Retrograde Axoplasmic Transport Research Articles

Bone marrow mesenchymal stem cell-derived exosome uptake and retrograde transport can occur at peripheral nerve endings

We investigated the occurrence of mesenchymal stem cell (MSC)-derived exosome uptake and retrograde transport at peripheral nerve endings using bone marrow MSCs (bMSCs) transduced with recombinant CD63-green fluorescent protein (GFP) lentiviral plasmid. GFP was used to track the release of bMSC-derived exosomes and the uptake and transport at peripheral nerve terminals, the dorsal root ganglion (DRG), and the spinal cord. In vitro cell culture and injection of a CD63-GFP exosome suspension into the right gastrocnemius muscle of an in vivo rat model were also performed. Fluorescence microscopy of co-cultured CD63-GFP exosomes and SH-SY5Y or BV2 cell lines and primary cultured DRG cells in a separate experiment demonstrated exosome uptake into DRG neurons and glia. Moreover, we observed both retrograde axoplasmic transport and hematogenous transport of exosomes injected into rat models at the DRG and the ipsilateral side of the anterior horn of the spinal cord using fluorescence microscopy, immunohistochemistry, and Western blot analyses. In conclusion, we showed that exosome uptake at peripheral nerve endings and retrograde transport of exosomes to DRG neurons and spinal cord motor neurons in the anterior horn can occur. In addition, our findings propose a novel drug delivery approach for treating neuronal diseases.

Fresh human amniotic membrane effectively promotes the repair of injured common peroneal nerve.

Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with α-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin-eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve; the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.

Reversible blockade of retrograde axonal transport in the rat sciatic nerve by vincristine.

Altered axoplasmic transport has been implicated as a contributing factor in neuropathic pain states. Treatments which produce transient axoplasmic transport blockade have shown initial promise as therapeutic procedures in the management of chronic neuropathic pain. The present study evaluated the capacity of the vinca alkaloid, vincristine to produce reversible blockade of retrograde axoplasmic transport. Results indicated that intraneural administration of 10(-5)M vincristine resulted in blockade of retrograde axoplasmic transport of [3H]leucine 24 h following administration. Importantly, the effect reversed over the next four days and axoplasmic transport was re-instated fully at day five. The implications of these results are discussed in relation to the potential use of axoplasmic transport blocking agents in the management of chronic neuropathic pain.

Mitigation of nociception via transganglionic degenerative atrophy: Possible mechanism of vinpocetine-induced blockade of retrograde axoplasmic transport

Vinpocetine, a derivative of vincamine, widely used in the clinical pharmacotherapy of cerebral circulatory diseases, inhibits retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve, resulting in transganglionic degenerative atrophy (TDA) in the related ipsilateral superficial spinal dorsal horn, as shown in our previous publications. TDA induced by vinpocetine has been demonstrated to be followed by depletion of the marker enzyme fluoride-resistant acid phosphatase (FRAP) and its isoenzyme thiamine monophosphatase (TMP), and by the decrease in the pain-related neuropeptide substance P from laminae I-II-(III) from the segmentally related, ipsilateral substance of Rolando of the spinal cord. In the present paper, we report on the behavioral effects of perineurally administered vinpocetine. Nociception, induced by intraplantar injection of formalin, was mitigated by vinpocetine; increased expression of c-fos in the ipsilateral, segmentally related upper dorsal horn was also prevented. Since vinpocetine is not a microtubule inhibitor, and its chemical structure differs from that of vincristin and vinblastin (used formerly by us in the therapy of intractable, chronic neuropathic pain), its mode of action is enigmatic. We assume that the effect of vinpocetine in blocking retrograde axoplasmic transport of NGF might be related to its interaction with membrane trafficking proteins, such as signalling endosomes and the endocytosis-mediating "pincher" protein. Temporary, locally restricted decrease of nociception, induced by vinpocetine, might be useful in the clinical treatment of intractable, chronic neuropathic pain, since vinpocetine can successfully be applied by transcutaneous iontophoresis.

Targeting Inflammatory Demyelinating Lesions to Sites of Wallerian Degeneration

In Theiler’s murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelinating lesions, and the distribution of axonal damage present during the early phase of infection corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal damage recruits inflammatory cells to sites of Wallerian degeneration, leading to demyelination. Three weeks after TMEV infection, axonal degeneration was induced in the posterior funiculus of mice by injecting the toxic lectin Ricinus communis agglutinin (RCA) I into the sciatic nerve. Neuropathology was examined 1 week after lectin injection. Control mice, infected with TMEV but receiving no RCA I, had inflammatory demyelinating lesions in the anterior/lateral funiculi. Other control mice that received RCA I alone did not develop inflammatory lesions. In contrast, RCA I injection into TMEV-infected mice induced lesions in the posterior funiculus in addition to the anterior/lateral funiculi. We found no differences in lymphoproliferative responses or antibody titers against TMEV among the groups. This suggests that axonal degeneration contributes to the recruitment of inflammatory cells into the central nervous system by altering the local microenvironment. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).

Optic Nerve Hydropic Axonal Degeneration and Blocked Retrograde Axoplasmic Transport

To describe histopathologic features of anterior optic nerves of 12 eyes enucleated for sustained high ocular pressure from iris-ciliary body melanomas in 10 and choroidal melanomas with chronic angle closure in 2. In this retrospective study, we analyzed cases indexed in 2 eye pathology laboratories and reviewed the pertinent literature. Cases were identified from diagnostic indexes; microscopic study of slides stained with hematoxylin-eosin and Verhoeff-van Gieson, Mallory trichrome, periodic acid-Schiff, alcian blue, or colloidal iron for acid mucopolysaccharide; review of available clinical documentation; and analysis of features and photography. The main outcome measures were description of optic nerve heads, prelaminar atrophy, laminar posterior bowing, locations and density of hydropic axonal degeneration, blocked retrograde axoplasmic transport, posterior atrophy, and optic nerve disorganization with glial proliferation. Hydropic axonal degeneration was present in front of, within, and posterior to the lamina cribrosa in all 12 eyes. This degeneration extended diffusely and posteriorly from the peripheral lamina and was most dense centrally in 10 eyes. Retrolaminar changes compatible with blockage of retrograde axoplasmic transport were seen in 9 eyes. Posterior atrophy with disorganization and glial proliferation was seen in 10 eyes. No eye had classic glaucomatous atrophic cupping. Diffuse and centrally intense hydropic axonal degeneration and central blocked retrograde axoplasmic transport explain loss of central acuity, generalized contraction of visual field, and generalized optic atrophy without glaucomatous cupping in eyes with prolonged high-pressure secondary glaucoma.

Effect of vinpocetine on retrograde axoplasmic transport

Vinpocetine, a derivate of vincamine, is widely used in the clinical pharmacotherapy of cerebral circulatory diseases. Herewith we report on a novel effect of vinpocetine: inhibition of retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve. Blockade of retrograde transport of NGF results in transganglionic degenerative atrophy (TDA) in the segmentally related ipsilateral superficial spinal dorsal horn, which is characterized by depletion of the marker enzymes fluoride-resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP). At the same time, pain-related neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), are depleted from lamina I-III from the segmentally related, ipsitateral Rolando substance of the spinal cord. On the basis of these experiments it is suggested that vinpocetine may result in a locally restricted decrease of nociception, that might be useful in clinical treatment of intractable pain. Pilot self-experiments support this assumption.

Understanding optic nerve degeneration: Key to neuroprotection in glaucoma

Glaucoma is a group of neurodegenerative disorders characterized by degeneration of optic nerve axons and retinal ganglion cell death. While the most common strategy in glaucoma therapy is aimed at lowering elevated intraocular pressure (IOP), an emerging field of research is currently focused on protecting the optic nerve from degeneration. Neuroprotection is directed at preventing or slowing the death of retinal ganglion cells, thereby decreasing the progression of visual field loss. In all optic neuropathies including glaucoma, the initial site of injury are the axons of retinal ganglion cells. Axonal injury triggers apoptotic mechanisms that ultimately culminate in retinal ganglion cell death. Neuroprotective approaches are varied: 1) the prevention of apoptosis by inhibiting TNF-α and caspase activity; 2) blocking excessive Ca 2+ overload due to overactivation of NMDA receptors; or 3) blocking nitric oxide toxicity. Axonal injury may cause the blockade of retrograde axoplasmic transport of neurotrophins essential for retinal ganglion cells. In this sense, neurotrophic factors such as brain-derived neurotrophic factor (BDNF) have been reported to prolong retinal ganglion cell survival, and α 2 -adrenoceptor agonists such as brimonidine are thought to exert their neuroprotective effects partly by upregulating BDNF expression. Modulation of the immunological response has also proved effective in reducing retinal ganglion cell loss, suggesting a possible use of vaccines to prevent glaucoma progression.

Vibration‐induced disruption of retrograde axoplasmic transport in peripheral nerve

Hand-arm vibration syndrome (HAVS) results from excessive exposure to hand-transmitted vibration. Whether the peripheral nerve damage characteristic of HAVS is a direct result of vibration or is secondary to vascular insufficiency remains unclear. The purpose of this study was to explore the effect of vibration exposure on axoplasmic transport in peripheral nerves and soleus motor neurons. Sciatic nerves and motor neurons from rats following two 5-h periods of vibration exposure demonstrated disruption in retrograde transport compared to normal. After 10 days of vibration (5 h/day), axoplasmic transport failed to recover within 24-48 h in most rats. This study demonstrates that disrupted axoplasmic transport is an early consequence of short-term vibration exposure. The effects of vibration on axoplasmic transport also appear to be cumulative. This study provides a new biological way to evaluate measures to prevent early vibration injury.

Ganglion cell death in rat retina by persistent intraocular pressure elevation.

Glaucoma is characterized by loss of retinal ganglion cells (RGCs) and their axons. Retrograde axoplasmic transport blockade and excitotoxicity were proposed to be a major cause of RGC apoptosis. We conducted this study to characterize the episcleral vessel cauterization glaucoma model in the rat with respect to decreased retrograde axoplasmic flow and subsequent apoptotic RGC death. After episcleral vessels were cauterized in Sprague-Dawley rats, Fluorogold was injected into their superior colliculi by stereotactic method. Retrograde axoplasmic flow and TUNEL-stained apoptotic dead cells were observed microscopically. Elevated intraocular pressure was maintained for up to 6 weeks during follow-up. Retrograde axoplasmic flow to the rat retina was significantly decreased. Apoptotic RGC was selectively TUNEL-stained in the retina, especially at the ganglion cell layers. We concluded that elevated intraocular pressure caused apoptotic RGC death through retrograde axoplasmic flow blockage. Further studies will elucidate the neuroprotection strategies in glaucoma patients.

Molecular plasticity of primary nociceptive neurons: relations of the NGF- c-jun system to neurotomy and chronic pain

Neurotomy is widely used as a model of chronic, intractable pain, the proverbial "crux medicorum". Immunohistochemical aspects of this chronic pain model are discussed in this paper, with the aim of shedding new light on the pathomechanism and possible therapeutical consequences. Central terminals of nociceptive neurons contain substance P, somatostatin and calcitonin generelated peptide or exhibit fluoride resistant acid phosphatase and thiamine monophosphatase enzyme reaction in the superficial dorsal horn of the spinal cord and in analogous structures of the brain stem. These neuropeptides and neuroproteins are expressed by the related dorsal root ganglion cells and transported via orthograde axoplasmic transport via dorsal roots to the central nervous system. Transection of the ipsilateral, segmentally related peripheral sensory nerve results in transganglionic degenerative atrophy of central terminals of primary nociceptive neurons. Transganglionic degenerative atrophy is characterized by marked ultrastructural alterations superficially similar to, but essentially differing from the signs of Wallerian degeneration which ensue after dorsal rhizotomy. Transganglionic degenerative atrophy is accompanied by depletion of marker neuropeptides and enzymes, and later by the expression of vicarious neuropeptides such as vasoactive intestinal polypeptide, neuropeptide Y and galanin and of the enzyme choline acetyl transferase. Consequences of blockade of retrograde axoplasmic transport of the nerve growth factor elicited either by perineural application of microtubule inhibitors or by perineural administration of anti-nerve growth factor are similar to peripheral neurotomy. According to recent studies described in this paper, the blockade of nerve growth factor supply to primary nociceptive neurons induces activation of c-jun in nuclei of primary nociceptive neurons probably responsible for the plasticity of the neuropeptide and neuroprotein synthesizing machinery. In contrast, invasion of and formation of pericellular baskets by noradrenergic axons can be elicited only by axotomy and not by blockade of retrograde axoplasmic transport. Involvement of nerve growth factor and the nerve growth factor-dependent immediate early genes in neuroplasticity of neuropeptidergic primary sensory neurons raise the possibility of a gene therapy of chronic intractable pain.

Intradermal doxorubicin reduces ganglionic reactivation of latent herpes simplex virus in mice after pretreatment with hypertonic saline.

Recurrent lesions from herpes simplex virus (HSV) occur after reactivation of latent HSV in neurons of sensory ganglion, axonal transport of reactivated virus, and HSV replication on the skin. A potential treatment strategy is to inject epithelial sites of frequent recurrences with anti-viral or cytotoxic agents that are taken up by nerve terminals and transported by axoplasmic flow to latently infected ganglionic neurons. Doxorubicin is transported by nerves and destroys the corresponding nerve cell bodies, but earlier attempts in HSV animal models required intraneural injection to eliminate HSV infection and this treatment destroyed the nerve and large portions of the ganglion. The present study used intradermal doxorubicin in latently infected mice that had been inoculated with HSV by the lip route and passively immunized at the time of inoculation. As found previously, doxorubicin injection in the lip 2 or more months after HSV inoculation did not eliminate HSV latency as evaluated by recovery of virus from trigeminal ganglionic explants. However, when hypertonic saline was injected in the same site 24 hr prior to doxorubicin, there was a 55% reduction of positive ganglionic explant cultures. Edema from the hypertonic saline may increase access of doxorubicin to nerve terminals. This technique with hypertonic saline, which has also been used to enhance virulence of HSV skin innoculation, may have general application of increasing axoplasmic transport of drugs or biologicals. Skin toxicity may preclude doxorubicin use for HSV recurrences in patients; however, the results of this study support the concept of anti-HSV treatment via retrograde axoplasmic transport.

Dorsal root ganglia sensory neurons.

The ability of lentivirus vectors to stably transduce postmitotic cells by genomic integration and early evidence of a capacity for retrograde axoplasmic transport, depending on the envelope pseudotype, underpin the immense potential of this vector system for neuronal transduction (, , ). The bulk of studies performed to date have focused on central nervous system (CNS) neurons despite the relative merits of peripheral nervous system (PNS) neurons as targets for gene transfer (, , ). The neural circuitry of the PNS is relatively simple, accessible, and anatomically discrete. Dorsal root ganglion (DRG) sensory neurons exemplify these properties, are readily cultured ex vivo after dissociation or as intact DRG explants, and as a consequence, have been extensively exploited as a model system by neurobiologists (). The demonstrated ability of human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors to stably transduce DRG sensory neurons () extends the utility of this model system for both basic science investigations of neuronal structure and function and for the development and evaluation of gene therapy strategies targeting PNS neuropathology.

Growth cone neuropilin-1 mediates collapsin-1/Sema III facilitation of antero- and retrograde axoplasmic transport.

Collapsin-1/Sema III, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cellsurface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose-response curve for collapsin-1 to induce growth cone collapse to higher concentrations. sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport.

Effects of ALCAR on the fast axoplasmic transport in cultured sensory neurons of streptozotocin-induced diabetic rats.

The effects of acetyl-L-carnitine (ALCAR) on fast axoplasmic transport were studied in cultured dorsal root ganglion (DRG) neurons of diabetic rats. Three-month-old male rats were used 7 days after streptozotocin injection. Neurons obtained from ganglia were cultured with a high concentration of glucose. The amount and the mean velocity of retrogradely transported particles, reduced in the diabetic animal, were transiently recovered by 1 mM ALCAR. The number of particles moving at 0.8-1.2 microm/s, considered to be lysosomes, increased in the velocity distribution. ALCAR did not modify the amount and mean velocity of anterograde particles which were unaffected by diabetes, or of bidirectional particles in neurons of control rats. This study suggests that diabetic neuropathy may be relieved by ALCAR via recovering retrograde axoplasmic transport.

A novel action of collapsin: collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse.

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons.

P-4-512 - Selective facial neuronotomy by way of the retrograde axoplasmic transport of adriamycin: Surgical experience in idiopathic blepharospasm

P-4-512 - Selective facial neuronotomy by way of the retrograde axoplasmic transport of adriamycin: Surgical experience in idiopathic blepharospasm

Role of neurotrophic factors in adaptational processes of the nervous system.

Neurotrophic factors (NTF) are low-molecular-weight proteins which epigenetically determine neuron survival during embryogenesis and the maintenance of their morphofunctional properties in the adult organism. NTF are located in mesenchymal tissues and reach neuron bodies by means of retrograde axoplasmic transport in nerve fibers; in cell bodies, NTF increase anabolic activity, neurotransmitter synthesis, and structural protein production. Neuron cell bodies have two types of specialized receptors for binding the most common NTF, nerve growth factor (NGF). In the brain, NGF does not affect adrenergic neurons, as it does in the periphery, but acts on cholinergic neurons in the basal part of the forebrain. The forebrain plays the major role in the processes of learning, memory, and plasticity, i.e., in the entire complex of adaptational responses of the nervous system, and these may thus depend on the biological activities of substances, including NTF, in forebrain tissues. An experimental model was developed consisting of organotypic co-cultivation of rat hippocampus fragments with chick embryo dorsal root ganglia. This model was used to demonstrate that epileptiform activity in the hippocampus is associated with increases in NTF release, which can be regarded as an adaptive compensatory response to nerve cell damage occurring during convulsive activity.

Differential suppression of axoplasmic transport: effects of light irradiation to the growth cone of cultured dorsal root ganglion neurons.

1. Growth cones of cultured dorsal root ganglion neurons from mice were irradiated using a mercury lamp. 2. The flux of particles of fast retrograde axoplasmic transport decreased promptly after light irradiation without a change in velocity. 3. That of anterograde transport decreased as well, but with a significant latency. The decrease in anterograde flux was attributed to decreased velocity of particles. 4. Video-enhanced contrast microscopy of growth cones revealed transient swelling of growth cones and transient stagnation of particles in growth cones. 5. The longer the neurite, the larger the latency of the change of the anterograde transport; peripheral information was calculated to be conveyed to the cell body at a speed of 6 microns/min. 6. The mechanism of this information conveyance and the export of materials from the cell body are discussed.

Gene delivery to spinal motor neurons

This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. β-Galactosidase expression was observed in α and γ motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).