Nerve growth factor regulates central terminals of primary sensory neurons

Transection of peripheral sensory axons results in transganglionic degenerative atrophy of central terminals of the affected primary sensory neurons. Nerve growth factor applied at the central stump of the transected nerve prevents or delays transganglionic degenerative atrophy. It is concluded that, under normal conditions, nerve growth factor taken up by receptors at peripheral sensory nerve endings and transported retrogradely to perikarya in dorsal root ganglia, regulates synthesis of neuroproteins destined for maintenance of central terminals of these neurons. Accordingly, transganglionic degenerative atrophy is the consequence of failure of nerve growth factor to reach perikarya of primary sensory neurons.     

Transganglionic regulation of the primary sensory neuron

Central terminals of the primary sensory neurons depend on the integrity of the retrograde transport mechanism within the peripheral axon. Whenever retrograde transport is impaired (either by injury or by blockade induced by perineural application of microtubule inhibitors) central terminals undergo transganglionic degenerative atrophy (TDA), characterized by depletion of substance P, somatostatin, FRAP (fluoride resistant acid phosphatase), TMPase (thiamine monophosphatase) and lectin-binding fucose-terminated glyco-conjugates. The TDA is essentially a failure of the central terminals to bind the above genuine marker substances. TDA-inflicted central terminals undergo a slowly proceeding ultrastructural deterioration, accompanied by derangement of the dorsal root potential, reflecting decreased functional activity of synaptic transmission between first and second-order cells. One of the important trophic substances carried by retrograde axoplasmic transport to dorsal root ganglion cells is nerve growth factor (NGF); blockade of NGF transport results in TDA; conversely, locally applied NGF delays or prevents TDA.     

Blockade of retrograde axoplasmic transport induces transganglionic degenerative atrophy of central terminals of primary nociceptive neurons

If applied locally around a peripheral sensory nerve, Formyl-Leurosin, a semi-synthetic diindol alkaloid of Vinca rosea--that, just like other mitotic spindle inhibitors, induces blockade of axoplasmic transport via inhibiting microtubular function--causes transganglionic degenerative atrophy of central terminals of primary nociceptive neurons in the substantia gelatinosa Rolandi of the spinal cord. In contrast, if applied to dorsal roots, Formyl-Leurosin fails to induce such alterations. Based upon these observations it is postulated that blockade of retrograde axoplasmic transport, rather than that of the orthograde one, is the decisive factor in the pathomechanism of transganglionic degenerative atrophy.     

Transganglionic degenerative atrophy in the substantia gelatinosa of the spinal cord after peripheral nerve transection in rhesus monkeys

Summary The effect of sciatic nerve transection on its centrally located terminals in the spinal cord was analyzed by electron microscopy in adult rhesus monkeys one and three months following lesion. Although the peripheral and intermediate portions of the dorsal roots, where the axons are enveloped by Schwann cells were normal, their central portion and their terminals in the substantia gelatinosa were remarkably altered. Transganglionic degenerative atrophy (TDA) is characterized by three distinct types of electronmicroscopic alterations. The first type exhibits a conspicuous electron density of the terminal and pre-terminal axoplasm. Importantly, shrinkage replaces fragmentation and glial engulfement of the terminal seen in the course of Wallerian degeneration. The second type is characterized by the disappearance of synaptic vesicles from the terminals. The third type of TDA consists of intricate labyrinthine structures, composed of flattened profiles of axonal, dendritic and glial elements. The complex and diverse cellular changes that occur in the upper dorsal horn following peripheral nerve injury may provide the structural basis of plasticity of the primary nociceptive system.     

Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons

Thiamine monophosphatase (TMPase) has been selectively localized in small dorsal root ganglion cells and in their central and peripheral terminals. Light microscopic localization of TMPase, and its alterations due to transganglionic effects, are identical with those of fluoride-resistant acid phosphatase (FRAP), but are not contaminated by the ubiquitous lysosomal reaction inevitable in trivial acid phosphatase-stained sections. TMPase is inhibited by 0.1 mM NaF, which is slightly less than the concentration needed to inhibit FRAP (0.2-0.4 mM). It is assumed that TMPase and FRAP are identical enzymes. In the perikaryon of small dorsal root ganglion cells, TMPase is located in the cisterns of the endoplasmic reticulum and in the Golgi apparatus. The central terminals of these cells are scalloped (sinusoid) axon terminals, surrounded by membrane-bound TMPase activity. Central terminals outline substantia gelatinosa Rolandi throughout the spinal cord, as well as the analogous nucleus spinalis trigemini in the medulla. TMPase-active central terminals outline "faisceau de la corne postérieure" in the sacral cord, as well as Lissauer's tract in the thoracic, upper lumbar, and sacral segments, and the paratrigeminal nucleus and the terminal (sensory) nucleus of the ala cinerea in the brainstem. Peripheral terminals displaying TMPase activity are fine nerve plexuses of C fibers. The TMPase activity of the central terminals disappears after dorsal rhizotomy in the course of Wallerian degeneration, and is depleted in the course of transganglionic degenerative atrophy (after transection of the related peripheral sensory nerve). TMPase is an outstanding genuine marker for the study of transganglionic regulation in Muridae.     

Depletion of substance P and somatostatin in the upper dorsal horn after blockade of axoplasmic transport

Summary In the upper dorsal horn of the rat lumbosacral spinal cord, substance P and somatostatin are present in two distinct and different populations of primary central afferent terminals. Substance-P-positive terminals are mainly concentrated in lamina I, while somatostatin-positive terminals are confined to lamina II. Although these two populations of primary afferent terminals differ at light- and electron-microscopic level, they are equally affected by transganglionic degenerative atrophy (TDA) which is induced by the blockade of axoplasmic transport in the segmentally related, ipsilateral sensory nerve by the local application of Vinblastin, a microtubule inhibitor. In consequence, substance P and somatostatin are depleted in the medial and intermediate portions of the upper dorsal horn, while the lateralmost area, which represents the postaxial portion of the dermatome, remains virtually intact. Substance P and somatostatin in propriospinal elements and the axonal meshwork within the dorsolateral funicle are not affected by TDA. Neurotensine, a propriospinal neuropeptide, does not show any alterations in the affected spinal segments.This work was supported by research grant no. 06/4-01/449 from the Hungarian Ministry of Health and no. 375/82/3.2 from the Hungarian Academy of Sciences     

Alterations of dorsal root potential in the course of transganglionic degenerative atrophy

Alterations in the dorsal root potential (DRP) which was evoked by stimulation of the common peroneal nerve of the rat, have been studied in the course of transganglionic degenerative atrophy (TDA) of primary sensory terminals in the upper dorsal horn. TDA was induced by perineural application of Vinca alkaloids around the sciatic nerve. In 9 to 30 days after this treatment, latency of DRP increased, whereas its amplitude and duration decreased. In this period, no C fibre response could be elicited. As a possible mechanism underlying the alterations of DRP, the functional consequences of atrophic changes of primary central afferent terminals are being discussed in terms of the close correlation between structure and function and the possible inferences of the electrophysiological reaction to the therapeutic application of Vinca alkaloids in the iontophoretic treatment of chronic intractable pain.     

Fine structure of growth cones in the upper dorsal horn of the adult primate spinal cord in the course of reactive synapto-neogenesis

Summary Following transganglionic degenerative atrophy of primary afferent terminals induced by a crush-injury of the sciatic nerve, a regenerative process takes places in the upper dorsal horn of the lumbar spinal cord in the primate Macacus rhesus. Axonal growth cones are characterized by cisterns of axoplasmic reticulum; filopodia emanating from growth cones are electron-optically translucent sheet-like expansions, often containing growth-cone vesicles. Axoplasmic reticulum appears also in preterminal portions of regenerating axons. Dendritic growth cones contain a fine, filamentous matrix; electron-dense membrane specializations can be seen in well-defined areas of their surfaces. Immature synapses are formed between filopodia of axonal growth cones and dendritic growth cones. Electron-microscopic structures of this unique CNS regeneration are similar to those seen in the course of embryonic development of the spinal cord.     

Extrinsic cellular and molecular mediators of peripheral axonal regeneration

The ability of injured peripheral nerves to regenerate and reinnervate their original targets is a characteristic feature of the peripheral nervous system (PNS). On the other hand, neurons of the central nervous system (CNS), including retinal ganglion cell (RGC) axons, are incapable of spontaneous regeneration. In the adult PNS, axonal regeneration after injury depends on well-orchestrated cellular and molecular processes that comprise a highly reproducible series of degenerative reactions distal to the site of injury. During this fine-tuned process, named Wallerian degeneration, a remodeling of the distal nerve fragment prepares a permissive microenvironment that permits successful axonal regrowth originating from the proximal nerve fragment. Therefore, a multitude of adjusted intrinsic and extrinsic factors are important for surviving neurons, Schwann cells, macrophages and fibroblasts as well as endothelial cells in order to achieve successful regeneration. The aim of this review is to summarize relevant extrinsic cellular and molecular determinants of successful axonal regeneration in rodents that contribute to the regenerative microenvironment of the PNS.     

Costorage of BDNF and neuropeptides within individual dense-core vesicles in central and peripheral neurons

Some central and peripheral neurons synthesize brain-derived neurotrophic factor (BDNF), and, after anterograde transport, release it at synapses. By immunocytochemistry, we examined, in rat and mouse, the subcellular localization of BDNF and BDNF/peptide coexistence, under normal conditions or after intrathecal infusion of nerve growth factor. In dorsal root ganglion neurons and afferent terminals, and in the parabrachial projection to amygdala, we show that BDNF is costored in individual dense-core vesicles (DCVs) with the neuropeptides calcitonin gene-related peptide (CGRP) and substance P. At both locations, nerve endings costoring all three peptides were fairly rare. Remarkably however, costorage occurred in a stoichiometric ratio of 0.7 BDNF:1 CGRP:1 substance P, and DCVs contained 31 (spinal cord) -36 (amygdala) times the amount of BDNF detected in agranular vesicles. This is the first direct demonstration in peripheral and central neurons from two different mammals, that a growth factor is selectively packaged together with neuropeptide transmitters within individual DCVs. It provides structural bases for differential release upon stimulation, and has important implications for understanding BDNF transmitter function.     

Exosomes from human adipose-derived stem cells promote sciatic nerve regeneration via optimizing Schwann cell function

Human adipose-derived stem cells (ASCs) have a potential for the treatment of peripheral nerve injury. Recent studies demonstrated that stem cells can mediate therapeutic effect by secreting exosomes. We aimed to investigate the effect of human ASCs derived exosomes (ASC-Exos) on peripheral nerve regeneration in vitro and in vivo. Our results showed after being internalized by Schwann cells (SCs), ASC-Exos significantly promoted SC proliferation, migration, myelination, and secretion of neurotrophic factors by upregulating corresponding genes in vitro. We next evaluated the efficacy of ASC-Exo therapy in a rat sciatic nerve transection model with a 10-mm gap. Axon regeneration, myelination, and restoration of denervation muscle atrophy in ASC-Exos treated group was significantly improved compared to vehicle control. This study demonstrates that ASC-Exos effectively promote peripheral nerve regeneration via optimizing SC function and thereby represent a novel therapeutic strategy for regenerative medicine and nerve tissue engineering.     

抗 P 物质单克隆抗体的特性研究

观察了细胞株(BTP-1)所分泌的抗 P 物质单克隆抗体与17种 P 物质类似物的交叉反应。该单克隆抗体所针对的是 P 物质的羧基末端,与羧基端的甲硫氨酸残基有较大的关系,而和P 物质类似物的拮抗作用的强弱无相关性。与9种小肽(亮氨酸脑啡肽、甲硫氨酸脑啡肽,内啡肽、皮啡肽、血管紧张素Ⅰ、Ⅱ、缓激肽,催产素、胰高血糖素)均无交叉反应,显示该单克隆抗体有较好的特异性。经免疫组织化学试验,中脑以下部位的脊髓后角Ⅱ层、黑质、三叉神经脊束核、脚间核等末梢纤维和第Ⅱ脑室腹侧灰质、被盖外侧核、中缝核等胞体核周质染成棕色。BTP-1属于大鼠 IgG 型单克隆抗体。描述了微载体大量培养杂交瘤和制备单克隆抗体的技术,并讨论了大量制备的可能性。     

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

Nitric oxide synthase, an essential factor in peripheral nerve regeneration.

Nitric oxide (NO) exerts both, pro-apoptotic and anti-apoptotic actions and appears to be acritical factor inneuronal degenerative and regenerative processes. NO is synthesized from L-arginine by NO synthase occurring in three isoforms of (neuronal, nNOS; endothelial, eNOS; inducible, iNOS). In a mice sciatic nerve model the regenerative outcome was assessed when the endogenous NO supply was deficient by knocking out the respective NOS isoform and compared to that of wild type mice after nerve transection. In nNOS knock-out mice a delay in regeneration, preceded by slowedWallerian degeneration and a disturbed pruning of uncontrolled sprouts, was observed. This was associated with a delayed recovery of sensory and motor function. Additionally, deficiency of nNOS led after nerve cut to a substantial loss of small and medium-sized dorsal root ganglia neurons, spinal cord interneurons and, to a lesser extent, spinal cord motor neurons. A lack of iNOS resulted in a delayed Wallerian degeneration and impaired regenerative outcome without consequences for neuronal survival. A lack of eNOS was well tolerated, although a delay in nerve revascularization was observed. Thus, after peripheral nerve lesion, regular NOS activity is essential for cell survival and recovery with reference to the nNOS isoform.     

Autotransplantation of previously denervated muscles in the rabbit

Whole gastrocnemius muscles of rabbits, preliminarily denervated, were grafted. At the moment of grafting (60 days after the operation) the muscles were in the state of deep atrophy attended by distrophic changes.The autotransplantated muscles took at the site of grafting, their further reorganization provided progressive development of the muscle tissue within the transplant, its growth, and formation of definitive muscle fibers with nerve terminals. After a definite time some degenerative changes were observed in the transplant muscle tissue; as a result the muscle tissue was substituted by connective tissue. These data support the statement founded before on feasible free grafting of preliminary denervated whole muscles. However, deep denervation atrophy seems to influence the remote results of the transplantation.     

CHANGES IN CENTRAL NORADRENALINE NEURONSADMINISTRATION AFTER SYSTEMIC 6-HYDROXYDOPAMINE ADMINISTRATION

—Intravenous injection of a large dose of 6-hydroxydopamine (100 mg/kg) to adult rats caused a significant and long-lasting reduction (about 30 per cent) of the in oirro uptake of [³H]NA in the cerebral cortex and spinal cord, while no changes were seen in the hypothalamus. The endogenous NA in whole brain was similarly reduced (about 20 per cent). Fluorescence histochemistry revealed catecholamine accumulations which are degenerative signs, induced by 6-hydroxydopamine, in axons of the dorsal NA bundle innervating the cerebral cortex. It is concluded that the blood–brain barrier in adult rats is not completely protective with respect to the neurotoxic action of systemically injected 6-hydroxydopamine, which can produce degeneration of a significant number of NA nerve terminals in the cerebral cortex and spinal cord. Previous studies have shown that 6-hydroxydopamine caused a permanent and selective degeneration of a large number of central NA nerve terminals when injected systemically up to 1 week after birth, due to an incompletely developed blood-brain barrier. This barrier for 6-hydroxydopamine develops between the 7th and 9th day after birth (Sachs , 1973). In the present study 6-hydroxydopamine was found to cause a small transient reduction in [³H]NA uptake in cerebral cortex of rats between 9 and 28 days of age, while in older rats the damage produced by 6-hydroxydopamine was long-lasting. Thus, the NA nerves ascending to the cerebral cortex seem to possess a regenerative capacity to a 6-hydroxydopamine-induced degeneration up to about 28 days postnatally, but which later disappears or is markedly retarded.     

A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration

As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an appropriate regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body’s natural regenerative microenvironment closely, we co-cultured Schwann cells (SCs) and adipose-derived stem cells (ADSCs) as seed cells and introduced them into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC). Twelve weeks after the three different grafts (plain SF/collagen scaffold, TENC, and autograft) were transplanted to bridge 1-cm long sciatic nerve defects in rats, a series of electrophysiological examinations and morphological analyses were performed to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration. The regenerative outcomes showed that the effect of treatment with TENCs was similar to that with autologous nerve grafts but superior to that with plain SF/collagen scaffolds. Meanwhile, no experimental animals had inflammation around the grafts. Based on this evidence, our findings suggest that the TENC we developed could improve the regenerative microenvironment and accelerate nerve regeneration compared to plain SF/collagen and may serve as a promising strategy for peripheral nerve repair.     

Localization of substance P- and enkephalin-like immunoreactivity in the human paravertebral sympathetic ganglia

The presence of substance P- and enkephalin-like immunoreactive nerve fibers and terminals is demonstrated in the human paravertebral sympathetic ganglia by an indirect immunofluorescence technique. Substance P-positive nerve structures appear in the form of fiber bundles, isolated varicose filaments and dot-like and basket-like nerve terminals around the neuronal cell bodies. Their density shows a remarkable individual variability. Enkephalin-positive nerve structures appear as isolated varicose filaments and dot-like nerve terminals, forming densely innervated patchy areas. No substance P- or enkephalin-containing cell bodies were detected. No overlapping seems to exist among the areas innervated by the two types of neuropeptides.     

Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals

Antibodies were raised in rabbits to synaptic vesicles purified to homogeneity from the electric organ of Narcine brasiliensis, a marine electric ray. These antibodies were shown by indirect immunofluorescence techniques to bind a wide variety of nerve terminals in the mammalian nervous system, both peripheral and central. The shared antigenic determinants are found in cholinergic terminals, including the neuromuscular junction, sympathetic ganglionic and parasympathetic postganglionic terminals, and in those synaptic areas of the hippocampus and cerebellum that stain with acetylcholinesterase. They are also found in some noncholinergic regions, including adrenergic sympathetic postganglionic terminals, the peptidergic terminals in the posterior pituitary, and adrenal chromaffin cells. They are, however, not found in many noncholinergic synapse-rich regions. Such regions include the molecular layer of the cerebellum and those laminae of the dentate gyrus that receive hippocampal associational and commissural input. We conclude that one or more of the relatively small number of antigenic determinants in pure electric fish synaptic vesicles have been conserved during evolution, and are found in some but not all nerve terminals of the mammalian nervous system. The pattern of antibody binding in the central nervous system suggests unexpected biochemical similarities between nerve terminals heretofore regarded as unrelated.     

Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse

The coordinated movement of many organisms relies on efficient nerve–muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non-myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ regeneration.