Degeneration and regeneration of some mechanoreceptors. An ultrastructural study. I. Ultrastructure of denervated Herbst corpusles

The degenerative changes in the HERBST corpusles have been investigated at ultrastructural level during the 3rd h to 1 year after nerve section. The earliest changes have been established in the nerve ending followed by the remaining nonmyelinated and myelinated portion of the receptor nerve fibre. The changes reflect the retrograde character of the degenerative process. The receptor cell elements pass through the stage of activation followed by their destruction and elimination. The Schwann receptor cells fullfill the pagocytotic role in the elimination of the axonal debris, after that they have been also completely eliminated. Their places are occupied by the activated fibroblasts and developed collagen fibrils. One part of the perineural cells persist a long time after denervation and they preserve the common although modified receptor configuration.     

Terminal Schwann cells guide the reinnervation of muscle after nerve injury

Schwann cells and axons labeled by transgene-encoded, fluorescent proteins can be repeatedly imaged in living mice to observe the reinnervation of neuromuscular junctions. Axons typically return to denervated junctions by growing along Schwann cells contained in the old nerve sheaths or “Schwann cell tubes”. These axons then commonly “escape” the synaptic sites by growing along the Schwann cell processes extended during the period of denervation. These “escaped fibers” grow to innervate adjacent synaptic sites along Schwann cells bridging these sites. Within the synaptic site, Schwann cells, originally positioned above the synaptic site continue to cover the acetylcholine receptors (AChRs) immediately following denervation, but gradually vacate portions of this site. When regenerating axons return, they first deploy along the Schwann cells and ignore sites of AChRs vacated by Schwann cells. In many cases these vacated sites are never reinnervated and are ultimately lost. Following partial denervation, Schwann cells grow in an apparently tropic fashion from denervated to nearby innervated synaptic sites and serve as the substrates for nerve sprouting. These experiments show that Schwann cells provide pathways that stimulate axon growth and insure the rapid reinnervation of denervated or partially denervated muscles.     

Mechanisms controlling axonal sprouting at the neuromuscular junction

This review considers the relative roles of sprouting stimuli, perisynaptic Schwann cells and neuromuscular activity in axonal sprouting at the neuromuscular junction in partially denervated muscles. A number of sprouting stimuli, including insulin-like growth factor II, which are generated from inactive muscle fibers in partially denervated and paralyzed skeletal muscles, has been considered. There is also evidence that perisynaptic Schwann cells induce and guide axonal sprouting in adult partially denervated muscles. Excessive neuromuscular activity significantly reduces bridging of perisynaptic Schwann cell processes between innervated and denervated endplates and thereby inhibits axonal sprouting in partially denervated adult muscles. Elimination of neuromuscular activity is also detrimental to sprouting in these muscles, suggesting that calcium influx into the nerve is crucial for axonal sprouting. The role of neuromuscular activity in axonal sprouting will be considered critically in the context of the roles of sprouting stimuli and perisynaptic Schwann cells in the process of axonal sprouting.     

Differentiation and apoptosis without DNA fragmentation in cultured Schwann cells derived from wallerian-degenerated nerve

The Schwann cell cables provide particularly favorable sites for the growth of regenerating axonal sprouts. However, if they remain denervated, endoneurial fibrosis takes place with the Schwann cells atrophying and total Schwann cell number gradually decrease with time. Even when regenerating axonal sprouts invade into the cables, Schwann cells do not survive for long periods if they fail to make axonal contact. These observations strongly suggest the involvement of apoptosis in peripheral nerve degeneration and regeneration. So, we investigated the behavior of Schwann cells prepared from walleriandegenerated adult rat sciatic nerve in vitro. The secondary cultured Schwann cells showed serial changes in morphology, mitotic activity and migratory activity as they do during Schwann cell cable formation in vivo. At the final stage of differentiation, the Schwann cells became rounded and detached from the flask with extensive blebbing. Electron micrographs clearly demonstrated typical cytoplasmic changes of apoptosis, but, nuclei of most of the cells retained their size and morphology with residual nucleolar structures. An agarose gel electrophoresis of DNA clearly demonstrated that there was not any DNA fragmentation up to 120 h after detachment. Results by in situ apoptosis detection assay did not show any DNA degradation despite the substantial decrease in Schwann cell number. In conclusion, during peripheral nerve degeneration and regeneration, supernumerary Schwann cells are removed by apoptosis, however, it lacks most of the nuclear events of usual apoptosis.     

Neuromuscular activity impairs axonal sprouting in partially denervated muscles by inhibiting bridge formation of perisynaptic Schwann cells

Following partial denervation of rat hindlimb muscle, terminal Schwann cells extend processes from denervated endplates to induce and guide sprouting from the remaining intact axons. Increased neuromuscular activity significantly reduces motor unit enlargement and sprouting during the acute phase of sprouting. These findings led to the hypothesis that increased neuromuscular activity perturbs formation of Schwann cell bridges and thereby reduces sprouting. Adult rat tibialis anterior (TA) muscles were extensively denervated by avulsion of L4 spinal root and were immediately subjected to normal caged activity or running exercise (8 h daily) for 3, 7, 14, 21, and 28 days. Combined silver/cholinesterase histochemical staining revealed that the progressive reinnervation of denervated endplates by sprouts over a 1 month period in the extensively partially denervated TA muscles was completely abolished by increased neuromuscular activity. Immunohistochemical staining and triple immunofluorescence revealed that the increased neuromuscular activity did not perturb the production of Schwann cell processes, but prevented bridging between Schwann cell processes at innervated and denervated endplates. Our findings suggest that failure of Schwann cell processes to bridge between endplates accounts, at least in part, for the inhibitory effect of increased neuromuscular activity on sprouting.     

A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1

Sciatic nerve axons in cyclin D1 knockout mice develop normally, become properly ensheathed by Schwann cells, and appear to function normally. However, in the Wallerian degeneration model of nerve injury, the mitotic response of Schwann cells is completely inhibited. The mitotic block is Schwann cell autonomous and developmentally regulated. Rescue analysis (by "knockin" of cyclin E) indicates that D1 protein, rather than regulatory elements of the D1 gene, provides the essential Schwann cell function. Genetic inhibition of the Schwann cell cycle shows that neuronal responses to nerve injury are surprisingly independent of Schwann cell mitotic responses. Even axonal regrowth into the distal zone of a nerve crush injury is not markedly impaired in cyclin D1-/- mice.     

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

THE INTRAEPIDERMAL INNERVATION OF THE SNOUT SKIN OF THE OPOSSUM : A Light and Electron Microscope Study, with Observations on the Nature of Merkel's Tastzellen

The intraepidermal innervation of the snout skin of the opossum has been studied with the light and electron microscope. Numerous large nerve fibers loose their myelin sheath in the superficial dermis and pass into the epidermis. The basement membranes of the epidermis and Schwann cell become continuous at the point of entry of the neurite into the epidermis. Within the epidermis, the neurite is associated with a specialized secretory epidermal cell, termed a Merkel cell. This cell has many secretory granules apposed to the neurite. The Merkel cells are epidermal cells since they have desmosomes between them and adjacent epidermal cells. The neurite in the stratum spinosum is enveloped by Schwann cells in a manner analogous to the Schwann cell investment of unmyelinated neurites. In the upper stratum spinosum the nerve fiber evidences changes which can be interpreted as degenerative. The Merkel cell-neurite complex is interpreted as representing a sensory receptor unit.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

FAK is required for axonal sorting by Schwann cells

Signaling by laminins and axonal neuregulin has been implicated in regulating axon sorting by myelin-forming Schwann cells. However, the signal transduction mechanisms are unknown. Focal adhesion kinase (FAK) has been linked to alpha6beta1 integrin and ErbB receptor signaling, and we show that myelination by Schwann cells lacking FAK is severely impaired. Mutant Schwann cells could interdigitate between axon bundles, indicating that FAK signaling was not required for process extension. However, Schwann cell FAK was required to stimulate cell proliferation, suggesting that amyelination was caused by insufficient Schwann cells. ErbB2 receptor and AKT were robustly phosphorylated in mutant Schwann cells, indicating that neuregulin signaling from axons was unimpaired. These findings demonstrate the vital relationship between axon defasciculation and Schwann cell number and show the importance of FAK in regulating cell proliferation in the developing nervous system.     

Endothelins control the timing of Schwann cell generation in vitro and in vivo

Schwann cell precursors, derivatives of the neural crest, generate Schwann cells in a process that is tightly timed, well characterized, and directly controlled by axonal signals, in particular beta-neuregulins. Here we provide evidence that endothelins (ETs) are also important for survival and lineage progression in this system. We show that ETs promote rat Schwann cell precursor survival in vitro without stimulation of DNA synthesis. Using ET receptor agonists and antagonists, we demonstrate that this action of ET is mediated by the ET(B) receptor. RT-PCR reveals the presence of ET and ET receptor mRNA in the developing rat PNS. We showed previously that in vitro beta-neuregulins promote the generation of Schwann cells from precursors on schedule and that this process can be accelerated by fibroblast growth factor 2. Here we show that although ETs promote long-term precursor survival the transition of precursors to Schwann cells is delayed. Moreover, ETs block the maturation effects of beta-neuregulins. In spotting lethal rats, in which functional ET(B) receptors are absent, we find accelerated expression of the Schwann cell marker S100 in developing nerves. These observations indicate that complex growth factor interactions control the timing of Schwann cell development in embryonic nerves and that ETs act as negative regulators of Schwann cell generation.     

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.     

Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF BB

Neurofibromatosis type 1 (NF1) is characterized by the formation of neurofibromas, benign tumors of the peripheral nerve consisting essentially of Schwann cells, which can sometimes turn malignant to form neurofibrosarcomas. The mechanism of progression toward a malignant phenotype remains largely unknown. In this report, we show that platelet-derived growth factor (PDGF) BB, and to a lesser extent fibroblast growth factor 2, are mitogenic for two neurofibrosarcoma-derived Schwann cell lines, but not for a Schwann cell line derived from a schwannoma (from a non-NF1 patient) or for transformed rat Schwann cells. Levels of expression of both PDGF receptor α and β are significantly increased in the two neurofibrosarcoma-derived cell lines compared to the non-NF1 Schwann cell lines. The level of tyrosyl-phosphorylated PDGF receptor β is strongly increased upon stimulation by PDGF BB. In comparison, only modest levels of tyrosyl-phosphorylated PDGF receptor α are observed, upon stimulation by PDGF AA or PDGF BB. Accordingly, PDGF AA is only a weak mitogen for the neurofibrosarcoma-derived cells by comparison to PDGF BB. These results indicate that the mitogenic effect of PDGF BB for the neurofibrosarcoma-derived Schwann cell lines is primarily transduced by PDGF receptor β. Neu differentiation factor β, a potent mitogen for normal Schwann cells, was unable to stimulate proliferation of the transformed Schwann cell lines, due to a dramatic down-regulation of the erbB3 receptor. Therefore, aberrant expression of growth factor receptors by Schwann cells, such as the PDGF receptors, could represent an important step in the process leading to Schwann cell hyperplasia in NF1. J. Cell. Physiol. 177:334–342, 1998. © 1998 Wiley-Liss, Inc. The information in the article does not reflect government policy and no official endorsement should be inferred.     

ELECTRON MICROSCOPIC OBSERVATIONS OF THE OLFACTORY MUCOSA AND OLFACTORY NERVE

The olfactory receptor cell is characterized by a distal process (the dendrite) which terminates in the olfactory passage as the olfactory rod. The olfactory rod is provided with numerous cilia which are similar in structure to those seen in other tissues. The central processes of the bipolar cell constitute the fila olfactoria. The cytoplasmic organelles of the sustentacular cell are concentrated at the apical and basal ends of the cell with a paucity of cytoplasmic elements in the region of the nucleus. The plasma membrane of the supporting cell forms a mesaxon for both the dendrite and axon of the bipolar cell. Terminal bars are present in the epithelial cells. The axons constituting the fila olfactoria form fascicles which are ensheathed by mesaxons of adjacent Schwann cells. Thus the olfactory neurons are ensheathed throughout their course by the membranes of sustentacular and Schwann cells. Observations of the olfactory mucosa with the electron microscope are discussed with respect to recent electrophysiological studies.     

Role of macrophages in peripheral nerve degeneration and repair.

A cut or crush injury to a peripheral nerve results in the degeneration of that portion of the axon isolated from the cell body. The rapid degeneration of this distal segment was for many years believed to be a process intrinsic to the nerve. It was believed that Schwann cells both phagocytosed degenerating axons and myelin sheaths and also provided growth factors to promote regeneration of the damaged axons. In recent years, it has become apparent that the degenerating distal segment is invaded by monocytes from the blood. We will review the evidence that these recruited macrophages play a role in both degeneration and regeneration of peripheral nerve axons after injury and consider whether the slow degeneration and poor monocyte recruitment in the central nervous system may contribute to the poor regeneration there.     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

Regulated reprogramming in the regeneration of sensory receptor cells

Vision, olfaction, hearing, and balance are mediated by receptors that reside in specialized sensory epithelial organs. Age-related degeneration of the photoreceptors in the retina and the hair cells in the cochlea, caused by macular degeneration and sensorineural hearing loss, respectively, affect a growing number of individuals. Although sensory receptor cells in the mammalian retina and inner ear show only limited or no regeneration, in many nonmammalian vertebrates, these sensory epithelia show remarkable regenerative potential. We summarize the current state of knowledge of regeneration in the specialized sense organs in both nonmammalian vertebrates and mammals and discuss possible areas where new advances in regenerative medicine might provide approaches to successfully stimulate sensory receptor cell regeneration. The field of regenerative medicine is still in its infancy, but new approaches using stem cells and reprogramming suggest ways in which the potential for regeneration may be restored in individuals suffering from sensory loss.