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Suicide Transport: Destruction of Neurons by Retrograde Transport of Ricin, Abrin, and Modeccin
Abstract
Certain toxic lectins, including ricin, are retrogradely transported along neuronal processes to the cell body where they inactivate ribosomes, resulting in neuronal death. This process of "suicide transport" suggests a powerful new experimental strategy for solving neurobiological problems.
... A trauma free approach to generate a motor neuron insult is toxin delivery via peripheral nerve suicide transport (Wiley et al., 1982). This strategy has been used to deliver ricin (Harper et al., 1980;Yamamoto et al., 1985;Wiley and Oeltmann, 1986;Liang et al., 2018), a broad scale toxin that generates both peripheral and central nerve damage and mortality. ...
... We examined the potential for retrograde transport (Wiley et al., 1982) via the Sn to deliver wortmannin and target the destruction of trophic factor dependent lumbar spinal cord motor neurons (MN). Wortmannin is a covalent inhibitor of PI3K, a key intermediate in the survival signaling pathway for of trophic factor dependent cells. ...
... For wortmannin to induce MN cell death it must enter the Sn axons then move into their soma in the central nervous system (CNS). DMSO in our injection bolus should allow axon penetration in the nerve, and the trauma of injection should promote reverse axonal transport (Bisby and Bulger, 1977) as seen with transport of lectins injected into peripheral nerves (Wiley et al., 1982). However, it was possible that wortmannin could block retrograde transport or cause damage within the peripheral nerve, as reported after ricin delivery (Liang et al., 2018), with MN loss being an indirect secondary effect. ...
We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents. Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3’-kinase inhibitor, wortmannin, via the sciatic nerve results in loss of ipsilateral lumbar motor neurons proportional to the level of drug administered. Motor neuron loss was detected by choline acetyltransferase (ChAT) immunostaining and with a transgenic thy1-eGFP marker. The short half-life of wortmannin generates minimal wound spread, and wortmannin does not affect axon transport, as determined by co-injection of a pseudorabies virus tracer. Using quantitative transcript analysis, we found that ChAT transcripts significantly decreased at 14 days post-delivery of 1 μg wortmannin, relative to sham controls, and remained low after 90 days. Smaller effects were observed with 200 ng and 100 ng wortmannin. Wortmannin also generated a transient and significant increase in astrocyte Gfap transcripts after 14 days with a return to control levels at 90 days. Treated mice had hind limb spasticity and a forced motor function defect that was quantified using a water exit test. Controls rapidly exit a shallow water tray, and wortmannin treated animals were up to 12-fold slower, a phenotype that persisted for at least 3 months. Thus the focal delivery of wortmannin to motor neurons generates a reproducible and scalable injury that can facilitate quantitative studies on neural regeneration and repair. The efficacy of sciatic nerve suicide transport can also explain neurotoxin-mediated selective loss of motor neurons in diseases such as amyotrophic lateral sclerosis. All procedures were performed at Rutgers under established Institutional Animal Care and Use protocols (eIACUC_TR201800022, approved on March 20, 2018).
... A more selective lesioning technique is based upon the so-called s¡¡i cide axonal retrograde transport of some lectins that are potent cytotoxins (5). Toxic lectins confer the experimental advantage ofproducing a specific lesion ofneurons that leaves int¿ct the axon terminals of their afferent neurons (6). ...
... Intemalization is carried out by receptor-mediated endocytosis. As described above, ricin binds to cell surface glycoconjugates containing N-acetylgalactosamine or galactose which are abundant in the peripheral nervous system (5). However,,when injected into a severed nerve, ricin is directly exposed to the axoplasm, which does not prevent its retrograde transport. ...
... The application ofricin-HRP conjugate into the vagus nerve is accompanied in 24-48 hours by an intense labeling ofneuronal perikarya and processes in the dorsal motor nucleus and the nodose ganglia (5). Ricin-HRP conjugate also labels the superior cervical ganglion cells in l8 hours whe¡ injected in the submandibular salivary gland (14). ...
Toxic lectins, such as ricin, are the state-of-the-art tool in neurobiology for selectively destroying neuronal populations. Strikingly, lectins from plants and the toxins from some pathogenic bacteria that produce enteric and renal diseases share many functional and structural properties. These toxins might mimic the endocytic pathways of constitutive proteins of the organism to gain access to and destroy the metabolic machinery of the cell. In the nervous system, lectins can be applied both peripherally and centrally. Lectins are internalized in axon terminals by receptormediated endocytosis and transported towards the soma using anterograde and/or retrograde transport pathways. Ricin is the toxic lectin of Ricinus communis. It has been shown to interfere irreversibly with the synthesis of proteins by catalytically inactivating the 60S eukaryotic ribosome subunit in such an efficient manner that a single molecule of ricin is enough to kill a cell. Therefore, it is possible to discriminate between the effects of selectively destroying a group of cells and the side-effects caused by other lesioning methods such as axotomy or electrocoagulation.
Ricin, as opposed to other lectins, seems to be completely ineffective within the central nervous system. Its effects, when injected into skeletal muscles or peripheral nerves, have been suggested to mimic the syndrome of human motor neuron disease since it affects only motoneurons and sensory neurons but not surrounding afferents or glia. Finally, a less exploited approach is the use of ricin for the study of the physiological consequences on central nervous system premotor neurons of the loss of their neuronal target during the execution of well defined motor tasks. In this regard, the oculomotor system is an ideal model, since the normative morphophysiological and behavioral data are already well known. This approach enables precise determination of the fate of target-deprived neurons in an attempt at exploring the regenerative and compensatory capabilities of the brain.
... In the case of C. elegans the only cells exposed are the amphid neurons. As a Type II RIP, EHL can be subject to retrograde transport from the cell surface along the neuronal processes, at which point the ribosomes are inactivated, causing translation to cease (Wiley, Blessing & Reis, 1982). As no post-embryonic somatic division occurs in mature individuals, and multiple chemoreceptors are expressed in a single neuron, ribosome inactivation of the neurons within the amphids would affect many functions derived from chemosensation (Sulston & Horvitz, 1977). ...
... The occurrence of dauer formation and a failure to recover in the presence of food supports the hypothesis that EHL is binding specifically to amphid neurons. Mutant screening has demonstrated that EHL can act as a neuronally specific cytotoxin, an effect which has previously been described with ricin and other RIPs on mammalian sensory neurons (Wiley, Blessing & Reis, 1982;Tong et al., 2012). Further studies will aim to determine if those individuals that remained as arrested L1s were doing so as a consequence of an inability to perceive the food or if an additional mechanism is at work. ...
The lectin found in the tubers of the Winter Aconite (Eranthis hyemalis) plant is an N-acetyl-D-galactosamine specific Type II Ribosome Inactivating Protein (RIP); Type II RIPs have shown anti-cancer properties, and hence have potential as therapeutic agents. Here we present a modified protocol for the extraction and purification of the E. hyemalis lectin (EHL) using affinity chromatography. De novo amino acid sequencing of EHL confirms its classification as a Type II Ribosome Inactivating Protein. The biocidal properties of EHL have been investigated against the nematode Caenorhabditis elegans. Arrested first stage larvae treated with EHL have shown some direct mortality, with surviving larvae subsequently showing a range of phenotypes including food avoidance, reduced fecundity, developmental delay and constitutive dauer larvae formation. Both inappropriate dauer larvae development and failure to locate to bacterial food source are consistent with the disruption of chemosensory function and the ablation of amphid neurons. Further investigation indicates that mutations that disrupt normal amphid formation can block the EHL-induced dauer larvae formation. In combination, these phenotypes indicate that EHL is cytotoxic and suggest a cell specific activity against the amphid neurons of C. elegans.
... In the case of C. elegans the only cells exposed are the amphid neurons. As a Type II RIP, EHL can be subject to retrograde transport from the cell surface along the neuronal processes, at which point the ribosomes are inactivated, causing translation to cease (Wiley, Blessing & Reis, 1982). As no post-embryonic somatic division occurs in mature individuals, and multiple chemoreceptors are expressed in a single neuron, ribosome inactivation of the neurons within the amphids would affect many functions derived from chemosensation (Sulston & Horvitz, 1977). ...
... The occurrence of dauer formation and a failure to recover in the presence of food supports the hypothesis that EHL is binding specifically to amphid neurons. Mutant screening has demonstrated that EHL can act as a neuronally specific cytotoxin, an effect which has previously been described with ricin and other RIPs on mammalian sensory neurons (Wiley, Blessing & Reis, 1982;Tong et al., 2012). Further studies will aim to determine if those individuals that remained as arrested L1s were doing so as a consequence of an inability to perceive the food or if an additional mechanism is at work. ...
... These are ribosome inactivating plant proteins that bind to certain oligosaccharides on the cell surface and irreversibly inhibit protein synthesis using binding subunits. 53,55 The neurons projecting axons through a treated nerve take up the toxin by endocytosis and through intracellular routing transport it Neuroma-in-continuity S45 to the trans-Golgi and endoplasmic reticulum at the perikaryon. Upon arrival in the perikaryon, the toxin's A subunit gains access to the cytoplasm where it inactivates ribosomes resulting in death of the neuron. ...
... This is probably due to obstruction of binding by addition of a penultimate sialic acid on the oligosaccharides of CNS neuronal plasma membranes. 53,55 Toxic lectins with different oligosaccharide binding specificities such as volkensin and modeccin are active as suicide transport agents to make selective CNS lesions on some CNS pathways. 16,39,51,54,57 Despite their effectiveness, other limitations of toxic lectins include unavoidable and indiscriminate destruction of many cell types at the injection site, high systemic toxicity and low neural specificity. ...
A neuroma-in-continuity is a neuroma that results from failure of the regenerating nerve growth cone to reach peripheral targets. It occurs within an intact nerve in response to internally damaged fascicles, resulting in a distal portion of the nerve that no longer functions properly. Management of neuromas-in-continuity is challenging. Chemical methods, and microsurgical techniques including fascicular ligation, and burying into muscle and bone have been reported to prevent neuroma-in-continuity formation. The purpose of this article is to present novel techniques for neuroma-in-continuity management, and to discuss the related literature.
... A variety of toxins have been used in studies of PNS axonal pathology and targeted neurotoxicity, and the resulting pathology is dependent on the insult used. Scholars focused on toxic lectins such as ricin (Wiley et al., 1982;Wiley and Oeltmann, 1986), which generate both peripheral and central nerve damage (Harper et al., 1980;Yamamoto et al., 1985;Wiley and Oeltmann, 1986). Ricin is a potent toxin that interferes with ribosomal protein synthesis (Lord et al., 1994) with broad scale tissue destructive effects, and this model has been limited by high lethality in rodents (Liang et al., 2018). ...
We explore the hypothesis that a potential explanation for the initiation of motor neuron disease is an unappreciated vulnerability in central nervous system defense, the direct delivery of neurotoxins into motor neurons via peripheral nerve retrograde transport. This further suggests a mechanism for focal initiation of neuro-degenerative diseases in general, with subsequent spread by network degeneration as suggested by the Frost-Diamond hypothesis. We propose this vulnerability may be a byproduct of vertebrate evolution in a benign aquatic environment, where external surfaces were not exposed to concentrated neurotoxins.
... Trauma to muscles also injures peripheral nerve bundles and this promotes retrograde transport in axons (Bisby and Bulger, 1977). If toxins enter such wounds, they would thus be delivered through axons directly into neuronal soma, a process termed suicide transport (Wiley et al., 1982). We delivered a neurotoxin into the rodent sciatic nerve and demonstrated that suicide transport caused specific loss of lumbar spinal cord MN (Liang et al., 2021). ...
... Some lectins transported along with neuronal processes inactivate the ribosomes resulting in neuronal death. An example includes ricin and abrin (Wiley et al. 1982). The toxin lectin consists of two polypeptide chains inter-linked by disulfide bonds. ...
Lectins are diverse group of carbohydrate binding proteins distributed ubiquitously in plant species. Lectins are the subject of intense investigation. Therefore, in the past few years studies on legume lectins with respect to their biochemical and pharmacological properties have been extensively carried out. Legume lectins are reported to contain antifungal, mitogenic, immunomodulatory, and antitumor properties. Some of the lectin also display anti-HIV-1 reverse transcriptase and antineoplastic activities. Plant lectins are expected to open new vistas for the design and development of drugs to be used against different serious diseases. This article aims to review up-to-date advances of legume lectins vis-à-vis structure, biological properties, and their practical applications.
... The discovery of the retrograde neuronal transport of ricin in 1980 has opened up a large area of research in neurobiology [92], mostly performed by Wiley at Vanderbilt University in Memphis, Tennessee. Similar to ricin and abrin, modeccin [93] and volkensin [94] may also be transported along neuronal processes, and this property has been exploited for the destruction of neuron bodies. The Adeniae toxins modeccin, volkensin [95] and stenodactylin [96] can be retrogradely transported not only in peripheral nerves but also in the rat central nervous system. ...
This review provides a historical overview of the research on plant ribosome-inactivating proteins (RIPs), starting from the first studies at the end of eighteenth century involving the purification of abrin and ricin, as well as the immunological experiments of Paul Erlich. Interest in these plant toxins was revived in 1970 by the observation of their anticancer activity, which has given rise to a large amount of research contributing to the development of various scientific fields. Biochemistry analyses succeeded in identifying the enzymatic activity of RIPs and allowed for a better understanding of the ribosomal machinery. Studies on RIP/cell interactions were able to detail the endocytosis and intracellular routing of ricin, thus increasing our knowledge of how cells handle exogenous proteins. The identification of new RIPs and the finding that most RIPs are single-chain polypeptides, together with their genetic sequencing, has aided in the development of new phylogenetic theories. Overall, the biological properties of these proteins, including their abortifacient, anticancer, antiviral and neurotoxic activities, suggest that RIPs could be utilized in agriculture and in many biomedical fields, including clinical drug development.
... Functional neuroanatomy research has long relied on the analysis of the effects by selective lesions to infer the function(s) of distinct neural structures. Type II RIPs ricin, abrin, modeccin and volkensin have been used to ablate neurons projecting through a particular nerve to investigate neuronal plasticity and neurotransmission mechanisms [119,120]. An important limitation of this approach relies on it's being based on retrograde axonal transport of the Type II toxins and included the unavoidable and indiscriminate destruction of different cell types at the injection site. ...
Toxin domains from plants or bacterial origin (such as Diphtheria toxin (DT) or Pseudomonas exotoxin A (PEA), which are endowed with ADP-ribosylation activity of the Eukaryotic elongation factor-2) have been extensively exploited for research and therapeutic purposes. Denileukin diftitox is the first FDA approved recombinant fusion toxin between a truncated diphtheria toxin and human interleukin-2 for the treatment of cutaneous T cell lymphomas. Ribosome-inactivating proteins (RIPs) are also a class of potent inhibitors of protein synthesis that act differently, by catalytically depurinating an adenine residue (A4324 in rat) exposed in an universally conserved stem-loop region of 23/26/28S ribosomal RNA (rRNA), also known as the α-sarcin loop. This causes an irreversible block in protein synthesis, leading to apoptotic cell death of mammalian target cells. RIP-containing conjugates have been used in the therapy of cancer and other incurable diseases with potential promising results and failures. In the last decade, many research efforts have been dedicated to the development of more efficient and less immunogenic chimeric fusions. In this chapter we will mainly focus on plant RIPs with the aim to report some of the most promising biomedical applications currently under investigation and further discuss their future perspectives. © 2013 Springer Science+Business Media Dordrecht. All rights are reserved.
... A number of studies have reported that previous episodes of intense physical activity are associated with a greater susceptibility to ALS later in life [55,63-66]. This may be because exercise increases the chance of “suicide transport” [67] of circulating toxins into LMNs. ...
Background
Environmental toxins are suspected to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). In an attempt to determine which pathways these toxins can use to enter motor neurons we compared the distribution of mercury in the CNS of a human and of mice that had been exposed to inorganic mercury.
Results
In the human who had been exposed to metallic mercury, mercury was seen predominantly in the locus ceruleus and corticomotor neurons, as well as in scattered glial cells. In mice that had been exposed to mercury vapor or mercuric chloride, mercury was present in lower motor neurons in the spinal cord and brain stem.
Conclusions
In humans, inorganic mercury can be taken up predominantly by corticomotor neurons, possibly when the locus ceruleus is upregulated by stress. This toxin uptake into corticomotor neurons is in accord with the hypothesis that ALS originates in these upper motor neurons. In mice, inorganic mercury is taken up predominantly by lower motor neurons. The routes toxins use to enter motor neurons depends on the nature of the toxin, the duration of exposure, and possibly the amount of stress (for upper motor neuron uptake) and exercise (for lower motor neuron uptake) at the time of toxin exposure.
... The lectins like ricin, abrin, and modeccine are toxic and move down through neuronal processes to the cell body where they inactivate ribosomes resulting in neuronal death. 50 The defensive effect of ricin, 51,52 Con A, 53 and Griffonia simplicifolia lectin 54 against tumor growth in experimental animals has been depicted. The shielding effect of ricin and abrin in humans against tumor growth and in grouping with anticancer drugs has also been illustrated. ...
Lectins are clusters of glycoproteins of nonimmune foundation that combine specifically and reversibly to carbohydrates, mainly the sugar moiety of glycoconjugates, resulting in cell agglutination and precipitation of glycoconjugates. They are universally distributed in nature, being established in plants, fungi, viruses, bacteria, crustacea, insects, and animals, but leguminacae plants are rich source of lectins. The present review reveals the structure, biological properties, and application of plant lectins.
... The peculiar features of some RIPs have been exploited in neuroscience research with experiments of ''suicide transport,'' since the first demonstration of retrograde transport along peripheral nerves of ricin (Dumas et al., 1979;Harper et al., 1980) and also of abrin and modeccin with consequent neuronal degeneration (Wiley et al., 1982). After unsuccessful attempts to use ricin for suicide transport in the central nervous system (Contestabile et al., 1984;Wiley et al., 1983), it was found that other type 2 RIPs, most notably volkensin, were effectively transported in a retrograde way and killed distant neurons projecting to the injected brain area (Contestabile et al., 1990;Harrison et al., 1990;Wiley and Stirpe, 1988). ...
Ricin and volkensin, two potent toxins belonging to the family of ribosome- inactivating proteins (RIPs), have been largely exploited in recent years in in vivo experiments of neuronal degeneration consequent to suicide transport or immunolesioning. We have determined both the toxicity of, and the inhibition of, protein synthesis by ricin and volkensin in in vitro cultures enriched in microglial cells, astrocytes, or neurons. In microglial cultures, 50% of toxicity (estimated by LDH released from dead cells) after 24 h exposure to RIPs was obtained with volkensin at 2.2×10−12 M concentration and 50% of protein synthesis inhibition at 2×10−14 M concentration. Both values were higher by about one order of magnitude in astrocyte-enriched cultures. Toxicity of, and inhibition of, protein synthesis by, ricin were lower for both cell types by about 1 order of magnitude as compared to volkensin. Cerebellar granule neurons in culture survived remarkably well to 24 h exposure to ricin or volkensin, although their protein synthesis was effectively inhibited by the two toxins with a potency similar to that found for astrocytes. These results demonstrate that glial cells, in particular microglia, are very sensitive to RIPs toxicity and should, therefore, be a primary target of these toxins when injected in vivo. Thus, the damage observed after in vivo experiments could be partly related to diffusion of toxic substances from early-affected glial cells. GLIA 20:203-209, 1997. © 1997 Wiley-Liss, Inc.
Fast axonal transport of radiolabeled protein was examined in lumbar and tail dorsal root ganglion (DRG) neurons at progressive stages of bullfrog tadpole metamorphosis. Accumulation of [³⁵S]methionine‐labeled protein proximal to a lumbar peripheral nerve ligature (at a fixed distance from the DRG) increased as tadpoles advanced from premetamorphosis through prometamorphosis to metamorphic climax. The rate of increase was steeper when expressed as a percentage of protein synthesized in the neurons of origin than when expressed as a percentage of total DRG protein synthesis. Further, the increase was not secondary to a rise in protein synthesis. In contrast, fast axonal transport decreased in DRG neurons of the tail at the onset of metamorphic climax, when tail resorption is initiated. The stage‐related increase in protein transport in lumbar nerves is due, at least in part, to an increased rate of transport. As determined from optically detected anterograde organelles in individual lumbar nerve axons, an approximate doubling of the fast transport rate occurred between the premetamorphic stage and metamorphic climax. In addition, the rates of organelle transport in lumbar axons of adult bullfrogs were significantly greater than in corresponding axons of tadpoles at metamorphic climax, further suggesting that organelle velocity is a developmentally regulated parameter of fast axonal transport.
Describimos el caso de una paciente femenina con signos y síntomas iniciales poco específicos, con alteración en el estado de conciencia. Se sospechó posible intoxicación, por nexo causal, probablemente por amitraz versus benzodiacepinas, sin embargo, sin confirmación de este. La paciente tenía alteración del sistema nervioso central con síntomas sugestivos de síndrome neurovascular, con posible compromiso de circulación posterior. La importancia de este caso es resaltar el amplio espectro de presentación clínica de las enfermedades neurológicas, la importancia de realizar diagnósticos diferenciales considerando los síntomas camaleónicos del accidente cerebrovascular, al igual que los “mimics stroke” y realizar enfoques clínicos integrales.
The CNS action of kainic acid (KA), quisqualic acid (QA) and 1-(4-chlorobenzoyl)-piperazine-2, 3-dicarboxylic acid (pCB-PzDA) was investigated in male Sprague Dawley rats, and their effects on monoamine metabolite levels in rat striatum were studied using brain dialysis.
Intracerebroventricularly injected KA and QA (100nmol) induced spike discharges, and pCB-PzDA (100nmol) suppressed electrocorticograms (ECoG) for 1 hour. pCB-PzDA aggravated KA induced spike discharges and inhibited QA-induced spike discharges.
Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels increased transitorily by injection of 10nmol and continuously by injection of 100nmol of KA. KA increased the 5-hydroxyindoleachtic acid (5-HIAA) level 2 hours after the administration dosedependently. Though 10nmol of QA increased the HVA level slightly, 100nmol of QA increased the DOPAC, HVA and 5-HIAA levels. Though 100nmol of pCB-PzDA increased the DOPAC and HVA levels, it inhibited the increases in DOPAC, HVA and 5-HIAA levels induced by KA. On the other hand, pCB-PzDA inhibited the increases in DOPAC, HVA and 5-HIAA levels induced by QA for 1 hour, after which the DOPAC and HVA levels increased additively.
These findings suggest that pCB-PzDA acts not only as a non-NMDA antagonist but also on dopaminergic neurons directly.
Zur Tribus gehört nur die früher zu den Vicieae gestellte pan(sub)tropische Gattung Abrus. Sie zählt je nach Autor 4, z.T. sehr polytypische, bis gegen 20 Arten. Am bekanntesten ist die heute überall in den Tropen und Subtropen wachsende Paternostererbse, Abrus precatorius, deren prächtig rote Samen mit schwarzen Flecken als Semen Jequirity oder Semen Abri bekannt sind. Die Taxonomie der Gattung ist noch keineswegs geklärt; dementsprechend ist auch die Nomenklatur z.T. unübersichtlich.
With gratitude I dedicate this review to Norman Aldridge and to all staff of the M.R.C. Toxicology Unit at Carshalton, who greatly contributed to my scientific formation.
Proteins and other materials synthesized in the cell bodies are transported into the axons and dendrites to support their functions: conduction of nerve impulses, release of neurotransmitters from terminals, and transduction in terminals. The structures of the axon continually turn over and are themselves maintained by transport: the cytoskeleton microtubules and neurofilaments, the membrane and its ion channels and pumps, endoplasmic reticulum, and other membrane-bound organelles (MBO) including transmitter vesicles, etc. It is apparent that toxicants affecting axonal transport can have profound effects on the function and structure of the neuron. A synopsis of transport properties and mechanisms is given in Sect. B to set the stage for the discussion of toxicants known to involve transport in Sect. C. The discussion is restricted for the most part to studies carried out in peripheral nerves taking a wider view of neurotoxicity, including the actions of toxicants on nerves in vitro as well as those following systemic administration. Transport is commonly held to consist of separate fast and slow mechanisms. Slow transport is associated with the maintenance of the cytoskeletal organelles. Their dramatic alteration by some key toxicants is usually accounted for by a selective action on the slow transport mechanism. In this presentation the alternate concept of a unitary mechanism for both slow and fast transport is advanced and neurotoxic actions discussed on that basis.
The pathology of motor neuron disease (MND) is well recognised, but poses many problems of interpretation and understanding. There is, as yet, no insight into the pathobiological mechanisms that underlie the development of the specific neuro–degenerative changes that must characterise the disease. In order to try to understand this problem a number of other approaches have been tried.
The toxic lectin ricin was applied to the hamster chorda tympani (CT), producing anterograde degeneration of its terminal boutons within the gustatory zone of the nucleus of the solitary tract (NST). Immunocytochemistry was subsequently performed with antiserum against tyrosine hydroxylase (TH), and the synaptic relationships between degenerating CT terminal boutons and either TH-immunoreactive or unlabeled dendritic processes were examined al the electron microscopic level. Degenerating CT terminal boutons formed asymmetric axodendritic synapses and contained small, clear, spherical synaptic vesicles that were densely packed and evenly distributed throughout the ending, with no accumulation at the active synaptic. The degenerating CT terminated on the dendrites of TH-immunoreactive neurons in 36% (35/97) of the cases. The most frequent termination pattern involved the CT and two or three other inputs in synaptic contact with a single immunoreactive dendrite, resulting in a glomerular-like structure that was enclosed by glial processes. In 64% (62/97) of the cases, the degenerating CT was in synaptic contact with unlabeled dendrites, often forming a calyx-like synaptic profile that surrounded much of the perimeter of a single unlabeled dendrite. These results indicate that the TH-immunoreactive neurons of the gustatory NST receive direct input from the CT and taste receptors of the anterior tongue and that the termination patterns of the CT vary with its target neuron in the gustatory NST. The glomerular-like structure that characterizes many of the terminations of the CT provides an opportunity for the convergence of several functionally distinct inputs (both gustatory and somatosensory) onto putative dopaminergic neurons that may shape their responsiveness to the stimulation of the oral cavity. (C) 1998 Wiley-Liss, Inc.
The electrical activity of antidromically identified abducens internuclear neurons selectively deprived of their target motoneurons was recorded in chronic alert cats. Target motoneurons were killed by the injection of the cytotoxic lectin of Ricinus communis into the medial rectus muscle. Following target removal, the discharge pattern of abducens internuclear neurons showed an overall decrease in firing rate, a significant reduction in their sensitivity to eye position and velocity, and the presence of anomalous responses such as bursts of spikes associated with off-directed saccades. The decreased excitability of abducens internuclear neurons correlated well with a marked reduction in the synaptic efficacy of their inputs. Thus, both excitatory and inhibitory synaptic potentials of vestibular origin showed a noticeable decrease in amplitude. The alterations in firing properties and synaptic transmission were only observed during an initial period of 3 weeks following ricin injection. Within 1 month the electrophysiological parameters returned to control values and remained unaltered for 1 year. Retrograde labelling of abducens internuclear neurons revealed that no cell death occurred after target loss. The anterograde axonal labelling of these neurons showed a progressive decrease in the density of their axonal terminals, and no sign of redistribution to other areas was found. These findings indicate that abducens internuclear neurons are not dependent on the presence of their natural target cells, either for the survival or for the maintenance of appropriate physiological signals.
Motor neuron disease (MND) or amyotrophic lateral sclerosis (ALS), the commonest form of motor system degeneration in man, occurs sporadically in middle or late adult life and can be divided into three clinical subtypes irrespective of bulbar or spinal predominance. This classification, convenient for conceptualization of the pathology germane to MND, includes: (1) lower motor neuron type, or primary muscular atrophy (PMA) (Aran’s disease 1850); (2) upper motor neuron type, or primary lateral sclerosis (PLS) (Erb’s disease 1891); and (3) upper-lower motor neuron type, or classic ALS. On the basis of some 20 clinical and 5 autopsy cases, Charcot (1869, 1885) coined the term “la sclerose laterale amyotrophique”, recognizing that the disease started in the lateral columns of the spinal cord, hence “lateral sclerosis”, propagated to the bulbar gray and anterior horns, and secondarily produced muscle atrophy as shown by the term “amyotrophic”. Many atypical forms of MND, all with a high familial incidence, have been recently added. They include: (a) familial ALS, (b) Guamanian ALS, (c) Kii-peninsula ALS in Japan, (d) ALS in Auyu and Jaki people of West New Guinea, and (e) familial juvenile ALS. Gowers (1888) believed that the degeneration in both upper and lower motor neurons might occur simultaneously or independently and, following this concept, pure lower motor neuron diseases have been regarded as separate entities. With this notion, one is obliged to compare the pathology of ALS with other MNDs such as Werdnig-Hoffmann disease, Fazio-Londe disease, Kugelberg-Welander disease, familial spastic paraplegia of Striimpell and the neuronal type of Charcot-Marie-Tooth disease. Striimpell disease is chosen for comparison in this chapter.
The plant toxin from Castor plant (Ricinus communis L.) is one of the most toxic toxins. Its structure is the key behind its unique mechanism of action, which can be also used in scientific research of endocytosis and intacellular transport, determination of source, development and function of individual cell type, and studies in neurobiology. Its use extends into cancer treatment, where imunotoxins have more and more important role. Because of its high toxicity and relatively easy production it is also subject of abuse as biological warfare.
After traumatic injury of peripheral nerve, regenerating axons escape and proliferate into the surrounding epineurial tissue in a disorganized fashion, forming whorls, spirals, and convolutions, and finally leading to the so called traumatic neuroma. About 10% of traumatic neuromas are companied by intractable neuropathic pain. In handsurgery, post-traumatic painful neuroma is known as a debilitating sequela of upper limb nerve injury and still remains a clinical challenge. Classical surgical interventions including nerve stump transposition to muscle, vein or bone provide limited efficacy. However, novel surgical techniques in recent years such as adipose autograft, tissue transfer, electrical stimulation, nerve guide tube, etc. have considerably enriched our armamentarium of neuroma treatment. In this article, we briefly review the common painful neuromas of upper extremity with special reference to new approaches emerging in recent years, which might lend support for future study in this area.
During the last two decades it has become clear that a number of protein toxins are able to penetrate the membrane of cells and get access to targets in the cytosol. It was first thought that these proteins enter the cytosol from the external surface of the cell, but it now appears that at least the major part of the toxins enter the cytosol from intracellular vesicles that they reach through receptor-mediated endocytosis.
The first edition of this textbook was published 11 years ago. It contained Chapter 170, entitled The Pathophysiology of Trigeminal Neuralgia written by William H. Sweet. His chapter is an exceptionally valuable treatise on this subject. His remarkable knowledge, wisdom, and vast experience treating trigeminal neuralgia (TN) patients remain unmatched. I am humbled by the task of updating our understanding of possible causes and mechanisms involved in this most painful affliction. My interest in this topic was kindled by 7 years of training under Dr. Sweet during neurosurgical residency. This was followed by my 30-year experience surgically treating over 700 patients with TN. The late Dr. Sweet, one of the great pillars of neurosurgery, had enormous influence on my life. His scholarship overshadows my own, and it would be impossible for me to stray far from his teachings in writing this chapter.
This chapter discusses the use of ribosome-inactivating protein (RIP) conjugates to understand the workings of the nervous systems. Different conjugates utilize different several different targeting agents, antibodies, peptide ligands, cytokines, or lectins, for elimination of specific cell types that express the target. This fits well with the history of neuroscientists who have lesioned a particular nervous system structure to determine its function from a loss or even gain of a behavioral process. We term the process Molecular Neurosurgery and here describe some of the ways researchers have used these materials in both research and therapeutic applications.
A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s-s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192-s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor-bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.
Objective: To describe our experience with adriamycin (ADM) selective chemical rhizotomy of facial nerves in a patient with hemifacial spasm (HFS) who refused neuro-vascular decompression surgery. This unique technique was less invasive than intracranial neuro-surgery. It was possible to perform a permanent nerve block under local anesthesia. Case report: The patient, a 73-year-old female, had difficulty opening her left eye due to HFS. After treatment with antiepileptic medicine (carbamazepine) was ineffective, the patient received selective intraneural injections of ADM under local anesthesia. One week after the surgery, the spasms had disappeared completely and slight muscle weakness was observed. No major complications were caused by this procedure and no recurrence of spasms was observed two years after the surgery. Main outcome: It is thought that recurrence of HFS should be observed after simple neurotomy due to re-growth of nerve fibers. However, this did not occur after selective ADM chemical rhizotomy of facial nerves. This method clearly differs from previously used methods of simple neurotomy because simple neurotomy does not cause severe destructive change in the facial motor nucleus. Conclusion: We conclude that selective facial nerve chemical rhizotomy with ADM under local anesthesia may be effective in treating a subgroup of patients with HFS, especially elderly patients and those in high risk groups for general anesthesia and intra cranial neuro-vascular decompression surgery.
Tryptamine-4,5-dione protected as the carbamate was prepared in excellent overall yield by the oxidation of serotonin carbamate with benzeneseleninic anhydride. This o-quinone, which was potently neurotoxic, readily added sulfur nucleophiles under basic conditions including cysteine and glutathione in 1,4-fashion, a reactivity pattern which may reflect the mechanism of the neurotoxicity.
: The authors previously have demonstrated axonal necrosis of autonomic nerves in the surgically resected ilea of patients with Crohn's disease both in grossly normal ileal resection margins and in diseased areas. The present study of ileal stomal biopsies was carried out to obviate the possibility that the observed axonal damage might be related to the prolonged surgical manipulations required for ileal resection. The authors present studies of biopsies of ileal stomas and of small bowel from patients with Crohn's disease and various control disorders, including ulcerative colitis. Stomal biopsies were fixed immediately after they were obtained. Widespread, severe axonal necrosis of autonomic nerves was present in all Crohn's disease specimens, regardless of the patient's clinical status or the gross or routine microscopic evaluation of the same specimen. Controls either had no necrosis or displayed a minor degree of focal necrosis involving single axons. The authors conclude that Crohn's disease is accompanied by a severe and extensive necrosis of gut axons, and that such electron microscopic findings may serve to differentiate Crohn's disease from other inflammatory disorders.
Up until about 15 years ago lectins were thought of as laboratory tools, useful for histochemistry and blood transfusion work. The fact that many common foods are rich sources of lectin was not considered by most biomedical scientists. In the last decade, however, there has been a flowering of knowledge about the interactions of lectins with body organs and tissues, and it has become clear that many lectins are resistant to cooking and digestion and are distributed to distant parts of the body after ingestion. There is now abundant evidence that dietary lectins can cause disease in Man and animals. This review will adduce evidence that such hitherto mysterious diseases as inflammatory bowel disease, diabetes mellitus, rheumatoid arthritis, glomerulonephri-tis, psoriasis, multiple sclerosis, retinitis and cataracts in the eye, are all explicable in terms of a lectin aetiology, as are congenital malformations, infertility, IgE-mediated allergies and autoimmune states. Complete proof is still lacking in most cases, but the new hypothesis, if true, offers scope for rational therapy in these hitherto intractable diseases
The i.v. 48 hour (and 7 day) LD50 and the minimum lethal dose (MLD) of ricin in male New Zealand White rabbits has been determined by the Up and Down procedure. A MLD and a toxic sub-lethal dose (TSD) lowered blood pressure after a 12 hour or greater lag period, but only the MLD did so significantly (p < 0.05). Heart rate was increased when blood pressure was reduced, which seems to be a reflex effect, but the ECG was not altered. Abnormal laboratory values correlated well with histological findings. Serum CPK, SGPT, LDH, and cholesterol concentrations were higher and serum calcium concentrations were lower in rabbits given ricin. Rabbits that died earliest (approximately 22 hours after ricin) had marked pulmonary damage, while those that died later (36-48 hours after ricin) showed much more heart and liver damage. Ricin increased total blood flow to most organs. Exceptions were the brain and lungs, where the MLD markedly reduced blood flow. Ricin administration decreased the sensitivity of the central ear artery to norepinephrine (NE) (i.e., increased the EC50). Ricin increased methacholine (endothelium-dependent) relaxations of aorta rings, but did not alter those to papaverine. Ricin did not alter the activity of monoamine oxidase or catechol-0-methyltransferase, which metabolize NE. Ricin in some studies increased the amount of NE released by nerve stimulation, but did not alter NE reuptake by the neuronal membrane. Ricin did not alter basal calcium uptake by the aorta, but depressed stimulated calcium uptake in some studies. ...Ricin, Ricin in rabbits, Ricin cardiovascular effects, Ricin vascular tissue, Ricin neuroeffector mechanism, Ricin toxicity, Ricin on blood vessels, RA I, BD.
discusses the morphological organization of monoaminergic projections to anterior area 24 and posterior areas 23, 30, and 29 of cingulate cortex in the rat, monkey, and human / the laminar distribution of presynaptic auto- and heteroreceptors, as well as postsynaptic somatodendritic receptors, is discussed in relation to the electrophysiological effects of monoamines on cortical neurons / by considering the pharmacological, anatomical, physiological, and biochemical interplay among 5-hydroxytryptamine (5-HT), [norepinephrine], and [dopamine] in cingulate cortex, a synthesis is pursued of their functional roles in behaviors that are regulated by cingulate cortex [motor functions, orientation and arousal, avoidance conditioning] and how dysfunction in these systems contribute to clinical manifestations of particular diseases [depression and Alzheimer's disease (AD)] (PsycINFO Database Record (c) 2012 APA, all rights reserved)
Certain plant lectins and bacterial toxins have proven to be useful axonally transported probes for studies of neuronal connectivity. The sensitivity and resolution provided by such compounds appears to be attributable to an efficient incorporation of tracers, which derives primarily from their capacity to bind specific sugar moieties of membrane-bound glycoproteins or glycolipids. Differential uptake, transport and cellular disposition of individual markers may now be exploited to advantage for retrograde and/or anterograde tracing studies, for transneuronal labeling, or to destroy the cells of origin of specific projections.
Sprouted collateral axons were observed in the electrosensory lateral line lobe (ELL) of gymnotiform teleosts (Apteronotus leptorhynchus:) following the ablation of the supraorbital branch of the anterior lateral line nerve. Ablation was accomplished by using microinjections of the toxic lectin ricin. Sprouted axons were followed for up to 26 weeks postablation. Ricin exposure severely reduced axonal numbers and the peripheral electroreceptors in the region innervated by these fibers. To visualize sprouted fibers, intact lateral line afferent nerve branches were anterogradely labelled with the neuronal tract tracers horseradish peroxidase or cobalt chloride, or the monoclonal antibody Q26A3.
Within the four somatotopically organized ELL segments, sprouted collaterals were first observed two weeks after ricin injection in the medial and centromedial segments, and four weeks postinjection in the centrolateral and lateral segments. Sprouting involved intrasegmental, horizontally directed axons from adjacent nerve branch terminal fields, and mixed intra-and extrasegmental, dorsally directed axons from the ELL deep fiber layer. The sprouting response was robust but variable in its timing, peaking between 6 and 12 weeks. Subsequently, the intrasegmental, horizontally directed fibers were retained but the mixed dorsally directed fibers, including all extrasegmental axons, were retracted. Therefore, this sprouting response appears to consist of a collateral overproduction followed by a selective axonal retraction. In our view, the most likely explanation for this axonal retraction is that the descending inputs from the isthmus and the cerebellum, as well as commissural fibers from the contralateral ELL, maintain established somatotopic relationships by eliminating somatotopically mismatched sprouted collaterals.
The term molecular neurosurgery has been applied to several different experimental strategies, including a variety of genetic manipulations. For the purposes
of this book, the term is used to refer to the use of targeted cytotoxins to produce highly selective neural lesions. Used
in this sense, the term is relevant to both experimental and potential clinical applications. The body of work addressed in
this volume grew out of initial experiments in the laboratory of Donald J. Reis in 1980–1981. The initial experimental challenge
was how to selectively destroy baroreceptor afferents that make up a small portion of the vagus and glossopharyngeal nerves.
The strategy chosen was to develop a technique using toxin retrogradely transported from an application site on the peripheral
baroreceptor nerves in the neck. First attempts used low-molecular-weight cytotoxic drugs, such as doxorubicin, and were unsuccessful.
Reasoning that the initial lack of success reflected inadequate delivery of toxin to the cell bodies, a plan was developed
to attach these drugs to a well-transported agent, such as wheat germ agglutinin, which at the time was introduced as a highly
effective anatomical tracer (1). However, a simpler option seemed attractive. If a lectin such as wheat germ agglutinin was well transported, then perhaps
a toxic lectin such as ricin or abrin would work. In retrospect, Harper and colleagues (2) had previously shown evidence for retrograde axonal transport of ricin, but this publication was discovered only after the
initial suicide transport experiments applied ricin to the vagus nerve (3).
One of the major goals of research in neurobiology is the characterization of neuronal circuits, which are the basis of the
functioning of the entire nervous system. Tract tracing methods are among the best approaches to elucidate the connections
between neurons located in distinct areas of the nervous system since they allow an easy recognition of large neuronal populations
and their structural characterization. Although the bulk of information is achieved at light microscope (LM) level, only electron
microscopy allows one to demonstrate of the occurrence of biological interaction between neurons and characterize the synaptic
circuitry involved. In the choice of tracers for ultrastructural analysis of neuronal connections, neurobiologists need to
consider specific characteristics of the tracer, in addition to those addressed for tracing at LM (see Chapter 12). High sensitivity
is an essential requisite for tracers in ultrastructural studies, since synaptic contacts are mainly established with small
diameter neuronal processes. Further structural analysis and that the tracing method can further be combined with other neurobiological
techniques aimed to achieve a more complete characterization of the neuronal circuit. Under these perspectives, the most suitable
tracers to perform retrograde, anterograde, or bidirectional tracing at the electron microscope (EM) level, are discussed.
The elected techniques are presented in a sole protocol that allows a successful combination of retrograde, anterograde, and
neurochemical analysis at LM and EM levels. Each technique can, however, be used separately or designed to achieve different
goals,as pointed out at the various steps of the protocol.
Fluorescent somatopetal tracers were used to infiltrate, by diffusion rather than injections, the dorsolateral cortex of one hemisphere in rats. In different animals the tracers penetrated into the cortex to different depths. We found several interesting features of the commissural system: first, there were no areas without commissural neurons. At least a few labelled cell bodies were present in a single-cell layer also in acallosal cortical areas. Secondly, there is a considerable variety of laminar distribution patterns of labelled perikarya in different areas. Thirdly, some cortical fields, which cytoarchitecturally appear uniform, can be subdivided according to different distributions of cell bodies with commissural projections. Fourthly, when only supragranular layers were infiltrated, labelled cell bodies were present mainly in the supragranular layers of the contralateral cortex. Infiltration of the first layer alone did not label any neurons in the contralateral cortex but did label neurons in layer VIb ipsilaterally. In the subcortex, the labelled perikarya were found in the structures already known to project directly to the cortex. In rats with the tracer restricted mainly to the supragranular layers, a conspicuously reduced labelling was found in the basal forebrain and the thalamus. In the thalami of those animals, labelled neurons were found only in paralamellar nuclei. The high sensitivity of the tracer used, together with infiltration of the entire dorsolateral cortex, allows us to conclude that probably all sources of innervation of the isocortex in rats have been seen.
Neurotoxicity of tryptamine-4,5-dione (4,5-DKT), a partially oxidized form of serotonin, was assessed after microinjection into the lateral ventricle, hippocampus, or cingulate cortex of rats followed by Fink-Heimer staining for axon terminal degeneration. Intracerebroventricular injections of 4,5-DKT resulted in terminal degeneration which was most dense in layers I and III of insular cortex, layer I of cingulate cortex, and the molecular layer of the dentate gyrus. Argyrophilic and probably degeneration neurons were most frequently subjacent to the granule cell layer of the dentate gyrus, layers II, III, and IV of entorhinal cortex, and throghout the insula. Injections of 5–20 μg of 4,5-DKT directly into the hippocampus indicated that toxicity was dose-dependent. These injections produced axon terminal degeneration and neuronal argyrophilia in sectors CA1 and CA3 and in the dentate gyrus. Argyrophilic neurons were also observed in layers II, III, and IV of ipsi- and contralateral entorhinal cortices. Injections into anterior and posterior cingulate cortices produced degeneration in the caudate and anterior thalamic nuclei, and contralateral cortex. These results indicate that 4,5-DKT is a novel indole neurotoxin which exhibits a striking propensity for medial limbic system structures including some of those affected in dementia of the Alzheimer type.
Exposure of serotonin (5-HT) to oxygen-derived free-radical-generating system, xanthine oxidase-hypoxanthine or to a Fenton reaction results in the formation of the neurotoxin, tryptamine-4,5-dione. In cultured embryonic chick brain neurons, incubation of tryptamine-4,5-dione or its ethyl carbonate derivative resulted in a dose-dependent neurotoxicity (1–100 μM). The addition of sulfhydryl compound, glutathione at 2 or 10 μM significantly enhanced the toxicity induced by 10 μM tryptamine-4,5-dione. On the contrary, glutathione at 10 μM decreased the neurotoxic effect caused by 10 μM 5,6- and 5,7-dihydroxytryptamine in the cultured neurons. The toxicity resulted from 5,6- and 5,7-dihydroxytryptamine could be fully prevented by a 5-HT uptake inhibitor, fluoxetine. However, the toxicity caused by tryptamine-4,5-dione and glutathione conjugate could not be blocked by fluoxetine (10 or 100 μM) or by a glutathione transferase inhibitor, boric acid/serine. The results indicate a different molecular mechanism among 5-HT derived neurotoxins and suggest that tryptamine-4,5-dione and/or its glutathione conjugate would cause neuronal damage, if they are formed in vivo.
Motor nerve transection in adult rats induce a series of metabolic and structural changes in the injured neurons as well as in surrounding glial cells; however, without substantial neuronal degeneration. In the present study we found, in contrast with axotomy, a massive neuronal death in the ipsilateral hypoglossal nucleus following injection of toxic ricin (RCA) into the hypoglossal nerve, which is in line with previous observations. Injection of RCA enables examination of the glial reaction in a situation where neuronal degeneration is profound, which has been the approach in the present study. We found an increase in OX42-, GFAP-, and transferrin-immunoreactivity in microglial, astroglial, and oligodendroglial cells respectively, in the ipsilateral hypoglossal nucleus three to seven days following injection of toxic ricin in the hypoglossal nerve. Proliferation was found in astrocytes as well as in microglial cells, as shown by uptake of bromodeoxyuridine. In addition, the complement cascade was activated locally in the ipsilateral hypoglossal nucleus, as demonstrated by immunohistochemical detection of complement components C3d and C9. Complement activation may serve several effects in the glial-neuronal interactions. Stimulation of phagocytosis by reactive microglia is probably the most important one. Furthermore, the degenerative neuronal somata showed increased immunoreactivity for clusterin, which is a known complement inhibitor, but a decrease in clusterin-mRNA. In conclusion, the glial cell response was in several aspects principally different following massive motorneuron degeneration induced by toxic ricin in comparison to previous findings reported after axotomy.
Laminin, an extracellular matrix glycoprotein rich in basement membrane, is a multifunctional molecule of approximately 1000 kDa and is known to possess a potent neurotrophic activity. Laminin-like immunoreactivity (LLI) was for the first time demonstrated in mouse and rat CNS neurons by a sensitive immunohistochemical technique. Transblotting of SDS-PAGE of the supernatant of the mouse and rat brain homogenate identified distinct 180 kDa and weak 380 kDa bands immunoreactive to anti-laminin and these molecules differed from authentic laminin subunits. The intraneuronal distribution of LLI disclosed two distinct patterns; LLI-1 (diffuse perikaryal stain) and LLI-2 (coarse granular stain). By immunoelectron microscopy, LLI was localized to the ERs in LLI-1 neurons, whereas it appeared to be confined to lysosomes in LLI-2 neurons. LLI-1 neurons were found predominantly in hippocampal pyramidal, granule and neocortical layers 1–3, 6 neurons, in most of the striatal and thalamic neurons, and Purkinje cells. The majority of neurons in neocortical layers 4–5, medial septal and Meynert neurons, somatic motor neurons, and neurons of the deep cerebellar nuclei were classified as LLI-2 cells. No LLI was found in hypothalamic mammillary, habenular and vagal dorsal motor neurons (LLI-3). These observations may indicate intraneuronal production of laminin-related molecules in central neurons. We speculate that the laminin-related molecules (neurolaminin) play important roles in trophic or servo mechanisms in the CNS.
The efficacy of the neurotoxic lectin volkensin to induce motoneuron loss in the lumbar spinal cord was investigated at different time-points following unilateral injection into the medial gastrocnemius muscle of newborn (postnatal day 1 (PD 1)) animals, using retrograde fluorescent neuron labelling and histochemical procedures to evaluate the extent of the toxin-induced depletion, in comparison with the effects produced by neonatal crushing of the sciatic nerve. The results show that very low doses (2.0 ng) of volkensin intramuscularly can produce extensive (about 90%) and long-lasting (up to at least 8 months post-lesion) motoneuronal loss in the lumbar spinal cord, whose magnitude is higher than that observed following mechanical injury of the developing peripheral nerve (50-60%). Volkensin-induced motoneuronal depletion may therefore represent a useful model for experimental studies aimed at functional cell replacement in the immature spinal cord.
Review of the literature relative to experimental peripheral injury to the nerves reveals many interesting results, interpretations and contentions, particularly concerning the effects produced on the body of the nerve cell. The subsequent changes in the neurocyte are exceedingly variable and present histologic pictures varying from one extreme to the other, namely, from that of no apparent change at all, to that of complete disintegration and disappearance of the cell.
These alterations or lack of alterations are dependent on at least four factors: first, the type and the age of the animal used; second, the distance from the central nervous system at which the nerve is severed, ligated or otherwise injured; third, the interval that is permitted to elapse between the time of operation on the nerve and that of removal and fixation of the tissue of the central nervous system, and, fourth, the histologic and functional type of neuron.
Changes in nerve cell cytoplasm with its correlated axon and dendritic processes, together with changes in the size, shape, position and general character of its various constituents—the Nissl bodies, canalicular apparatus, mitochondria and the nucleus with its contained chromatin material and nucleolus—have been investigated repeatedly in connection with a number of variously induced methods of injury. For the most part, the changes described have been swelling, shrinkage, vacuolation and pigmentation of the cytoplasm; chromatolysis of the Nissl substance; fragmentation and dispersion of the canalicular apparatus; stability of the mitochondria; swelling, shrinkage, distortion, eccentricity, nuclear wall degeneration, vacuolation and increase or decrease in chromatin content of the nucleus; and increase or decrease in the number or size of the paranucleoli and nucleoli. The methods of inducing injury have been, in general, either direct, such as sectioning, tearing or stimulating electrically the axon processes, or indirect, as in inflammations, infections, chemical poisonings
The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.
The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.
Rats treated with ricin, an inhibitor of protein synthesis, at the dose of 1 μg/100 g body weight, developed within 72 hours
a severe liver necrosis. Protein synthesis was practically unchanged. The ultrastructural changes that ricin induces in rat
liver were investigated. The earliest changes were observed in sinusoidal cells which were progressively damaged until they
became necrotic. Only after the development of these lesions hepatocytes appeared to be damaged. The hypothesis is formulated
that hepatocyte necrosis is a consequence of the disappearance of sinusoidal cells. This might explain why protein synthesis
was unaffected in liver: since ricin exerted its toxic effect on the sinusoidal cells the inhibition was not detectable, these
cells being “diluted” by the mass of parenchymal cells. Ricin at the dose of 10 μg/100 g body weight did not affect protein
synthesis in the liver, but exerted a marked and precocious inhibition of protein synthesis in the spleen, which is very rich
in reticuloendothelial cells. Moreover, a severe necrosis of the red pulp of the spleen was observed in rats poisoned with
this dose of ricin.
Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.
The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.
The uptake of macromolecules by nerve terminals which is followed by retrograde axonal transport seems to occur by two different mechanisms, a specific and a nonspecific one. The nonspecific uptake depends on the presence of macromolecules (e.g., horseradish peroxidase) in the vicinity of the nerve terminals at very high concentrations and is enhanced by neuronal activity. In contrast, the specific uptake and subsequent retrograde axonal transport becomes apparent at much lower concentrations of the appropriate macromolecules, depends on the affinity of these ligands for specific binding sites on the surface of the neuronal membrane, and is independent of neuronal activity. The fact that lectins and some bacterial toxins bind to specific membrane glycoproteins or glycolipids allows conclusions to be drawn regarding qualitative and even quantitative aspects of the composition of the plasma membrane of the nerve terminals.
Counts of neuronal nucleoli were made in the dorsal motor vagal nucleus (DMV) of the adult rabbit 10, 18, 70 and 90 days following unilateral cervical vagotomy. The structural characteristics of nerve cell bodies in the DMV were studied electron microscopically 2--90 days after cervical vagotomy. The nucleolar counts indicated that 20% of the large DMV neurones had disappeared ipsilateral to the operation 10 days postoperatively (p.o.), 65% 18 days p.o. and 70% 70 and 90 days p.o. No loss of small neurones was found. Large neurones ipsilateral to the operation showed nuclear displacement, infoldings of the nuclear membrane and disappearance of granular endoplasmic reticulum beginning 4 days p.o. and being prominent 6--18 days p.o. At the peak of the response, 10--18 days p.o., reacting neurones showed nucleolar condensation and vacuolation, the appearance of intranuclear electron-dense particles, extensive accumulation of intracytoplasmic lipid droplets, increased numbers of microtubules and neurofilaments, focal mitochondrial aggregates, and widespread mitochondrial degeneration. Ten to 21 days p.o. degenerating neurones were observed. After 30 days p.o. survival a partial recovery of surviving large DMV neurones seemed to have taken place. The findings are interpreted as indications of distubed protein metabolism, oxidative metabolism and intraneuronal transport in the axotomized DMV neurones. The unique response of these neurones compared to previously studied peripherally projecting neurones is emphasized.
The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31-33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.
The mechanism of action of the plant toxins abrin and ricin is described. The toxins inhibit protein synthesis probably by inactivating the 60S ribosomal subunits, interfering with chain elongation. Only eukaryotic ribosomes are attacked by the toxins.
Publisher Summary
This chapter provides an overview of the principal features of perikaryal responses to axon injury. The neuron is an unusual cell. Its axon terminals may be situated at what in cellular terms is an enormous distance from the cell body (perikaryon); the volume of the latter may be but a small fraction of the total cellular volume. Yet the neuronal processes are maintained and their substance is constantly renewed from the perikaryon. The separation of an axon from its cell body results (in vertebrates) in the degeneration of the separated portion and is followed by a series of morphological changes in the perikarya. The most conspicuous of these is the disintegration, redistribution, and apparent disappearance from the cell body of cytoplasmic basophil material. Changes in axotomized neurons are generally assessed by comparison with the corresponding contralateral neurons of the experimental animal.
In attempts to assess how many molecules of the toxic lectins abrin, ricin and modeccin are needed in the cytosol to kill HeLa cells the effect of these toxins on protein synthesis and plating efficiency was studied. The incubation time of the cells after a 1 h exposure to the toxins influenced strongly the extent of inhibition of protein synthesis. The full toxic effect was expressed about 20 h of incubation after the exposure. On further incubation, protein synthesis again increased at a rate comparable to that in the control cells. After exposure to increasing concentrations of toxins the inhibition of cellular protein synthesis measured after 20 h showed excellent agreement with the inhibition of plating efficiency, indicating that the inhibition of protein synthesis can be used as a measure of cell killing. The inhibition of protein synthesis by toxins was found to follow first order kinetics, indicating that the cells are killed by an all- or none-effect. Autoradiographic studies indicated that after exposure to intermediate toxin concentrations protein synthesis was completely abolished in some cells, whereas it appeared to proceed at a normal rate in the remaining cells. The results provide evidence that penetration of one molecule of abrin, ricin or modeccin into cytosol is lethal to HeLa cells and that the efficiency of toxin entry into the cytoplasm is very low compared to the rate of bulk toxin uptake.