The curious ability of polyethylene glycol fusion technologies to restore lost behaviors after nerve severance.

Traumatic injuries to PNS and CNS axons are not uncommon. Restoration of lost behaviors following severance of mammalian peripheral nerve axons (PNAs) relies on regeneration by slow outgrowths and is typically poor or nonexistent when after ablation or injuries close to the soma. Behavioral recovery after severing spinal tract axons (STAs) is poor because STAs do not naturally regenerate. Current techniques to enhance PNA and/or STA regeneration have had limited success and do not prevent the onset of Wallerian degeneration of severed distal segments. This Review describes the use of a recently developed polyethylene glycol (PEG) fusion technology combining concepts from biochemical engineering, cell biology, and clinical microsurgery. Within minutes after microsuturing carefully trimmed cut ends and applying a well-specified sequence of solutions, PEG-fused axons exhibit morphological continuity (assessed by intra-axonal dye diffusion) and electrophysiological continuity (assessed by conduction of action potentials) across the lesion site. Wallerian degeneration of PEG-fused PNAs is greatly reduced as measured by counts of sensory and/or motor axons and maintenance of axonal diameters and neuromuscular synapses. After PEG-fusion repair, cut-severed, crush-severed, or ablated PNAs or crush-severed STAs rapidly (within days to weeks), more completely, and permanently restore PNA- or STA-mediated behaviors compared with nontreated or conventionally treated animals. PEG-fusion success is enhanced or decreased by applying antioxidants or oxidants, trimming cut ends or stretching axons, and exposure to Ca(2+) -free or Ca(2+) -containing solutions, respectively. PEG-fusion technology employs surgical techniques and chemicals already used by clinicians and has the potential to produce a paradigm shift in the treatment of traumatic injuries to PNAs and STAs.

Polyethylene glycol-fused allografts produce rapid behavioral recovery after ablation of sciatic nerve segments.

Restoration of neuronal functions by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed peripheral nerves takes many weeks or months, if it occurs at all, especially after ablation of nerve segments. Distal segments of severed axons typically degenerate in 1-3 days. This study shows that Wallerian degeneration can be prevented or retarded, and lost behavioral function can be restored, following ablation of 0.5-1-cm segments of rat sciatic nerves in host animals. This is achieved by using 0.8-1.1-cm microsutured donor allografts treated with bioengineered solutions varying in ionic and polyethylene glycol (PEG) concentrations (modified PEG-fusion procedure), being careful not to stretch any portion of donor or host sciatic nerves. The data show that PEG fusion permanently restores axonal continuity within minutes, as initially assessed by action potential conduction and intracellular diffusion of dye. Behavioral functions mediated by the sciatic nerve are largely restored within 2-4 weeks, as measured by the sciatic functional index. Increased restoration of sciatic behavioral functions after ablating 0.5-1-cm segments is associated with greater numbers of viable myelinated axons within and distal to PEG-fused allografts. Many such viable myelinated axons are almost certainly spared from Wallerian degeneration by PEG fusion. PEG fusion of donor allografts may produce a paradigm shift in the treatment of peripheral nerve injuries.

PET imaging demonstrates histone deacetylase target engagement and clarifies brain penetrance of known and novel small molecule inhibitors in rat.

Histone deacetylase (HDAC) enzymes have been demonstrated as critical components in maintaining chromatin homeostasis, CNS development, and normal brain function. Evidence in mouse models links HDAC expression to learning, memory, and mood-related behaviors; small molecule HDAC inhibitor tool compounds have been used to demonstrate the importance of specific HDAC subtypes in modulating CNS-disease-related behaviors in rodents. So far, no direct evidence exists to understand the quantitative changes in HDAC target engagement that are necessary to alter biochemistry and behavior in a living animal. Understanding the relationship between target engagement and in vivo effect is essential in refining new ways to alleviate disease. We describe here, using positron emission tomography (PET) imaging of rat brain, the in vivo target engagement of a subset of class I/IIb HDAC enzymes implicated in CNS-disease (HDAC subtypes 1, 2, 3, and 6). We found marked differences in the brain penetrance of tool compounds from the hydroxamate and benzamide HDAC inhibitor classes and resolved a novel, highly brain penetrant benzamide, CN147, chronic treatment with which resulted in an antidepressant-like effect in a rat behavioral test. Our work highlights a new translational path for understanding the molecular and behavioral consequences of HDAC target engagement.

Neurite transection produces cytosolic oxidation, which enhances plasmalemmal repair.

To survive, cells must rapidly repair (seal) plasmalemmal damage. Cytosolic oxidation has been shown to increase cell survival in some cases and produce cell death in other protocols. An antioxidant (melatonin; Mel) has been reported to decrease the probability of sealing plasmalemmal damage. Here we report that plasmalemmal damage produces cytosolic oxidation, as assayed by methylene blue (MB) color change in rat B104 hippocampal cells. Plasmalemmal sealing is affected by duration of Ca²âº deprivation and length of exposure to, and concentration of, oxidizing agents such as H2O2 and thimerosal (TH). Cytosolic oxidation by 10 µM to 50 mM H2O2 or 100 µM to 2 mM TH increases the probability of Ca²âº-dependent plasmalemmal sealing, whereas higher concentrations of H2O2 decrease sealing probability and also damage uninjured cells. We also show that antioxidants (Mel, MB) or reducing agents (dithiothreitol) decrease sealing. Proteins, such as protein kinase A, SNAP-25, synaptobrevin, and N-ethylmaleimide-sensitive factor (previously reported to enhance sealing in other pathways), also enhance sealing in this oxidation pathway. In brief, our data show that plasmalemmal damage produces cytosolic oxidation that increases the probability of plasmalemmal sealing, which is strongly correlated with cell survival in other studies. Our results may provide new insights into the etiology and treatment of oxidation-dependent neurodegenerative disorders, such as Parkinson's, Huntington's, and Alzheimer's diseases.

Cellular mechanisms of plasmalemmal sealing and axonal repair by polyethylene glycol and methylene blue.

Mammalian neurons and all other eukaryotic cells endogenously repair traumatic injury within minutes by a Ca²âº-induced accumulation of vesicles that interact and fuse with each other and the plasmalemma to seal any openings. We have used uptake or exclusion of extracellular fluorescent dye to measure the ability of rat hippocampal B104 cells or rat sciatic nerves to repair (seal) transected neurites in vitro or transected axons ex vivo. We report that endogenous sealing in both preparations is enhanced by Ca²âº-containing solutions and is decreased by Ca²âº-free solutions containing antioxidants such as dithiothreitol (DTT), melatonin (MEL), methylene blue (MB), and various toxins that decrease vesicular interactions. In contrast, the fusogen polyethylene glycol (PEG) at 10-50 mM artificially seals the cut ends of B104 cells and rat sciatic axons within seconds and is not affected by Ca²âº or any of the substances that affect endogenous sealing. At higher concentrations, PEG decreases sealing of transected axons and disrupts the plasmalemma of intact cells. These PEG-sealing data are consistent with the hypothesis that lower concentrations of PEG directly seal a damaged plasmalemma. We have considered these and other data to devise a protocol using a well-specified series of solutions that vary in tonicity, Ca²âº, MB, and PEG content. These protocols rapidly and consistently repair (PEG-fuse) rat sciatic axons in completely cut sciatic nerves in vivo rapidly and dramatically to restore long-lasting morphological continuity, action potential conduction, and behavioral functions.

Rapid, effective, and long-lasting behavioral recovery produced by microsutures, methylene blue, and polyethylene glycol after completely cutting rat sciatic nerves.

Behavioral function lost in mammals (including humans) after peripheral nerve severance is slowly (weeks to years) and often poorly restored by 1-2-mm/day, nonspecifically directed outgrowths from proximal axonal stumps. To survive, proximal stumps must quickly repair (seal) plasmalemmal damage. We report that, after complete cut- or crush-severance of rat sciatic nerves, morphological continuity, action potential conduction, and behavioral functions can be consistently (>98% of trials), rapidly (minutes to days), dramatically (70-85% recovery), and chronically restored and some Wallerian degeneration prevented. We assess axoplasmic and axolemmal continuity by intra-axonal dye diffusion and action potential conduction across the lesion site and amount of behavioral recovery by Sciatic Functional Index and Foot Fault tests. We apply well-specified sequences of solutions containing FDA-approved chemicals. First, severed axonal ends are opened and resealing is prevented by hypotonic Ca²âº-free saline containing antioxidants (especially methylene blue) that inhibit plasmalemmal sealing in sciatic nerves in vivo, ex vivo, and in rat B104 hippocampal cells in vitro. Second, a hypotonic solution of polyethylene glycol (PEG) is applied to open closely apposed (by microsutures, if cut) axonal ends to induce their membranes to flow rapidly into each other (PEG-fusion), consistent with data showing that PEG rapidly seals (PEG-seals) transected neurites of B104 cells, independently of any known endogenous sealing mechanism. Third, Ca²âº-containing isotonic saline is applied to induce sealing of any remaining plasmalemmal holes by Ca²âº-induced accumulation and fusion of vesicles. These and other data suggest that PEG-sealing is neuroprotective, and our PEG-fusion protocols that repair cut- and crush-severed rat nerves might rapidly translate to clinical procedures.

[The Da Vinci Medical-mental motivation program for supporting lifestyle changes in patients with type 2 diabetes]. / Medizinisch-mentales Motivationstraining Da Vinci zur Unterstützung einer Lebensstilumstellung bei Typ-2-Diabetes.

BACKGROUND AND OBJECTIVE: Healthy diet and physical activity can improve metabolic control in patients with type 2 diabetes mellitus. However, lifestyle change without external help is difficult: an alteration of mental attitude is necessary to achieve long-term success. A computer-based motivational program ("Da Vinci") has been developed to help patients to change their mental attitudes and beliefs. METHODS: Patients with type 2 diabetes were supervised by psychological trainers in four sessions at ten study centers. The interactive computer program allowed for identification of motivation restraints and overcoming them. Parameters of carbohydrate metabolism were measured at the beginning and end of the three-months program as well as three and six months after end of program. RESULTS: All participants (n = 61) developed a positive attitude towards the range of their action and by themselves changed their lifestyle. After three months their weight (-4.6 kg; p < 0.0001), body mass index (-1.1 kg/m2; p < 0.0001), waist circumference (-3.5 cm; p < 0.0001), HbA1c (-0.6 %; p < 0.0001), triglycerides (-31.1 mg/dl; p = 0.033), systolic (-4.0 mmHg; p = 0.005) and diastolic blood pressure (-3.0 mmHg; p = 0.006) had been reduced. Short duration of diabetes and high baseline HbA1c values were predictive for successful HbA1c reduction. Three and six months after end of the program participants were able to maintain or even augment achieved improvements. CONCLUSION: During the motivational program, which is intended to alter mental attitude and beliefs, but not to teach knowledge about diabetes, participants were able to significantly improve their metabolic control. As these improvements were maintained long-term, this points to sustainable lifestyle change.

Utilizing multiple vital status tracing services optimizes mortality follow-up in large cohort studies.

PURPOSE: To compare the three national-scale death identification services used in our two-stage vital status tracing protocol, Pension Benefit Information Company (PBI), Social Security Administration (SSA), and the Health Care Financing Administration (HCFA), with respect to death identification and confirmation rate, and relevant demographic variables. METHODS: Information on 31,223 subjects with unconfirmed vital status in an ongoing occupational cohort mortality study was simultaneously submitted to PBI, SSA, and HCFA to identify subjects deceased as of December 31, 1992. Subjects whose dates of death were between 1979 and 1992 were then sent to the National Death Index (NDI) to obtain death certificate numbers and supplemental states of death. RESULTS: PBI identified and confirmed the highest number deaths in this cohort. PBI and SSA identified a higher proportion of deaths for persons who died in earlier years and/or who died at a younger age, for both confirmed and unconfirmed deaths. HCFA identified fewer deaths overall and had a smaller proportion of unconfirmed deaths. These deaths occurred in later years among older subjects and had the highest proportion of females. NDI provided exact matches for 92-96% of deaths identified by each of the three services. CONCLUSIONS: PBI was the most comprehensive service, especially for identifying younger subjects and those with an earlier date of death, while HCFA may help to identify deceased female subjects. SSA data can be purchased and used for periodic updates or interactively to identify deaths among subjects with poor identifiers (such as incorrect or missing social security numbers or misspelled names). Because each service makes a valuable contribution to the identification of deceased cohort subjects, all three should be considered for optimal mortality follow-up.

Plasmalemmal repair of severed neurites of PC12 cells requires Ca(2+) and synaptotagmin.

Ca(2+) and synaptotagmin (a Ca(2+)-binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24-hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6-hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS(++)) also exclude entry of an externally placed hydrophilic fluorescent dye at 15-min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation-free solution (PBS(=)) or in PBS(=) + Mg(2+), respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody-loaded neurites do not form a dye barrier, whereas most (>/=81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca(2+) is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically.

Barrier permeability at cut axonal ends progressively decreases until an ionic seal is formed.

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.

Axolemmal repair requires proteins that mediate synaptic vesicle fusion.

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.

Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic outgrowth.

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2-10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time-lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur.

[Marburg modular prone positioning system (MBS) for positioning therapy]. / Modulares Marburger Bauchlagerungssystem (MBS) für die dorsoventrale Wechsellagerung.

Despite the many benefits of prone positioning in critically ill patients with respiratory failure and ARDS in the ICU, its technical problems have not yet been adequately resolved. Different approaches with special beds for prone positioning do exist, but these devices are difficult to handle, often not available and involve high costs. With this in mind, we developed an easy handling prone positioning system (MBS) that requires no special beds and runs at low cost. The MBS is a cost-effective device, yielding many benefits for prone positioning in critically ill patients with severe athelectasis and ARDS.

Calcium entry initiates processes that restore a barrier to dye entry in severed earthworm giant axons.

After severance, axons can restore structural barriers that are necessary for recovery of their electrical function. In earthworm myelinated axons, such a barrier to dye entry is mediated by many vesicles and myelin-derived membranous structures. From time-lapse confocal fluorescence and DIC images, we now report that Ca2+ entry and not axonal injury per se initiates the processes that form a dye barrier, as well as the subsequent structural changes in this barrier and associated membranous structures. The time required to restore a dye barrier after transection also depends only on the time of Ca2+ entry.

Rapid induction of functional and morphological continuity between severed ends of mammalian or earthworm myelinated axons.

The inability to rapidly restore the loss of function that results from severance (cutting or crushing) of PNS and CNS axons is a severe clinical problem. As a novel strategy to help alleviate this problem, we have developed in vitro procedures using Ca2+-free solutions of polyethylene glycol (PEG solutions), which within minutes induce functional and morphological continuity (PEG-induced fusion) between the cut or crushed ends of myelinated sciatic or spinal axons in rats. Using a PEG-based hydrogel that binds to connective tissue to provide mechanical strength at the lesion site and is nontoxic to nerve tissues in earthworms and mammals, we have also developed in vivo procedures that permanently maintain earthworm myelinated medial giant axons whose functional and morphological integrity has been restored by PEG-induced fusion after axonal severance. In all these in vitro or in vivo procedures, the success of PEG-induced fusion of sciatic or spinal axons and myelinated medial giant axons is measured by the restored conduction of action potentials through the lesion site, the presence of intact axonal profiles in electron micrographs taken at the lesion site, and/or the intra-axonal diffusion of fluorescent dyes across the lesion site. These and other data suggest that the application of polymeric fusiogens (such as our PEG solutions), possibly combined with a tissue adherent (such as our PEG hydrogels), could lead to in vivo treatments that rapidly and permanently repair cut or crushed axons in the PNS and CNS of adult mammals, including humans.

Potentiation of platinum antitumor effects in human lung tumor xenografts by the angiogenesis inhibitor squalamine: effects on tumor neovascularization.

Squalamine is a novel anti-angiogenic aminosterol that is postulated to inhibit neovascularization by selectively inhibiting the sodium-hydrogen antiporter exchanger. To determine how to most effectively use this agent in patients with cancer, we examined the antitumor effects of squalamine with or without cytotoxic agents in human lung cancer xenografts and correlated these observations with the degree of tumor neovascularization. No direct cytotoxic effects of squalamine against tumor cells were observed in vitro with or without cisplatin. Squalamine was effective in inhibiting the establishment of H460 human tumors in BALBc nude mice but was ineffective in inhibiting the growth of H460, CALU-6, or NL20T-A human tumor xenografts when administered i.p. to mice bearing established tumors. However, when combined with cisplatin or carboplatin, squalamine increased tumor growth delay by > or =1.5-fold in the three human lung carcinoma cell lines compared with cisplatin or carboplatin alone. No enhancement of antitumor activity was observed when squalamine was combined with paclitaxel, vinorelbine, gemcitabine, or docetaxel. Repeated cycles of squalamine plus cisplatin administration delayed H460 tumor growth >8.6-fold. Squalamine plus cisplatin reduced CD31 vessel formation by 25% compared with controls, squalamine alone, or cisplatin alone; however, no inhibition in CD31 vessel formation was observed when squalamine was combined with vinorelbine. These data demonstrate that the combination of squalamine and a platinum analog has significant preclinical antitumor activity against human lung cancer that is related to the anti-angiogenic effects of squalamine.

Anomalies associated with dye exclusion as a measure of axolemmal repair in invertebrate axons.

After axonal injury, dye exclusion is often used as a measure of the re-establishment of a structural barrier. We now report that this use of dye exclusion is equivocal in two situations. (1) When a negatively-charged hydrophilic fluorescent dye (HFD) was placed in the physiological saline (PS) surrounding a crayfish medial giant axon (CMGA) before transection, this dye did not readily diffuse into the cut ends after transection whereas uncharged or neutralized dyes did do so. (2) When axoplasm flowed out of the cut ends of a transected squid giant axon (SGA), this outflow markedly slowed hydrophilic fluorescent dyes from diffusing into the cut ends. These anomalies suggest that dye exclusion by an injured axon does not always indicate that a structural barrier has formed. Therefore, dye assessments of axonal repair require control experiments that rule out anomalous exclusion due to dye interactions (biochemical and fluid dynamics) with components (axoplasm, axolemma, glial sheath, etc.) of the particular axon under study.

Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.