Typical and atypical properties of peripheral nerve allografts enable novel strategies to repair segmental-loss injuries.

We review data showing that peripheral nerve injuries (PNIs) that involve the loss of a nerve segment are the most common type of traumatic injury to nervous systems. Segmental-loss PNIs have a poor prognosis compared to other injuries, especially when one or more mixed motor/sensory nerves are involved and are typically the major source of disability associated with extremities that have sustained other injuries. Relatively little progress has been made, since the treatment of segmental loss PNIs with cable autografts that are currently the gold standard for repair has slow and incomplete (often non-existent) functional recovery. Viable peripheral nerve allografts (PNAs) to repair segmental-loss PNIs have not been experimentally or clinically useful due to their immunological rejection, Wallerian degeneration (WD) of anucleate donor graft and distal host axons, and slow regeneration of host axons, leading to delayed re-innervation and producing atrophy or degeneration of distal target tissues. However, two significant advances have recently been made using viable PNAs to repair segmental-loss PNIs: (1) hydrogel release of Treg cells that reduce the immunological response and (2) PEG-fusion of donor PNAs that reduce the immune response, reduce and/or suppress much WD, immediately restore axonal conduction across the donor graft and re-innervate many target tissues, and restore much voluntary behavioral functions within weeks, sometimes to levels approaching that of uninjured nerves. We review the rather sparse cellular/biochemical data for rejection of conventional PNAs and their acceptance following Treg hydrogel and PEG-fusion of PNAs, as well as cellular and systemic data for their acceptance and remarkable behavioral recovery in the absence of tissue matching or immune suppression. We also review typical and atypical characteristics of PNAs compared with other types of tissue or organ allografts, problems and potential solutions for PNA use and storage, clinical implications and commercial availability of PNAs, and future possibilities for PNAs to repair segmental-loss PNIs.

Coding transcriptome analyses reveal altered functions underlying immunotolerance of PEG-fused rat sciatic nerve allografts.

BACKGROUND: Current methods to repair ablation-type peripheral nerve injuries (PNIs) using peripheral nerve allografts (PNAs) often result in poor functional recovery due to immunological rejection as well as to slow and inaccurate outgrowth of regenerating axonal sprouts. In contrast, ablation-type PNIs repaired by PNAs, using a multistep protocol in which one step employs the membrane fusogen polyethylene glycol (PEG), permanently restore sciatic-mediated behaviors within weeks. Axons and cells within PEG-fused PNAs remain viable, even though outbred host and donor tissues are neither immunosuppressed nor tissue matched. PEG-fused PNAs exhibit significantly reduced T cell and macrophage infiltration, expression of major histocompatibility complex I/II and consistently low apoptosis. In this study, we analyzed the coding transcriptome of PEG-fused PNAs to examine possible mechanisms underlying immunosuppression. METHODS: Ablation-type sciatic PNIs in adult Sprague-Dawley rats were repaired using PNAs and a PEG-fusion protocol combined with neurorrhaphy. Electrophysiological and behavioral tests confirmed successful PEG-fusion of PNAs. RNA sequencing analyzed differential expression profiles of protein-coding genes between PEG-fused PNAs and negative control PNAs (not treated with PEG) at 14 days PO, along with unoperated control nerves. Sequencing results were validated by quantitative reverse transcription PCR (RT-qPCR), and in some cases, immunohistochemistry. RESULTS: PEG-fused PNAs display significant downregulation of many gene transcripts associated with innate and adaptive allorejection responses. Schwann cell-associated transcripts are often upregulated, and cellular processes such as extracellular matrix remodeling and cell/tissue development are particularly enriched. Transcripts encoding several potentially immunosuppressive proteins (e.g., thrombospondins 1 and 2) also are upregulated in PEG-fused PNAs. CONCLUSIONS: This study is the first to characterize the coding transcriptome of PEG-fused PNAs and to identify possible links between alterations of the extracellular matrix and suppression of the allorejection response. The results establish an initial molecular basis to understand mechanisms underlying PEG-mediated immunosuppression.

Polyethylene glycol-fusion repair of sciatic allografts in female rats achieves immunotolerance via attenuated innate and adaptive responses.

Ablation/segmental loss peripheral nerve injuries (PNIs) exhibit poor functional recovery due to slow and inaccurate outgrowth of regenerating axons. Viable peripheral nerve allografts (PNAs) as growth-guide conduits are immunologically rejected and all anucleated donor/host axonal segments undergo Wallerian degeneration. In contrast, we report that ablation-type sciatic PNIs repaired by neurorrhaphy of viable sciatic PNAs and a polyethylene glycol (PEG)-fusion protocol using PEG immediately restored axonal continuity for many axons, reinnervated/maintained their neuromuscular junctions, and prevented much Wallerian degeneration. PEG-fused PNAs permanently restored many sciatic-mediated behaviors within 2-6 weeks. PEG-fused PNAs were not rejected even though host/donors were neither immunosuppressed nor tissue-matched in outbred female Sprague Dawley rats. Innate and adaptive immune responses to PEG-fused sciatic PNAs were analyzed using electron microscopy, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction for morphological features, T cell and macrophage infiltration, major histocompatibility complex (MHC) expression, apoptosis, expression of cytokines, chemokines, and cytotoxic effectors. PEG-fused PNAs exhibited attenuated innate and adaptive immune responses by 14-21 days postoperatively, as evidenced by (a) many axons and cells remaining viable, (b) significantly reduced infiltration of cytotoxic and total T cells and macrophages, (c) significantly reduced expression of inflammatory cytokines, chemokines, and MHC proteins, (d) consistently low apoptotic response. Morphologically and/or biochemically, PEG-fused sciatic PNAs often resembled sciatic autografts or intact sciatic nerves. In brief, PEG-fused PNAs are an unstudied, perhaps unique, example of immune tolerance of viable allograft tissue in a nonimmune-privileged environment and could greatly improve the clinical outcomes for PNIs relative to current protocols.

Natural mechanisms and artificial PEG-induced mechanism that repair traumatic damage to the plasmalemma in eukaryotes.

Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.

Ca2+/calmodulin-dependent protein kinase II and Dimethyl Sulfoxide affect the sealing frequencies of transected hippocampal neurons.

Traumatic injury often results in axonal severance, initiating obligatory Wallerian degeneration of distal segments, whereas proximal segments often survive. Calcium ion (Ca2+ ) influx at severed proximal axonal ends activates pathways that can induce apoptosis. However, this same Ca2+ -influx also activates multiple parallel pathways that seal the plasmalemma by inducing accumulation and fusion of vesicles at the lesion site that reduce Ca2+ -influx and enhance survival. We examined whether various inhibitors of Ca2+ /calmodulin-dependent protein kinases (CaMKs), and/or dimethyl sulfoxide (DMSO), a common solvent for biologically active substances, affected the ability of a hippocampal-derived neuronal cell line (B104 cells) to seal membrane damage following axotomy. Axolemmal sealing frequencies were assessed at different transection distances from the axon hillock and at various times after Ca2+ -influx (PC times) by observing whether transected cells took-up fluorescent dyes. Inhibition of CaMKII by tatCN21 and KN-93, but not inhibition of CaMKI and CaMKIV by STO-609, affected axonal sealing frequencies. That is, CaMKII is a component of previously reported parallel pathways that induce membrane sealing, whereas CaMKI and CaMKIV are not involved. The effects of these CaMKII inhibitors on plasmalemmal sealing depended on their mechanism of inhibition, transection distance, and PC time. DMSO at low concentrations (90 µM-28 mM or 0.00064%-0.2% v/v) significantly increased membrane-sealing frequencies at most PC times and transection distances, possibly by permeabilizing the plasmalemma to Ca2+ . Inhibition of CaMKII, DMSO, PC time, and the transection distance significantly affect plasmalemmal sealing that is critical to somal survival in traumatic lesions.

Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats.

Many publications report that ablations of segments of peripheral nerves produce the following unfortunate results: (1) Immediate loss of sensory signaling and motor control; (2) rapid Wallerian degeneration of severed distal axons within days; (3) muscle atrophy within weeks; (4) poor behavioral (functional) recovery after many months, if ever, by slowly-regenerating (∼1mm/d) axon outgrowths from surviving proximal nerve stumps; and (5) Nerve allografts to repair gap injuries are rejected, often even if tissue matched and immunosuppressed. In contrast, using a female rat sciatic nerve model system, we report that neurorrhaphy of allografts plus a well-specified-sequence of solutions (one containing polyethylene glycol: PEG) successfully addresses each of these problems by: (a) Reestablishing axonal continuity/signaling within minutes by nonspecific ally PEG-fusing (connecting) severed motor and sensory axons across each anastomosis; (b) preventing Wallerian degeneration by maintaining many distal segments of inappropriately-reconnected, PEG-fused axons that continuously activate nerve-muscle junctions; (c) maintaining innervation of muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (d) inducing remarkable behavioral recovery to near-unoperated levels within days to weeks, almost certainly by CNS and PNS plasticities well-beyond what most neuroscientists currently imagine; and (e) preventing rejection of PEG-fused donor nerve allografts with no tissue matching or immunosuppression. Similar behavioral results are produced by PEG-fused autografts. All results for Negative Control allografts agree with current neuroscience data 1-5 given above. Hence, PEG-fusion of allografts for repair of ablated peripheral nerve segments expand on previous observations in single-cut injuries, provoke reconsideration of some current neuroscience dogma, and further extend the potential of PEG-fusion in clinical practice.

Polyethylene glycol solutions rapidly restore and maintain axonal continuity, neuromuscular structures, and behaviors lost after sciatic nerve transections in female rats.

Complete severance of major peripheral mixed sensory-motor nerve proximally in a mammalian limb produces immediate loss of action potential conduction and voluntary behaviors mediated by the severed distal axonal segments. These severed distal segments undergo Wallerian degeneration within days. Denervated muscles atrophy within weeks. Slowly regenerating (∼1 mm/day) outgrowths from surviving proximal stumps that often nonspecifically reinnervate denervated targets produce poor, if any, restoration of lost voluntary behaviors. In contrast, in this study using completely transected female rat sciatic axons as a model system, we provide extensive morphometric, immunohistochemical, electrophysiological, and behavioral data to show that these adverse outcomes are avoided by microsuturing closely apposed axonal cut ends (neurorrhaphy) and applying a sequence of well-specified solutions, one of which contains polyethylene glycol (PEG). This "PEG-fusion" procedure within minutes reestablishes axoplasmic and axolemmal continuity and signaling by nonspecifically fusing (connecting) closely apposed open ends of severed motor and/or sensory axons at the lesion site. These PEG-fused axons continue to conduct action potentials and generate muscle action potentials and muscle twitches for months and do not undergo Wallerian degeneration. Continuously innervated muscle fibers undergo much less atrophy compared with denervated muscle fibers. Dramatic behavioral recovery to near-unoperated levels occurs within days to weeks, almost certainly by activating many central nervous system and peripheral nervous system synaptic and other plasticities, some perhaps to a greater extent than most neuroscientists would expect. Negative control transections in which neurorrhaphy and all solutions except the PEG-containing solution are applied produce none of these remarkably fortuitous outcomes observed for PEG-fusion.

Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves.

Complete crush or cut severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons is restored only after months, if ever, by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1-3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca(2+) -free saline, methylene blue, PEG in distilled water, and finally Ca(2+) -containing isotonic saline. This study examines factors that affect the PEG fusion restoration of axonal continuity within minutes, as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut or crush severed in the midthigh. Also examined are factors that affect the longer-term PEG fusion restoration of lost behavioral functions within days to weeks, as measured by the sciatic functional index. We report that exposure of cut-severed axonal ends to Ca(2+) -containing saline prior to PEG fusion and stretch/tension of proximal or distal axonal segments of cut-severed axons decrease PEG fusion success. Conversely, trimming cut-severed ends in Ca(2+) -free saline just prior to PEG fusion increases PEG fusion success. PEG fusion prevents or retards the Wallerian degeneration of cut-severed axons, as assessed by measures of axon diameter and G ratio. PEG fusion may produce a paradigm shift in the treatment of peripheral nerve injuries. © 2016 Wiley Periodicals, Inc.

Estrogenic chemicals often leach from BPA-free plastic products that are replacements for BPA-containing polycarbonate products.

BACKGROUND: Xenobiotic chemicals with estrogenic activity (EA), such as bisphenol A (BPA), have been reported to have potential adverse health effects in mammals, including humans, especially in fetal and infant stages. Concerns about safety have caused many manufacturers to use alternatives to polycarbonate (PC) resins to make hard and clear, reusable, plastic products that do not leach BPA. However, no study has focused on whether such BPA-free PC-replacement products, chosen for their perceived higher safety, especially for babies, also release other chemicals that have EA. METHODS: We used two, well-established, mammalian cell-based, assays (MCF-7 and BG1Luc) to assess the EA of chemicals that leached into over 1000 saline or ethanol extracts of 50 unstressed or stressed (autoclaving, microwaving, and UV radiation) BPA-free PC-replacement products. An EA antagonist, ICI 182,780, was used to confirm that agonist activity in leachates was due to chemicals that activated the mammalian estrogen receptor. RESULTS: Many unstressed and stressed, PC-replacement-products made from acrylic, polystyrene, polyethersulfone, and Tritan™ resins leached chemicals with EA, including products made for use by babies. Exposure to various forms of UV radiation often increased the leaching of chemicals with EA. In contrast, some BPA-free PC-replacement products made from glycol-modified polyethylene terephthalate or cyclic olefin polymer or co-polymer resins did not release chemicals with detectable EA under any conditions tested. CONCLUSIONS: This hazard assessment survey showed that many BPA-free PC- replacement products still leached chemicals having significant levels of EA, as did BPA-containing PC counterparts they were meant to replace. That is, BPA-free did not mean EA-free. However, this study also showed that some PC-replacement products did not leach chemicals having significant levels of EA. That is, EA-free PC-replacement products could be made in commercial quantities at prices that compete with PC-replacement products that were not BPA-free. Since plastic products often have advantages (price, weight, shatter-resistance, etc.) compared to other materials such as steel or glass, it is not necessary to forgo those advantages to avoid release into foodstuffs or the environment of chemicals having EA that may have potential adverse effects on our health or the health of future generations.

Chemicals having estrogenic activity can be released from some bisphenol A-free, hard and clear, thermoplastic resins.

BACKGROUND: Chemicals that have estrogenic activity (EA) can potentially cause adverse health effects in mammals including humans, sometimes at low doses in fetal through juvenile stages with effects detected in adults. Polycarbonate (PC) thermoplastic resins made from bisphenol A (BPA), a chemical that has EA, are now often avoided in products used by babies. Other BPA-free thermoplastic resins, some hypothesized or advertised to be EA-free, are replacing PC resins used to make reusable hard and clear thermoplastic products such as baby bottles. METHODS: We used two very sensitive and accurate in vitro assays (MCF-7 and BG1Luc human cell lines) to quantify the EA of chemicals leached into ethanol or water/saline extracts of fourteen unstressed or stressed (autoclaving, microwaving, UV radiation) thermoplastic resins. Estrogen receptor (ER)-dependent agonist responses were confirmed by their inhibition with the ER antagonist ICI 182,780. RESULTS: Our data showed that some (4/14) unstressed and stressed BPA-free thermoplastic resins leached chemicals having significant levels of EA, including one polystyrene (PS), and three Tritan™ resins, the latter reportedly EA-free. Exposure to UV radiation in natural sunlight resulted in an increased release of EA from Tritan™ resins. Triphenyl-phosphate (TPP), an additive used to manufacture some thermoplastic resins such as Tritan™, exhibited EA in both MCF-7 and BG1Luc assays. Ten unstressed or stressed glycol-modified polyethylene terephthalate (PETG), cyclic olefin polymer (COP) or copolymer (COC) thermoplastic resins did not release chemicals with detectable EA under any test condition. CONCLUSIONS: This hazard survey study assessed the release of chemicals exhibiting EA as detected by two sensitive, widely used and accepted, human cell line in vitro assays. Four PC replacement resins (Tritan™ and PS) released chemicals having EA. However, ten other PC-replacement resins did not leach chemicals having EA (EA-free-resins). These results indicate that PC-replacement plastic products could be made from EA-free resins (if appropriate EA-free additives are chosen) that maintain advantages of re-usable plastic items (price, weight, shatter resistance) without releasing chemicals having EA that potentially produce adverse health effects on current or future generations.

Polyethylene Glycol-Fusion Repair of Peripheral Nerve Injuries.

Axons successfully repaired with polyethylene glycol (PEG) fusion tecnology restored axonal continuity thereby preventing their Wallerian degeneration and minimizing muscle atrophy. PEG fusion studies in animal models and preliminary clinical trials involving patients with digital nerve repair have shown promise for this therapeutic approach. PEG fusion is safe to perform, and given the enormous potential benefits, there is no reason not to explore its therapeutic potential.

Polyethylene glycol fusion repair of severed rat sciatic nerves reestablishes axonal continuity and reorganizes sensory terminal fields in the spinal cord.

ABSTRACT: Peripheral nerve injuries result in the rapid degeneration of distal nerve segments and immediate loss of motor and sensory functions; behavioral recovery is typically poor. We used a plasmalemmal fusogen, polyethylene glycol (PEG), to immediately fuse closely apposed open ends of severed proximal and distal axons in rat sciatic nerves. We have previously reported that sciatic nerve axons repaired by PEG- fusion do not undergo Wallerian degeneration, and PEG-fused animals exhibit rapid (within 2-6 weeks) and extensive locomotor recovery. Furthermore, our previous report showed that PEG-fusion of severed sciatic motor axons was non-specific, i.e., spinal motoneurons in PEG- fused animals were found to project to appropriate as well as inappropriate target muscles. In this study, we examined the consequences of PEG-fusion for sensory axons of the sciatic nerve. Young adult male and female rats (Sprague-Dawley) received either a unilateral single cut or ablation injury to the sciatic nerve and subsequent repair with or without (Negative Control) the application of PEG. Compound action potentials recorded immediately after PEG-fusion repair confirmed conduction across the injury site. The success of PEG-fusion was confirmed through Sciatic Functional Index testing with PEG-fused animals showing improvement in locomotor function beginning at 35 days postoperatively. At 2-42 days postoperatively, we anterogradely labeled sensory afferents from the dorsal aspect of the hindpaw following bilateral intradermal injection of wheat germ agglutinin conjugated horseradish peroxidase. PEG-fusion repair reestablished axonal continuity. Compared to unoperated animals, labeled sensory afferents ipsilateral to the injury in PEG-fused animals were found in the appropriate area of the dorsal horn, as well as inappropriate mediolateral and rostrocaudal areas. Unexpectedly, despite having intact peripheral nerves, similar reorganizations of labeled sensory afferents were also observed contralateral to the injury and repair. This central reorganization may contribute to the improved behavioral recovery seen after PEG-fusion repair, supporting the use of this novel repair methodology over currently available treatments.

Polyethylene glycol fusion repair of severed sciatic nerves accelerates recovery of nociceptive sensory perceptions in male and female rats of different strains.

ABSTRACT: Successful polyethylene glycol fusion (PEG-fusion) of severed axons following peripheral nerve injuries for PEG-fused axons has been reported to: (1) rapidly restore electrophysiological continuity; (2) prevent distal Wallerian Degeneration and maintain their myelin sheaths; (3) promote primarily motor, voluntary behavioral recoveries as assessed by the Sciatic Functional Index; and, (4) rapidly produce correct and incorrect connections in many possible combinations that produce rapid and extensive recovery of functional peripheral nervous system/central nervous system connections and reflex (e.g., toe twitch) or voluntary behaviors. The preceding companion paper describes sensory terminal field reorganization following PEG-fusion repair of sciatic nerve transections or ablations; however, sensory behavioral recovery has not been explicitly explored following PEG-fusion repair. In the current study, we confirmed the success of PEG-fusion surgeries according to criteria (1-3) above and more extensively investigated whether PEG-fusion enhanced mechanical nociceptive recovery following sciatic transection in male and female outbred Sprague-Dawley and inbred Lewis rats. Mechanical nociceptive responses were assessed by measuring withdrawal thresholds using von Frey filaments on the dorsal and midplantar regions of the hindpaws. Dorsal von Frey filament test was a more reliable method than plantar von Frey filament test to assess mechanical nociceptive sensitivity following sciatic nerve transections. Baseline withdrawal thresholds of the sciatic-mediated lateral dorsal region differed significantly across strain but not sex. Withdrawal thresholds did not change significantly from baseline in chronic Unoperated and Sham-operated rats. Following sciatic transection, all rats exhibited severe hyposensitivity to stimuli at the lateral dorsal region of the hindpaw ipsilateral to the injury. However, PEG-fused rats exhibited significantly earlier return to baseline withdrawal thresholds than Negative Control rats. Furthermore, PEG-fused rats with significantly improved Sciatic Functional Index scores at or after 4 weeks postoperatively exhibited yet-earlier von Frey filament recovery compared with those without Sciatic Functional Index recovery, suggesting a correlation between successful pPEG-fusion and both motor-dominant and sensory-dominant behavioral recoveries. This correlation was independent of the sex or strain of the rat. Furthermore, our data showed that the acceleration of von Frey filament sensory recovery to baseline was solely due to the PEG-fused sciatic nerve and not saphenous nerve collateral outgrowths. No chronic hypersensitivity developed in any rat up to 12 weeks. All these data suggest that PEG-fusion repair of transection peripheral nerve injuries could have important clinical benefits.

Sealing of transected neurites of rat B104 cells requires a diacylglycerol PKC-dependent pathway and a PKA-dependent pathway.

To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing. By means of dye exclusion to assess Ca(2+)-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, we now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1 and PKA. Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes η (nPKCη) and θ (nPKCθ) and Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or protein kinase A (PKA). Sealing probability is further decreased by simultaneously inhibiting nPKCη, nPKCθ, and PKA. Extracellular Ca(2+), DAG, or cAMP analogs do not affect this decrease in sealing. These and other data suggest that DAG increases sealing through MARCKS and that nPKCη, nPKCθ, and PKA are all required to seal plasmalemmal damage in B104 and likely all eukaryotic cells.

Blocking the P2X7 receptor improves outcomes after axonal fusion.

BACKGROUND: Activation of the P2X7 receptor on peripheral neurons causes the formation of pannexin pores, which allows the influx of calcium across the cell membrane. Polyethylene glycol (PEG) and methylene blue have previously been shown to delay Wallerian degeneration if applied during microsuture repair of the severed nerve. Our hypothesis is that by modulating calcium influx via the P2X7 receptor pathway, we could improve PEG-based axonal repair. The P2X7 receptor can be stimulated or inhibited using bz adenosine triphosphate (bzATP) or brilliant blue (FCF), respectively. METHODS: A single incision rat sciatic nerve injury model was used. The defect was repaired using a previously described PEG methylene blue fusion protocol. Experimental animals were treated with 100 µL of 100 µM FCF solution (n = 8) or 100 µL of a 30 µM bzATP solution (n = 6). Control animals received no FCF, bzATP, or PEG. Compound action potentials were recorded prior to transection (baseline), immediately after repair, and 21 d postoperatively. Animals underwent behavioral testing 3, 7, 14, and 21 d postoperatively. After sacrifice, nerves were fixed, sectioned, and immunostained to allow for counting of total axons. RESULTS: Rats treated with FCF showed an improvement compared with control at all time points (n = 8) (P = 0.047, 0.044, 0.014, and 0.0059, respectively). A statistical difference was also shown between FCF and bzATP at d 7 (P < 0.05), but not shown with d 3, 14, and 21 (P > 0.05). CONCLUSIONS: Blocking the P2X7 receptor improves functional outcomes after PEG-mediated axonal fusion.

Repair of traumatic lesions to the plasmalemma of neurons and other cells: Commonalities, conflicts, and controversies.

Neuroscientists and Cell Biologists have known for many decades that eukaryotic cells, including neurons, are surrounded by a plasmalemma/axolemma consisting of a phospholipid bilayer that regulates trans-membrane diffusion of ions (including calcium) and other substances. Cells often incur plasmalemmal damage via traumatic injury and various diseases. If the damaged plasmalemma is not rapidly repaired within minutes, activation of apoptotic pathways by calcium influx often results in cell death. We review publications reporting what is less-well known (and not yet covered in neuroscience or cell biology textbooks): that calcium influx at the lesion sites ranging from small nm-sized holes to complete axonal transection activates parallel biochemical pathways that induce vesicles/membrane-bound structures to migrate and interact to restore original barrier properties and eventual reestablishment of the plasmalemma. We assess the reliability of, and problems with, various measures (e.g., membrane voltage, input resistance, current flow, tracer dyes, confocal microscopy, transmission and scanning electron microscopy) used individually and in combination to assess plasmalemmal sealing in various cell types (e.g., invertebrate giant axons, oocytes, hippocampal and other mammalian neurons). We identify controversies such as plug versus patch hypotheses that attempt to account for currently available data on the subcellular mechanisms of plasmalemmal repair/sealing. We describe current research gaps and potential future developments, such as much more extensive correlations of biochemical/biophysical measures with sub-cellular micromorphology. We compare and contrast naturally occurring sealing with recently-discovered artificially-induced plasmalemmal sealing by polyethylene glycol (PEG) that bypasses all natural pathways for membrane repair. We assess other recent developments such as adaptive membrane responses in neighboring cells following injury to an adjacent cell. Finally, we speculate how a better understanding of the mechanisms involved in natural and artificial plasmalemmal sealing is needed to develop better clinical treatments for muscular dystrophies, stroke and other ischemic conditions, and various cancers.

Repair and regeneration of peripheral nerve injuries that ablate branch points.

The peripheral nervous system has an extensive branching organization, and peripheral nerve injuries that ablate branch points present a complex challenge for clinical repair. Ablations of linear segments of the PNS have been extensively studied and routinely treated with autografts, acellular nerve allografts, conduits, wraps, and nerve transfers. In contrast, segmental-loss peripheral nerve injuries, in which one or more branch points are ablated so that there are three or more nerve endings, present additional complications that have not been rigorously studied or documented. This review discusses: (1) the branched anatomy of the peripheral nervous system, (2) case reports describing how peripheral nerve injuries with branched ablations have been surgically managed, (3) factors known to influence regeneration through branched nerve structures, (4) techniques and models of branched peripheral nerve injuries in animal models, and (5) conclusions regarding outcome measures and studies needed to improve understanding of regeneration through ablated branched structures of the peripheral nervous system.

Survival of rat sciatic nerve segments preserved in storage solutions ex vivo assessed by novel electrophysiological and morphological criteria.

Most organ or tissue allografts with viable cells are stored in solutions ex vivo for hours to several days. Most allografts then require rapid host revascularization upon transplantation to maintain donor-cell functions (e.g., cardiac muscle contractions, hepatic secretions). In contrast, peripheral nerve allografts stored ex vivo do not require revascularization to act as scaffolds to guide outgrowth by host axons at 1-2 mm/d, likely aided by viable donor Schwann cells. Using current storage solutions and protocols, axons in all these donor organ/tissue/nerve transplants are expected to rapidly become non-viable due to Wallerian degeneration within days. Therefore, ex vivo storage solutions have not been assessed for preserving normal axonal functions, i.e., conducting action potentials or maintaining myelin sheaths. We hypothesized that most or all organ storage solutions would maintain axonal viability. We examined several common organ/tissue storage solutions (University of Wisconsin Cold Storage Solution, Normosol-R, Normal Saline, and Lactated Ringers) for axonal viability in rat sciatic nerves ex vivo as assessed by maintaining: (1) conduction of artificially-induced compound action potentials; and (2) axonal and myelin morphology in a novel assay method. The ten different storage solution conditions for peripheral nerves with viable axons (PNVAs) differed in their solution composition, osmolarity (250-318 mOsm), temperature (4°C vs. 25°C), and presence of calcium. Compound action potentials and axonal morphology in PNVAs were best maintained for up to 9 days ex vivo in calcium-free hypotonic diluted (250 mOsm) Normosol-R (dNR) at 4°C. Surprisingly, compound action potentials were maintained for only 1-2 days in UW and NS at 4°C, a much shorter duration than PNVAs maintained in 4°C dNR (9 days) or even in 25°C dNR (5 days). Viable axons in peripheral nerve allografts are critical for successful polyethylene glycol (PEG)-fusion of viable proximal and distal ends of host axons with viable donor axons to repair segmental-loss peripheral nerve injuries. PEG-fusion repair using PNVAs prevents Wallerian degeneration of many axons within and distal to the graft and results in excellent recovery of sensory/motor functions and voluntary behaviors within weeks. Such PEG-fused PNVAs, unlike all other types of conventional donor transplants, are immune-tolerated without tissue matching or immune suppression. Preserving axonal viability in stored PNVAs would enable the establishment of PNVA tissue banks to address the current shortage of transplantable nerve grafts and the use of stored PEG-fused PNVAs to repair segmental-loss peripheral nerve injuries. Furthermore, PNVA storage solutions may enable the optimization of ex vivo storage solutions to maintain axons in other types of organ/tissue transplants.