Axolemmal Membrane Research Articles

Closing the Gap Between Mammalian and Invertebrate Peripheral Nerve Injury: Protocol for a Novel Nerve Repair.

BackgroundOutcomes after peripheral nerve injuries are poor despite current nerve repair techniques. Currently, there is no conclusive evidence that mammalian axons are capable of spontaneous fusion after transection. Notably, certain invertebrate species are able to auto-fuse after transection. Although mammalian axonal auto-fusion has not been observed experimentally, no mammalian study to date has demonstrated regenerating axolemmal membranes contacting intact distal segment axolemmal membranes to determine whether mammalian peripheral nerve axons have the intrinsic mechanisms necessary to auto-fuse after transection.ObjectiveThis study aims to assess fusion competence between regenerating axons and intact distal segment axons by enhancing axon regeneration, delaying Wallerian degeneration, limiting the immune response, and preventing myelin obstruction.MethodsThis study will use a rat sciatic nerve model to evaluate the effects of a novel peripheral nerve repair protocol on behavioral, electrophysiologic, and morphologic parameters. This protocol consists of a variety of preoperative, intraoperative, and postoperative interventions. Fusion will be assessed with electrophysiological conduction of action potentials across the repaired transection site. Axon-axon contact will be assessed with transmission electron microscopy. Behavioral recovery will be analyzed with the sciatic functional index. A total of 36 rats will be used for this study. The experimental group will use 24 rats and the negative control group will use 12 rats. For both the experimental and negative control groups, there will be both a behavior group and another group that will undergo electrophysiological and morphological analysis. The primary end point will be the presence or absence of action potentials across the lesion site. Secondary end points will include behavioral recovery with the sciatic functional index and morphological analysis of axon-axon contact between regenerating axons and intact distal segment axons.ResultsThe author is in the process of grant funding and institutional review board approval as of March 2020. The final follow-up will be completed by December 2021.ConclusionsIn this study, the efficacy of the proposed novel peripheral nerve repair protocol will be evaluated using behavioral and electrophysiologic parameters. The author believes this study will provide information regarding whether spontaneous axon fusion is possible in mammals under the proper conditions. This information could potentially be translated to clinical trials if successful to improve outcomes after peripheral nerve injury.International Registered Report Identifier (IRRID)PRR1-10.2196/18706

Damage and repair of the axolemmal membrane: From neural development to axonal trauma and restoration.

Abstract Integrity of the plasma membrane is essential for the maintenance of physiological conditions, metabolic activity and the shape of eukaryotic cells. In neurons, the plasma membrane surrounding the axon—the axolemma—fulfills all these functions plus those inherent to the specific function of the neuron: maintaining the membrane potential by the regulated and concerted operation of ion-selective channels. Membrane expansion and neurite growth are directly linked through intricate cellular signaling mechanisms during the early stages of embryonic development. During axonal development there is an increase in the surface area of the axolemma which provokes an increase in membrane tension. Membrane insertion involved in axonal growth reduces the membrane tension, and this in turn allows distal membrane expansion and axonal extension. Under certain pathological conditions, such as spinal cord and traumatic brain injuries, the axolemmal damage results in different degrees of neuronal degeneration due to unregulated ionic influx, followed by oxidative damage, finally triggering neuronal apoptosis. Neurons possess counteractive mechanisms to arrest these degenerative processes which involve sealing the axolemma as a first step toward membrane repair, followed by attempts at axonal extension. In this review we address the main molecular actors and mechanisms involved in axonal growth during embryonic development and the recapitulation of these mechanisms during the post-traumatic regeneration process. We also discuss the efficacy of some classical and novel therapeutic approaches to axolemmal sealing and repair in different pathologies.

The N‐glycan profile of mouse myelin, a specialized central nervous system membrane

Understanding the rich complement of sugar chains found in cellular membranes is impeded by the complexity of cell types and membrane diversity. To overcome this, we have analyzed the N-linked sugar chain composition of the glycoproteins of CNS myelin, an elaboration of the plasma membranes of oligodendrocytes (OLs) that result in a multilamellar wrapping of neuronal axons, facilitating nerve conduction with dramatic savings of space and energy. Due to an usually high lipid to protein ratio, myelin can be separated readily from other heavier membranes on sucrose gradients and further fractionated into subdomains related to myelin structure and function, including compact myelin and myelin-associated axolemmal membrane (Menon et al. 2003). We analyzed these fractions for N-linked sugar chains, using 2D HPLC following hydrazinolysis and pyridylamination. Our results indicate that compared with total brain homogenate, the amount of N-glycans is 1.3-fold higher in the myelin-associated axolemmal membranes, but it is 0.5-fold less in CM. M5 [Manalpha1-3((Manalpha1-3)(Manalpha1-6)Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc] is the most abundant sugar chain in total brain homogenate, compact myelin, and myelin-associated axolemma, constituting approximately 20% of sugar chains. Although the types of sugar chains are similar among the fractions, their expression levels vary significantly. In addition to high mannose type oligosaccharides, the core fucosylated, biantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc) residue, A2G1(3)FB [Galbeta1-4GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc], A2G1(6)FB [GlcNAcbeta1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4 (Fucalpha1-6)GlcNAc] and BA-1 [Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc], and A1(6)G0F [Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6) GlcNAc] are also present in relatively large proportions in compact myelin. We suggest that these differences may be related to myelin-axolemmal function.

Infectious causes of acute flaccid paralysis.

Although acute flaccid paralysis (AFP) is more frequently seen in the tropics than in temperate regions, recent outbreaks of West Nile virus (WNV) in North America have drawn attention to this important presentation. Starting with anatomical and neurophysiological considerations, this article examines data on AFP caused by WNV, and considers recent data on paralysis caused by enteroviruses, and Guillain-Barré syndrome (GBS). Neurophysiological, radiological and pathological studies suggest WNV causes AFP by damaging anterior horn cells in the spinal cord. The clinical presentation is probably best described as a 'poliomyelitis-like illness', and the disease as 'WNV myelitis'. Other findings during the recent outbreaks include increasing recognition of a Parkinson's-disease like presentation, and descriptions of virus transmission in blood transfusions and transplanted organs. GBS is now recognized as several disorders characterized by immune-mediated attack on peripheral nerves: in acute inflammatory demyelinating polyneuropathy the myelin sheath and Schwann cell of sensory and motor nerves are targeted; acute motor axonal and acute motor and sensory axonal neuropathy often follow Campylobacter jejuni enteritis and are associated with antibodies against the ganglioside component of the nerve axolemmal membrane. In Asia-Pacific, enterovirus 71 has caused outbreaks of neurological diseases with AFP and encephalitis, but no single genogroup of virus appears responsible for severe disease. Despite the near eradication of poliomyelitis, AFP caused by viruses remains an important clinical presentation. Distinguishing direct viral causes from GBS is important for public health reasons, and to avoid inappropriate therapies.

Extent and mechanism of sealing in transected giant axons of squid and earthworms

Transected axons are often assumed to seal at their cut ends by the formation of continuous membrane barriers that allow for the restoration of function in the axonal stumps. We have used several electrophysiological measures (membrane potential, input resistance, injury current density) and several morphological measures (phase-contrast, video-enhanced differential interference contrast, light, and electron microscopies) of living and fixed material to assess the extent and mechanism of sealing within hours after transecting giant axons of squid (Loligo pealei and Sepioteuthis lessoniana) and earthworms (Lumbricus terrestris). Our electrophysiological data suggest that the proximal and distal ends of transected squid giant axons do not completely seal within 2.5 hr in physiological saline. In contrast, the same set of measures suggest that proximal and distal ends of transected earthworm giant axons seal within 1 hr in physiological saline. Our morphological data show that the cut ends of both squid and earthworm axons constrict, but that a 20-70-microns-diameter opening always remains at the cut end that is filled with vesicles. Axonal transection induces the formation of vesicles that are observed in the axoplasm within minutes in standard salines and that rapidly migrate to the cut ends. These injury-induced vesicles are loosely packed near the cut ends of squid giant axons, which do not functionally seal within 2.5 hr of transection. In contrast, vesicles formed a tightly packed plug at the cut ends of earthworm medial giant axons, which do functionally seal within 1 hr of transection in physiological saline. Since we detect no single continuous membrane that spans the cut end, sealing does not appear to occur by the fusion of constricted axolemmal membrane or the formation of a membranous partition at the cut end. Rather, our data are consistent with the hypothesis that a tightly packed vesicular plug is responsible for sealing of earthworm giant axons.

Axolemmal and septal conduction in the impedance of the earthworm medial giant nerve fiber

Ionic conduction in the axolemmal and septal membranes of the medial giant fiber (MGF) of the earthworm (EW) Lumbricus terrestris was assessed by impedance spectroscopy in the frequency range 2.5-1000 Hz. Impedance loci in the complex plane were described by two semi-circular arcs, one at a lower characteristic frequency (100 Hz) and the other at a higher frequency (500 Hz). The lower frequency arc had a chord resistance of 53 k omega and was not affected by membrane potential changes or ion channel blockers [tetrodotoxin (TTX), 3,4-diaminopyridine (3,4-DAP), 4-aminopyridine (4-AP), and tetraethylammonium (TEA)]. The higher frequency arc had a chord resistance of 274 k omega at resting potential, was voltage-dependent, and was affected by the addition of TTX, 3,4-DAP, 4-AP, and TEA to the physiological EW salines. When all four blockers were added to the bathing solution, the impedance locus was described by two voltage-independent arcs. Considering the effects of these and other (i.e., Cd and Ni) ion channel blockers, we conclude that: 1) the higher frequency locus reflects conduction by voltage-sensitive ion channels in the axolemmal membrane, which contains at least four ion channels selective for sodium, calcium, and potassium (delayed rectifier and calcium-dependent), and 2) the lower frequency locus reflects voltage-insensitive channels in the septal membrane, which separates adjacent MGFs.

Lipid peroxidation inhibits oleoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase in rat CNS axolemma-enriched fractions.

The effect of phospholipid peroxidation on the acylation of lysoPtdCho (lysophosphatidylcholine) by axolemma-enriched fraction prepared from rat brain stem was investigated. After two types of peroxidative treatments, the in vitro induction of malondialdehyde and conjugated dienes formation in axolemmal membranes correlated to a shift in the ratio of saturated/unsaturated fatty acids. By using an Fe2+ (20 microM)-ascorbate (0.25 mM) peroxidation system, the residual acyltransferase activity was 55% of the initial one. No change in Km value for either oleoyl-CoA or lysoPtdCho was found, whereas a loss of 24% in Vmax was observed. After 5 min preincubation with 150 mM t-BuOOH, 70% inactivation of the acylation reaction was observed. A near suppression of enzyme activity was reached with 400 mM. The apparent Km for oleoyl-CoA decreased sharply (from 6.6 microM in control preparations to 4.1 microM in t-BuOOH-treated membranes), indicating a 2-fold increase in the enzymatic affinity for this substrate. The apparent Km for lysoPtdCho increased markedly (from 1.56 microM in the control preparations to 5.88 microM in t-BuOOH-treated membranes) whereas a decrease of Vmax (from 1.65 to 0.80 nmol/min/mg protein) for the same substrate was observed. Significant enzyme inactivation (loss of 60% of initial activity) was seen when 10 mumol of photooxidized phospholipids were preincubated with axolemmal membranes. Significant dose-dependent enzyme inactivation was brought about by addition of 10-60 mumol of peroxidized PtdEtn/100 micrograms axolemmal protein. The percent enzyme inhibition by peroxidized PtdCho at equivalent amounts was lower than that by PtdEtn.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of phospholipase A 2 and acyltransferase activities in squid ( loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions

Phospholipase A 2 and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A 2 activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A 2 was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating. The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K m (apparent) and V max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide. Phospholipase A 2 activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A 2 activity was stimulated by Ca ++ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca ++-independent phospholipase A 2, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.

Isolation and characterization of periaxolemmal and axolemmal enriched membrane fractions from the rat central nervous system.

In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.0M sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K+ ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.0M fraction was also highly enriched in Na+, K+ ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage-dependent Na+ channel, axon-specific fodrin, and MAP-1B. Based on these characteristics we concluded that the 0.8M and 1.0M subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipin10 was unique with equally high levels in myelin and in the 0.8M and 1.0M subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GA/1. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K+ and Cl- channels, which based on channel sizes and open times, are largely distinct from one another.

Giant axonal neuropathy (GAN): an immunohistochemical and ultrastructural study report of a Latin American case

Giant axonal neuropathy (GAN), a progressive childhood disorder of intermediate filaments (IF), is characterized by a peripheral neuropathy and central nervous system involvement. Twenty-eight cases have been reported while several pathogenic hypotheses have been proposed. Sural nerve biopsy of a 10-year-old Argentinian girl showed a reduced number of myelinated fibers as well as several enlarged axons up to 30 microns in diameter, thinly myelinated or devoid of myelin sheath, displaying accumulation of neurofilaments (NF), but few microtubules (MT) beneath the axolemmal membrane. There was IF accumulation in Schwann and perineural cells as well as in melanocytes, fibroblasts, pericytes, endothelial and epithelial cells in both nerve and skin biopsy. Our findings strongly support GAN as a generalized IF disorder with MT segregation from NF in giant axons. Abnormal NF phosphorylation is suggested by heavy immunostaining of enlarged axons by a monoclonal antibody to NF phosphorylated determinants (SMI 31-Sternberger's) and lack of reaction with a monoclonal antibody with different phosphoepitopes affinity (SMI 34-Sternberger's).

Enzyme translocation in the course of regeneration of central primary afferent terminals in the substantia gelatinosa of the adult rodent spinal cord

Synaptic circuitry in the upper dorsal horn, disorganized due to transganglionic degenerative atrophy (evoked by blockade of retrograde axoplasmic transport in the related peripheral nerve), begins to be reestablished by a process of regenerative synaptoneogenesis as soon as retrograde transport is resumed. Central axons of type C primary sensory neurons, terminating in substantia gelatinosa Rolandi, as well as their cells of origin in dorsal root ganglia are specifically and selectively labeled by the enzyme thiamine monophosphatase (TMPase). Under normal conditions, TMPase is localized in axolemmal membranes at the electron histochemical level. In axonal growth cones of regenerating central terminals, only negligible TMPase reaction was found. In axonal filopodia and young nerve sprouts, there appears an increasing number of intraaxonal grains of the reaction product. Varicous swellings (beads) of regenerating axonal sprouts are transformed into scalloped (sinusoid) en passant terminals. TMPase reaction end product, present initially in the axoplasm of beads and scalloped terminals, is successively translocated to the axolemmal membrane in the course of cytochemical maturation. Structural regeneration and cytochemical maturation of central terminals of primary sensory neurons are completed 60 days after crush injury to the related peripheral axons, i.e., about 7 weeks after the peripheral nerve has regenerated.

Ankyrin and spectrin associate with voltage-dependent sodium channels in brain

The segregation of voltage-dependent sodium channels to specialized regions of the neuron is crucial for propagation of an action potential. Studies of their lateral mobility indicate that sodium channels are freely mobile on the neuronal cell body but are immobile at the axon hillock, presynaptic terminal and at focal points along the axon. To elucidate the mechanisms that regulate sodium channel topography and mobility, we searched for specific proteins from the brain that associate with sodium channels. Here we show that sodium channels labelled with 3H-saxitoxin (STX) are precipitated in the presence of exogenous brain ankyrin by anti-ankyrin antibodies and that 125I-labelled ankyrin binds with high affinity to sodium channels reconstituted into lipid vesicles. The cytoplasmic domain of the erythrocyte anion transporter competes for the latter interaction. Neither the neuronal GABA (gamma-aminobutyric acid) receptor channel complex nor the dihydropyridine (DHP) receptor bind brain ankyrin. The results indicate that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeleton and may help to maintain axolemmal membrane heterogeneity and control sodium channel mobility.

Myelin-associated glycoprotein: electron microscopic immunocytochemical localization in compact developing and adult central nervous system myelin.

Light microscopic immunocytochemical studies have shown that myelin-associated glycoprotein (MAG) is localized in myelin of the developing CNS; but in the adult, MAG appears to be restricted to periaxonal regions of myelinated fibers. To extend these observations, we embedded optic nerves of 15-day-old rats, adult rats, and an adult human in epon after aldehyde and osmium tetroxide fixation. After 5% H2O2 pretreatment, thin sections were immunostained with 1:250-1:5,000 rabbit antiserum to rat CNS MAG according to the avidin-biotin-peroxidase complex (ABC) method. Dense deposits of reaction product covered compact myelin in both developing and adult optic nerves. When we used 1:500, 1:1,000, and 1:2,000 anti-MAG, less intense immunostaining of myelin was found. We also obtained the same localization in compact myelin with the peroxidase-antiperoxidase (PAP) method. With 1:250 anti-MAG, dense deposits of reaction product were not observed on axolemmal membranes or on oligodendroglial membranes located periaxonally and paranodally. In thin sections of adult human optic nerve, anti-MAG also stained compact myelin intensely. When thin sections of rat and human optic nerves were treated with preimmune or absorbed serum, no immunostaining was observed. Immunoblot tests showed that our MAG antisera did not react with any non-MAG myelin proteins. In contrast with earlier light microscopic data, this study shows that MAG localization does not change during CNS development; both developing and adult compact myelin sheaths contain MAG. As many biochemical studies also show that MAG is present in compact myelin, we suggest that this 100,000 dalton glycoprotein now be called myelin glycoprotein (MGP) instead of MAG.

Calcium distribution and transport in myelinated nerve

Ca in desheathed trunks of sciatic nerve ofRana pipiens is completely exchangeable with radioactive45Ca.45Ca distributes in three compartments, one intracellular compartment and two extracellular compartments, which exchange Ca with greatly different rate coefficients. Using impermeable solutes (sucrose, inulin, SO4) and electron microprobe techniques, we have identified the rapidly exchanging extracellular compartment as the interstitial space and the slowly exchanging extracellular compartment, accessible to solutes up to a molecular weight of 5,500, as the mesaxonal gap in the myelin sheath. Ca exists in both extracellular compartments as free Ca and in bound form. After equilibration in solutions containing 1 mM Ca, the contents of the extracellular compartments in mmole/kg wet weight are: 1) Interstitial space: free Ca=0.52, bound Ca=0.19; 2) Myelin region: free Ca=0.12, bound Ca 0.27. The intracellular space contains 0.11 mmole/kg wet weight of Ca. After 4 h of washout,45Ca release represents the Ca flux from the intracellular compartment since it can be inhibited 95% by the combined action of Na and Ca removal and the addition of La. Our results suggest further that45Ca release after this time represents a close approximation of the Ca transport across the axolemmal membrane.45Ca, therefore, represents a suitable tracer probe to investigate Ca compartmentalization and Ca exchange in this tissue.

Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction

1. Reversible depletion of synaptic vesicles from frog cutaneous pectoris neuromuscular junctions was studied by application of a Ringer solution containing 115 mM-K propionate.2. During the release of transmitter, the synaptic vesicle membrane is added to the axolemmal membrane. Under the conditions of high K(+)-induced release, the synaptic vesicle membrane accumulates as folds formed in the region of the axolemmal membrane between the active zones. In depleted terminals, large vesicular structures appear and the evidence shows that some of them (possibly all) are formed as axolemmal infoldings. During formation of such infoldings the active zones remain fixed in position with respect to the post-junctional membrane.3. During recovery in normal Ringer solution, which followed 30 min depolarization in high K(+) Ringer solution, spontaneous m.e.p.p.s were detected as early as 9 min after the start of the recovery period and the average time for their reappearance was 17 min.4. At the end of a 20 min recovery period which followed K(+) depolarization, small accumulations of synaptic vesicles were again found within the terminal close to the active zones. At this time coated vesicles and coated pits were seen associated with the prejunctional axolemma and its infoldings. It appears that synaptic vesicles are re-formed directly from these coated vesicles.5. After 60 min recovery from K(+) depolarization, at which time stimulation of the motor nerve induced a muscle twitch, the structure of the terminals closely resembled that of control preparations.6. The entire synaptic vesicle recycling process can take place in the absence of the neurone soma.

Ultrastructural localization of acetylcholinesterase in autonomic postganglionic axons

Acetylcholinesterase activity of autonomic nerve fibers in ther dilatator area of the rat iris was located light- and electronmicroscopically by means of am, dified thiocholin technique. Some axons exhibit acetylcholinesterase activity, other ones are devoid of this enzyme. The enzyme is located in the axolemmal membrane of cholinergic nerve fibers. On the basis of these studies, the idea is refuted that adrenergic postganglionic axons also exert acetylcholinesterase activity.

Electron microscope study of the human neuromuscular junction.

A preliminary electron microscope study of human neuromuscular junction is presented. The biopsy material was taken from the palmarus longus, and fixed routinely in osmium tetroxide and embedded in methacrylate. The structure of the motor endings and the relationship of the synaptic vesicles to the axolemmal membrane are described. The synaptic clefts are filled with an homogeneous material in continuity with the basement membrane covering the muscle fiber. The subneural apparatus is described, and special attention is paid to a vesicular component present in the sarcoplasm of the junctional area, which differs from synaptic vesicles and is presumed to be a derivate of the sarcoplasmic reticulum.