Transganglionic Degenerative Atrophy Research Articles

Iontophoretically applied microtubule inhibitors induce transganglionic degenerative atrophy of primary central nociceptive terminals and abolish chronic autochtonous pain.

Transcutaneous iontophoresis of microtubule inhibitors (Vinblastin, Vincristin, Formyl-Leurosin) in rats induces depletion of fluoride-resistant acid phosphatase (FRAP) and transganglionic degenerative atrophy (trggl. deg. atr.) of the central terminals of primary nociceptive neurons, probably via blockade of axoplasmic transport in the peripheral sensory nerves. Radiochemical experiments prove that about 0.2% of the microtubule inhibitors applied iontophoretically at the skin reach the level of nociceptive axon terminals. 40 out of 48 patients suffering from chronic intractable pain of diverse etiology (postherpetic, paresthetic, ischaemic and trigeminal neuralgia, alcoholic and diabetic polyneuropathy, meralgia, brachialgia, discopathia, arthropathia and terminal pain) were successfully treated with Vinblastin or Vincristin iontophoresis. Iontophoretically applied microtubule inhibitors do not affect the blood cell count, have no side-effects and do not impair the skin at the site of application.

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.

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.

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.

Fine structural correlates of VIP-like immunoreactivity in the upper spinal dorsal horn after peripheral axotomy: possibilities of a neuro-glial translocation of a neuropeptide.

After transection of the peripheral nerve, VIP-like immunoreactivity (VIPLI) increases markedly in the ipsilateral upper dorsal horn of the spinal cord. Immunoreactivity has been studied by means of light- and electron microscopic immunocytochemistry. Under normal conditions, there is little VIPLI present in the superficial dorsal horn, confined to small dot-like elements corresponding to axonal and glial profiles. At the electron microscopic level, immunostaining was found mainly in preterminal and, partly, also in en passant terminal swellings or varicosities. The reaction was confined to the axoplasm and, to a lesser extent, to large dense core vesicles. VIPLI is also present in several astroglial processes. 13, 19, and 25 d after transection of the sciatic nerve, increased immunoreactivity was present in the medial 2/3 of the superficial dorsal horn. Electron microscopically, VIPLI was seen mainly in preterminal axons and in many astroglial processes surrounding axon terminals while VIPLI in the en passant axon terminals themselves decreases. 2 months after peripheral axotomy, the amount of axonally localized VIPLI decreases considerably and most of the immunocytochemically detectable VIPLI is found in expansions and processes of astroglial cells. Perikarya of glial cells rarely exhibit VIPLI. VIPLI also increased after crushing the related peripheral nerve; however, as soon as the nerve fibers regenerate, VIPLI decreases again to normal levels. It appears that blockade of the retrograde axoplasmic transport induces a switch in the neuropeptide synthesizing machinery of dorsal root ganglion cells which results in the expression of VIP instead of substance P, somatostatin and CGRP. It is proposed that VIP is released from axon terminals affected by transganglionic degenerative atrophy. Subsequently, astroglial cells equipped with receptors for VIP, might bind and internalize the released VIP.

Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration.

Crush or transection of a peripheral nerve is known to induce transganglionic degenerative atrophy (TDA) in the segmentally related, ipsilateral Rolando substance of the spinal cord. When the lost peripheral connectivity is reestablished, the consecutive regenerative synaptoneogenesis results in restoration of the circuitry in the formerly deteriorated upper dorsal horn. Enhanced expression of the growth-associated protein (GAP43) B-50 occurs during neuronal differentiation, axon outgrowth, and peripheral nerve regeneration. This study documents changes in immunocytochemical distribution of B-50 in the regions of the lumbar spinal cord which are segmentally related to the axotomized sciatic nerve. At the light microscopic level, a weak B-50 immunoreactivity (BIR) is present in the neuropil of the upper dorsal horn of control animals. After unilateral transection and ligation of the sciatic nerve, BIR increased in the ipsilateral upper dorsal horn at 17 days postinjury, but decreased again after 24 days with respect to the contralateral side. Differences between effects of crush and transection were prominent in combined crush-cut experiments as well (i.e., after unilateral crush and contralateral transection and ligation of the sciatic nerve). Electron microscopic studies show that in the uninjured and injured spinal cord, BIR is detected in axons and axon terminals, but not all are stained. After transection of the sciatic nerve, BIR is found in afflicted primary sensory axon terminals, including those contacting substantia gelatinosa neurons and in axon terminals undergoing glial phagocytosis. The localization of BIR seen after crushing the sciatic nerve is similar. However, at 24 days after crush, BIR is detected also in axonal growth cones. In the ventral horn of control animals, synaptic boutons impinging upon motor neurons exhibited weak BIR. At 17 days after unilateral transection of the sciatic nerve, the pericellular BIR surrounding motor neurons is decreased at the ipsilateral with respect to the contralateral side, whereas 24 days after crush injury it increased considerably. Our results show that peripheral nerve injury inducing TDA also affects BIR distribution in the spinal gray matter. Successful regeneration of the peripheral nerve after crush lesion is associated with enhanced expression of B-50 in growth cones of sprouting central axons. The neuroplastic response of B-50 is in line with a function of B-50 in axonal sprouting and reactive synaptogenesis.

Alterations in glial fibrillary acidic protein immunoreactivity in the upper dorsal horn of the rat spinal cord in the course of transganglionic degenerative atrophy and regenerative proliferation

Transection of a peripheral nerve induces marked increase in the glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral, segmentally related upper dorsal horn. Increase of GFAP immunoreaction is similar to, but not identical with, that observed after dorsal rhizotomy. If the peripheral nerve succeeds in regenerating, GFAP immunoreactivity in the upper dorsal horn returns to normal. It is concluded that the amount and distribution of GFAP is determined by transganglionic degenerative atrophy, Wallerian degeneration and regenerative proliferation of dorsal root axon terminals, respectively.

Transneuronal degeneration in the Rolando substance of the primate spinal cord evoked by axotomy-induced transganglionic degenerative atrophy of central primary sensory terminals.

Transection of the sciatic nerve in Rhesus monkeys and the consequent transganglionic degenerative atrophy (TDA) of central terminals of primary afferents result in transneuronal degeneration of substantia gelatinosa (SG) cells. Severe degeneration is characterized by an increased electron density of the nucleus and by conspicuous shrinkage of the cytoplasm, mitochondrial swelling, dilation of cisterns of the rough-surfaced endoplasmic reticulum, accumulation of free ribosomes and an electron-dense material in the cytoplasm. In the mild form, dilation of cisternal elements of the endoplasmic reticulum, swollen mitochondria and accumulation of free ribosomes takes place. About 10% of SG cells in segment L5 undergo the severe form whereas the rest shows signs of the mild form. Cytoplasmic alterations that occur during transneuronal degeneration seem to start at the level of subsurface cisterns. Dendrites and axons of transneuronally degenerating SG cells also show a conspicuous electron density. By analyzing the synaptic relationships of such "darkened" dendrites, connections in the upper dorsal horn can be deciphered. Modular units of the primary nociceptive analyzer that evaluate noxious and innocuous inputs on the basis of thin versus thick (A delta C/A beta) afferent activity and subjecting them to descending control appear to be recruited from structurally dispersed elements of synaptic glomeruli. These are arranged alongside dendritic processes of large antenna cells which relay impulses to projection cells of the spinothalamic tract.

Cobalt-complex ATP enhanced regeneration in the dorsal horn of the rat spinal cord.

ATP, the energy source for axoplasmic transport, is indispensable for the transport of nerve growth factor (NGF). NGF regulates the regeneration of central processes of the primary sensory neurons, by means of transganglionic regulatory mechanisms. This central regeneration was investigated with the help of the histochemical detection of fluoride-resistant acid phosphatase (FRAP), one of the marker enzymes of the primary nociceptive neurons. Transganglionic degenerative atrophy (TDA) of the central terminals of primary sensory neurons was induced with sciatic nerve crush. Dynamics of central regeneration was studied in rats treated with a cobalt-ATP complex, and with commercially available Na-ATP, respectively, by means of histochemical detection of the restitution of FRAP activity in the Rolando substance. Disappearance of FRAP activity was complete on the 6th postoperative day in the medial two-thirds of the upper dorsal horn in segments L2-L6. The regeneration (i.e. replenishment of FRAP activity) began on the 14th day and was complete by the 31st day in animals treated with cobalt-ATP, while in the animals treated with Na-ATP the replenishment of FRAP activity began on the 20th day and was complete only by the 60th day. It is concluded that the cobalt-ATP-complex significantly enhances central regeneration.

Cytochemical restoration in the upper dorsal horn after transganglionic degenerative atrophy: Temporospatial and fine structural correlates

Reorganization of synaptic circuitry has been studied in the upper dorsal horn (Lamina II, substantia gelatinosa Rolandi) of the lumbar spinal cord in the adult rat, by means of electron histochemical visualization of thiamine monophosphatase after transganglionic degenerative atrophy. Thiamine monophosphatase, a highly specific and selective marker of Type C (small) dorsal root neurons, was demonstrated at light and electron microscopic levels by means of a Gömöri-type cytochemical reaction, using thiamine monophosphate chloride (Sigma) as substrate and Pb 2+ as a capturing agent. Transganglionic degenerative atrophy, induced by a crush injury of the sciatic nerve, results in partial depletion of thiamine monophosphatase from ipsilateral segments L2-S1. The extent of depletion was determined in a complete series of frozen cross-sections, by means of measuring the projections of active and depleted areas and their distances from the midline. Values were fed into a personal computer and maps demonstrating the distribution of intact and impaired areas were generated. The V-shaped area of depletion starts to shrink due to incipient regeneration on the 23rd postoperative day, in caudorostral and mediolateral gradients. Replenishment of thiamine monophosphatase is completed on the 60th postoperative day. Electron microscopic cytochemistry revealed the presence of the thiamine monophosphatase reaction end product in axonal growth cones, filopodia, young axons and their varicous swellings that are transformed into scallopped en passant terminals in the later course of regeneration. Axonal growth cones and regenerating sprouts undergo Wallerian degeneration and simultaneous redepletion of the marker enzyme after transection of dorsal roots L3, L4 and L5. Thiamine monophosphatase, located initially within the axoplasms of regenerating fibers, is successively translocated to the external axolemmal surfaces. Functional maturity of the terminals is achieved only later. The resulting redundant and transient wiring is thinned out in a following maturation period, in a manner resembling the sequence of events in embryonic development. The regenerative potency of central terminals of primary sensory neurons is not restricted to a single regeneration. By repeatedly crushing the sciatic nerve, four successive degenerative-regenerative cycles have been evoked, resulting in replenishment of the marker enzyme thiamine monophosphatase.

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.

Transganglionic regulation and fine structural localization of lectin-reactive carbohydrate epitopes in primary sensory neurons of the rat

Light- and electron microscopic lectin histochemical studies showed that small dorsal root ganglion cells of the rat projecting to substantia gelatinosa Rolandi (Lamina II) contain terminal alpha-D-galactose carbohydrate epitopes; while those projecting to Waldeyer's marginal zone (Lamina I) and the outer part of Lamina II contain terminal beta-D-galactose residues. These glycoconjugates are manufactured in the Golgi apparatus and transported to preterminal and terminal axoplasmic surface membranes. Both of the axolemmal carbohydrate moieties were shown to be subjected to transganglionic regulation, even though the effects of transganglionic degenerative atrophy become evident considerably later than the depletion of axoplasmic marker substances like fluoride resistant acid phosphatase and thiamine monophosphatase.

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

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 electron-microscopic 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.

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF)

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 μg) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [ 125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.

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

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.

Treatment of terminal pain in cancer patients by means of iontophoresis of vinca alkaloids.

One of the most delicate tasks of the medical profession is caring for the terminal cancer patient suffering from intolerable pain with no prospect of recovery. However desperate the situation is, the physician is always bound by the Hippocratic oath to alleviate pain by all efforts, even when the inevitable end is approaching. A recently (re)discovered reaction of primary sensory neurons, transganglionic degenerative atrophy (see Discussion), offers a new, noninvasive technique of alleviating pain in terminal cancer patients by means of iontophoretic application of microtubule inhibitors to the skin. The result of this treatment in seven cancer patients is described and the possible mechanisms underlying these therapy results are discussed.

Reversibility of microtubule inhibitor-induced transganglionic degenerative atrophy of central terminals of primary nociceptive neurons : Neuroscience, 7 (1982) 1149–1154

Reversibility of microtubule inhibitor-induced transganglionic degenerative atrophy of central terminals of primary nociceptive neurons : Neuroscience, 7 (1982) 1149–1154

Treatment of chronic pain syndromes with iontophoresis of vinca alkaloids to the skin of patients

Repeated iontophoretic administration of the microtubule inhibitors vinblastic or vincristine to the segmentally related dermatomes of patients suffering from postherpetic, trigeminal and other neuralgias permanently alleviates chronic, autochthonoue pain. The beneficial effect of this therapy is probably due to transganglionic degenerative atrophy of primary central sensory terminals in the Rolando substance by blockade of retrograde axoplasmic transport in sensory nerves.

Reversibility of microtubule inhibitor-induced transganglionic degenerative atrophy of central terminals of primary nociceptive neurons

Microtubule inhibitor Vinca alkaloids applied around a peripheral nerve induce transganglionic degenerative atrophy of the central terminals of primary nociceptive neurons. This effect is reversible: 40–50 days later the original histochemical structure of the central terminals is restored. Restoration of fluoride-resistant acid phosphatase activity (the marker enzyme of primary nociceptive neurons) in the Rolando substance is due to regenerative sprouting of the formerly atrophied central terminals. Since peripherally-applied Vinca alkaloids induce transganglionic degenerative atrophy of the central terminals without inducing Wallerian degeneration of the peripheral nerve, and since this effect (virtually a synaptic discoupling) is only temporary, this approach may be used in the treatment of otherwise intractable neuralgias without inducing irreparable alterations.