Peter S. Spencer

Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves

We re-examined the hypothesis of Cajal3, later refuted by Weiss and Taylor20, that cells in distal stumps of transected peripheral nerves exert an attractive (tropic) effect on regenerating axons. This question was re-assessed in vivo using surgical materials and assay procedures not available to those workers. Proximal stumps of transected rat sciatic or cat peroneal nerves were inserted into the single inlet end of a hollow, Y-shaped Silastic implant. Regenerating axons were provided with alternative targets consisting of a vacant arm vs one occupied by a sciatic nerve graft (rat), or a tibial (Tout) vs peroneal (Pout) distal nerve stump (cat). In some cases Pout was rendered metabolically compromised relative to Tout by exposing the former to dry ice and inhibitors of DNA and RNA synthesis. At 4.5 or 6 weeks postoperatively, the number of regenerating axons in each fork of the implant was assessed by morphometric analysis (total number of non-myelinated and myelinated axons greater than 1 micron in diameter at 4.5 weeks, and total number of myelinated axons at 6 weeks postoperatively), or by quantification of an axonally transported label. Rat sciatic nerve fibers exclusively regenerated toward the nerve graft, suggesting the existence of a neurotropic lure. In cats, morphometric analysis revealed a 10-(4.5 week) and 6-fold (6 week) greater number of axons growing towards untreated Tout vs treated Pout. When both distal stumps were untreated, more axons were seen in forks leading to Pout. Analysis of transported label confirmed the preferential growth of axons towards untreated Tout vs treated Pout for both motor and sensory axons. In separate experiments, Nuclepore filters (0.2 microns, pore size) were inserted between distal nerve stumps and outlet ends of Silastic implants. Preferential regeneration toward untreated stumps was observed if the distance between proximal and distal nerve stumps was equal to but not greater than 4-5 mm. These results suggest that peripheral nerve fiber regeneration in vivo can be directed by cells in distal stumps of transected nerves, and that this effect can be mediated over distances of several millimeters via diffusible factors.

Ultrastructural studies of the dying-back process. IV. Differential vulnerability of pns and cns fibers in experimental central-peripheral distal axonopathies

A companion paper in this issue (46) described the evolution of peripheral nervous system dying-back disease of the giant axonal type in animals chronically intoxicated with the neurotoxic hexacarbons n-hexane (CH3CH2CH2CH2CH2CH3), methyl n-butyl ketone or MBK (CH3COCH2CH2CH2CH3), and 2,5-hexanedione (CH3COCH2CH2COCH3). The present study compares the distribution and pattern of peripheral (PNS) and central nervous system (CNS) dying-back disease produced by these three neurotoxic hexacarbons with that produced by acrylamide (CH2CHCONH2), and, in addition, employs these compounds to address unresolved issues in the dying-back process. In the PNS, large myelinated fibers in tibial nerve branches supplying calf muscles were especially sensitive in rats intoxicated with hexacarbons. These nerve branches and sensory plantar nerves in the hindfeet were equally vulnerable in acrylamide-treated rats. In both conditions, fibers located at these sites commenced degeneration before the distal regions of much longer and smaller diameter nerve fibers in nerve branches supplying the flexor digitorum brevis muscle and, in rats intoxicated with hexacarbons, before equivalent regions of plantar sensory branches to the digits. Pacinian corpuscles sited in the hindfeet of intoxicated cats were much less vulnerable to MBK than to acrylamide. Rats and cats intoxicated with hexacarbons displayed giant axonal swellings in vulnerable regions of the PNS degeneration in these animals was accompanied by pronounced endoneurial edema. In the CNS, rostral regions of long, ascending tracts (dorso-spino-cerebellar, gracile and, later, the cuneate) and the caudal end of long, descending tracts (lateral colums, ventrolateral and ventromedial tracts) of hexacarbon-treated animals were especially vulnerable. After prolonged intoxication of cats with MBK, giant axonal swelling was also found in preterminal and terminal axons in Rexed laminae V-VII at spinal levels C4 through S3-Neurofilament proliferation without giant axonal swelling was seen in CNS fibers of rats intoxicated with acrylamide. Taken in concert, the findings underline the importance of axon diameter and length in determining the hierarchy of fiber vulnerability and indicate the common sensitivity of selected regions of the PNS and CNS. The term central-peripheral distal axonopathy is introduced to emphasize the widespread, distal distribution of disease in these and in similar experimental conditions. It is suggested that certain human neuropathies (toxic, nutritional, uremic, diabetic and some hereditary polyneuropathies, and the neuropathy associated with multiple myeloma) are additional examples of central-peripheral distal axonopathies.

Studies on the control of myelinogenesis. II. Evidence for neuronal regulation of myelin production

Tritiated thymidine has been used as a nuclear marker to trace the origin of Schwann cells, sited in the distal stump of a severed unmyelinated nerve, which are able to elaborate myelin around axons regenerating from an anastomosed proximal stump of a severed myelinated nerve. Two types of cross-anastomosis experiments were performed in young, adult rats: (1) the proximal stump of a myelinated sternohyoid nerve was labeled (5 mCi/kg body weight) selectively over a 4-day period of predetermined maximal thymidine uptake and two days later, after flushing the animal repeatedly with cold thymidine, the unmyelinated cervical sympathetic trunk was transected and its unlabeled distal stump linked to the proximal stump of the labeled sternohyoid nerve; (2) the distal stump of an unmyelinated cervical sympathetic trunk was labeled selectively over a 5-day period of predetermined maximal uptake and two days later, after flushing with cold thymidine, the myelinated sternohyoid nerve was severed and its unlabeled proximal stump linked to the labeled distal stump of the cervical sympathetic trunk. The fate of the labeled cells in each type of anastomosis was determined 3 weeks later by autoradiography and liquid scintillation spectrometry. In the first type, a small amount of label had migrated from proximal stumps but labeled Schwann cells were not found in successfully anastomosed distal stumps. In the second type, labeled Schwann cells were seen in the cervical sympathetic trunk in association with myelinated and non-myelinated axons regenerating from the sternohyoid nerve. These data suggest that the presence or absence of myelin formation by a Schwann cell is controlled by some property of the axon with which it is associated. Putative mechanisms underlying neuronal control of myelinogenesis are discussed.

The fate of Schwann cells isolated from axonal contact

SummaryChronically denervated rat and rabbit tibial nerve distal stumps were studied 3–58 weeks following nerve transaction. Schwann cells, macrophages and possibly fibroblasts participated in myelin removal which was largely complete by seven weeks. Degenerating myelinated and unmyelinated fibres developed respectively into circular and flattened columns of Schwann cell processes each delimited by a basal lamina. Schwann cell columns became encircled by fibroblasts and later by cells of perineurial type, underwent shrinkage with time and eventually were replaced by connective tissue. In another experiment, endoneurial tissue was removed from rabbit tibial nerve stumps seven weeks after transection and transplanted between the corneal stroma of the same animal for 2–6 weeks. In this locus, Schwann cells developed a thickened basal lamina and then underwent necrosis. It was concluded that the maintenance of Schwann cells in bands of Büngner is in part dependent on axonal contact and that failure of reinnervation eventually causes the columns of Schwann cells to disappear.

Ultrastructural studies of the dying-back process II. The sequestration and removal by Schwann cells and oligodendrocytes of organelles from normal and diseased axons

SummaryA mechanism is postulated and described for the sequestration and phagocytosis of unusual and abnormal axoplasmic organelles by Schwann and oligodendroglial cells. Axonal organelles involved in this process are clear and dense-core vesicles, membrane-bounded dense membranous bodies reminiscent of secondary lysosomes, enlarged mitochondria, glycogen-like granules and glycogen-filled mitochondrial remnants. The process of sequestration of these organelles begins with the formation of a ridge of ensheathing cell adaxonal cytoplasm adjacent to an internally coated region of axolemma. The ridge of adaxonal cytoplasm enlarges to form a thin sheet which indents the axon surface adjacent to the abnormal axonal organelles. The invaginating adaxonal cytoplasmic sheet surrounds the abnormal axonal organelles and segregates them from the remainder of the axon. The cytoplasmic sheet infolds on itself and sequesters groups of axoplasmic organelles to form an interdigitated profile when viewed in cross-section. Electron lucent areas correspond to sequestered axoplasm and electron dense areas to ensheathing cell cytoplasm. The membranes separating axoplasm and ensheathing cell cytoplasm in the interdigitated networks break down allowing the abnormal axoplasmic organelles to be phagocytosed by the ensheathing cell cytoplasm. The process occurs to a limited degree in the normal nervous system at paranodes but is much more developed in pathologic situations where there is early axonal disease. The process is maximally developed in situations where there is centripetal axonal degeneration such as occurs in dying-back toxic disease and in the proximal stump of an amputated nerve.

Experimental neuropathy produced by 2,5-hexanedione--a major metabolite of the neurotoxic industrial solvent methyl n-butyl ketone.

Chronic exposure of rats to 2,5-hexanedione (CH3COCH2CH2COCH3), a major metabolite of the neurotoxic industrial solvent methyl n-buryl ketone (CH3COCH2CH2CH2CH3), has been shown to cause a clinical peripheral neuropathy with dying-back peripheral and central nervous system degeneration characterized by giant axonal swellings filled with neurofilaments. This pattern of disease is similar to that produced by methyl n-butyl ketone.

Specific antagonism of excitotoxic action of 'uncommon' amino acids assayed in organotypic mouse cortical cultures

Beta-N-Methylamino-L-alanine (BMAA) and beta-N-oxalylamino-L-alanine (BOAA) are chemically related excitant amino acids present in the seeds of Cycas circinalis and Lathyrus sativus, respectively. Consumption of these seeds has been linked to Guam amyotrophic lateral sclerosis (BMAA) and lathyrism (BOAA) (a form of primary lateral sclerosis). We report that the acute neuronotoxic actions of these amino acids are blocked selectively by specific glutamate receptor antagonists. Administration of BOAA and BMAA to neonatal mouse cortex explants (EC100 = 28 microM and 1.6 mM, respectively) rapidly induces postsynaptic vacuolation (PSV) and neuronal degeneration characterized by dark/shrunken (D/S) cells. BOAA-mediated neuronotoxic effects are attenuated in a concentration-dependent manner by cis-2,3-piperidine dicarboxylic acid (PDA), an antagonist of quisqualate (QA)-preferring and kainate (KA)-preferring glutamate receptors. PDA maximally protected against BOAA-induced PSV by 84% at 1 mM and D/S cells by 80% at 0.5 mM. BMAA-induced cellular changes were antagonized selectively in a concentration-dependent manner by 2-amino-7-phosphono-heptanoic acid (AP7), an N-methyl-D-aspartate (NMDA) glutamate-receptor antagonist. AP7 maximally protected against BMAA-induced PSV and D/S by 88% at 1.0 and 0.5 mM, respectively. These protective actions were selective and specific since AP7 failed to attenuate BOAA-induced alterations, and PDA was ineffective in ameliorating BMAA-induced changes. Other glutamate receptor antagonists (glutamic diethyl ester and streptomycin) failed to protect the explants from the destructive action of either toxin. Taken collectively, our data indicate that the acute neuronotoxic actions of BOAA and BMAA (or a metabolite) operate through different glutamate receptor species.(ABSTRACT TRUNCATED AT 250 WORDS)

An ultrastructural study of the inner core of the Pacinian corpuscle

SummaryPacinian corpuscles from the hindfeet and mesentery of cats have been examined by light and electron microscopy. The study focuses on the inner core region which houses the single, non-myelinated terminal of the afferent axon. This region of the axon possesses specialized axon processes which enormously increase the surface area of the axolemma. The axon processes are long, branched, filiform structures, containing exclusively ~6 nm microfilaments, and are reminiscent of filopodia on the tips of growth cones of axons and dendrites. These axon processes emanate from the two poles of the elliptical terminal axon, from several sites in the transitional zone, and from the entire surface of the ultraterminal axon, the bulbous, branched, distal extremity of the nerve fibre. Each branch of an axon process articulates with an inner core cell hemilamella except at the distal end of the ultraterminal where axon processes may also approximate the inner edge of the outer core. The base of each axon process contains an elaborate array of organelles including clear and dense-core vesicles of synaptic vesicle size, all enmeshed in ~6 nm microfilaments. The structure and location of the axon processes appear to be eminently suitable for detecting pressure transients transmitted through the outer core of the corpuscle. It is suggested that the mechano-electrical transduction system has, as its morphological substrate, multiple units, each formed by a branched axon process and its specialized basal region. The existence of sympathetic axon terminals abutting the central axon is disputed but the presence of unusual, dense-centred and elongated vesicles within cellular profiles close to the axon is confirmed.

Tropical myeloneuropathies The hidden endemias

Tropical myeloneuropathies include tropical ataxic neuropathy and tropical spastic paraparesis. These disorders occur in geographic isolates in several developing countries and are associated with malnutrition, cyanide intoxication from cassava consumption, tropical malabsorption (TM), vegetarian diets, and lathyrism. TM-malnutrition was a probable cause of myeloneuropathies among Far East prisoners of war in World War 11. Clusters of unknown etiology occur in India, Africa, the Seychelles, several Caribbean islands, Jamaica, and Colombia. Treponemal infection (yaws) could be an etiologic factor in the last two. Tropical myeloneuropathies, a serious health problem, are multifactorial conditions that provide unsurpassed opportunities for international cooperation and neurologic research.

Peripheral nerve abnormalities in aging rats.

The sciatic/tibial/plantar nerve complex of normal aged rats displays striking morphological changes that are most pronounced distally. Nerve fiber abnormalities include: (a) large numbers of axonal glycogenosomes, mitochondria, dense membranous bodies, and decorated particles; (b) adaxonal Schwann cell processes sequestering portions of axoplasm; (c) swollen demyelinated and remyelinated axons, some encircled by supernumerary cellular processes; and (d) collagen pockets, denervated Schwann cell columns, and empty basal laminae. Abnormalities a and b were encountered with increasing frequency on descent through the tibial and plantar nerves. Abnormalities b, c, and d were found in lateral and medial plantar nerves, where they were associated with an enlarged endoneurial space. Found in animals kept in cages with smooth or wire-mesh floors, the incidence of these changes increased with advancing age. They are attributed to trauma and ischemia from chronic pressure on the plantar nerve.