examines via RNA sequencing the coding transcriptome of PEG-fused PNAs to determine which biological processes, protein families, pathways, and protein-protein interaction networks differentiate PEG-fused PNAs from negative control PNAs not treated with PEG with the goal of identifying potential mechanisms underlying immunotolerance. This work provides a critical molecular foundation for future studies investigating PEG-fusion-mediated immunosuppression in PNAs. Chapter 4 investigates whether treatment of PNAs with PEG alone without axonal fusion induces similar immunosuppressive effects. Our results suggest that PEG treatment alone does not prevent Wallerian degeneration or attenuation of innate and adaptive immune responses. Chapter 5 provides a summary of the dissertation work and describes future directions for research.Item Polyethylene glycol fusion repair of peripheral nerve injuries(2021-07-26) Ghergherehchi, Cameron Lee; Bittner, George D.; Atkinson, Nigel; Tucker, Haley; Sakiyama-Elbert, ShellyPeripheral nerve injury results in debilitating physical impairments, including loss of motor, sensory, and autonomic functions, which are often permanent. The greatest advancement to clinical repair of traumatic peripheral nerve injury came in the 19th century with the implementation of microsurgical suturing to re-approximate severed nerve ends. However, even upon surgical repair, the distal nerve segment undergoes degeneration and therefore requires the slow regeneration of proximal axons (at a rate of 1-2mm per day) for restoration of functions. This results in slow and often unsatisfactory recovery. To overcome these obstacles, we have developed a method of nerve repair utilizing the fusogen polyethylene glycol (PEG) to fuse severed axons and rapidly restore lost functions. The work within this dissertation characterizes the mechanisms underlying PEG-fusion restoration of function for injuries to peripheral nerves. Chapter 1 provides background on the events that occur after nerve injury, current clinical strategies aimed at improving nerve function after injury, and the development of the PEG-fusion procedure from previously published work. Chapter 2 examines the functional and morphological changes that occur after PEG-fusion of transected peripheral nerves, extending our mechanistic understanding of PEG-fusion mediated repair. Here, we found PEG-fusion restores axonal continuity across the lesion site, Wallerian degeneration is prevented for many distal axons, and muscle innervation is maintained. Chapter 3 examines alterations in motoneuron morphology and circuity after PEG-fusion repair of peripheral nerves. PEG-fusion repair results in no changes to motoneuron soma size or number but does alter dendritic arborizations. Surprisingly, PEG-fusion results in the mismatch of proximal axons and distal muscle and sensory targets yet results in significant functional recovery. Chapter 4 highlights studies to translate our findings to the clinical setting. Here we have optimized the PEG-fusion procedure using a series of FDA approved solutions and precise surgical resection of axons after initial severance, leading to increased functional recovery and easy adaptation of methods for clinical trials. Chapter 5 provides a summary of the dissertation and future directions of the research, including the adaptation of PEG-fusion technology for vascularized composite allotransplantation and spinal cord injury.Item Rapid repair of severed mammalian axons via polyethylene glycol-mediated cell fusion(2014-05) Britt, Joshua Martin; Schallert, TimothyThe ability to repair damaged mammalian axons to re-establish functional connections continues to be a goal for neuroscientists. Following axonal severance, proximal segments of mammalian axons seal themselves rapidly at the lesion site. Distal segments of severed mammalian axons undergo Wallerian degeneration within 24-72 hours. Prior to the onset of degeneration, distal axonal segments remain electrically excitable. The work described in this dissertation demonstrates that polyethylene glycol (PEG), a hydrophilic polymer, can rapidly repair severed axons by fusing the plasmalemmas of two closely apposed distal and proximal axonal segments. This plasmalemmal fusion restores morphological integrity of severed axons and their ability to conduct action potentials across the injury site. The ability to fuse proximal and distal severed axonal segments using PEG is improved when the axonal segments are exposed to antioxidants, such as melatonin and methylene blue, and also when microsutures provide additional support in transected sciatic nerves. The restoration of axonal continuity by PEG-fusion restores function, improving behavioral recovery in rats with crush-injured sciatic nerves, as well as those in which the sciatic is complete transected.