CX3CL1-mediated macrophage activation contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy

Painful peripheral neuropathy is a dose-limiting side effect of paclitaxel therapy, which hampers the optimal clinical management of chemotherapy in cancer patients. Currently the underlying mechanisms remain largely unknown. Here we showed that the clinically relevant dose of paclitaxel (3 × 8 mg/kg, cumulative dose 24 mg/kg) induced significant upregulation of the chemokine CX3CL1 in the A-fiber primary sensory neurons in vivo and in vitro and infiltration of macrophages into the dorsal root ganglion (DRG) in rats. Paclitaxel treatment also increased cleaved caspase-3 expression, induced the loss of primary afferent terminal fibers and decreased sciatic-evoked A-fiber responses in the spinal dorsal horn, indicating DRG neuronal apoptosis induced by paclitaxel. In addition, the paclitaxel-induced DRG neuronal apoptosis occurred exclusively in the presence of macrophage in vitro study. Intrathecal or systemic injection of CX3CL1 neutralizing antibody blocked paclitaxel-induced macrophage recruitment and neuronal apoptosis in the DRG, and also attenuated paclitaxel-induced allodynia. Furthermore, depletion of macrophage by systemic administration of clodronate inhibited paclitaxel-induced allodynia. Blocking CX3CL1 decreased activation of p38 MAPK in the macrophage, and inhibition of p38 MAPK activity blocked the neuronal apoptosis and development of mechanical allodynia induced by paclitaxel. These findings provide novel evidence that CX3CL1-recruited macrophage contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy.     

Vasodilatation in the rat dorsal hindpaw induced by activation of sensory neurons is reduced by paclitaxel

Peripheral neuropathy is a major side effect following treatment with the cancer chemotherapeutic drug paclitaxel. Whether paclitaxel-induced peripheral neuropathy is secondary to altered function of small diameter sensory neurons remains controversial. To ascertain whether the function of the small diameter sensory neurons was altered following systemic administration of paclitaxel, we injected male Sprague Dawley rats with 1mg/kg paclitaxel every other day for a total of four doses and examined vasodilatation in the hindpaw at day 14 as an indirect measure of calcitonin gene related peptide (CGRP) release. In paclitaxel-treated rats, the vasodilatation induced by either intradermal injection of capsaicin into the hindpaw or electrical stimulation of the sciatic nerve was significantly attenuated in comparison to vehicle-injected animals. Paclitaxel treatment, however, did not affect direct vasodilatation induced by intradermal injection of methacholine or CGRP, demonstrating that the blood vessels' ability to dilate was intact. Paclitaxel treatment did not alter the compound action potentials or conduction velocity of C-fibers. The stimulated release of CGRP from the central terminals in the spinal cord was not altered in paclitaxel-injected animals. These results suggest that paclitaxel affects the peripheral endings of sensory neurons to alter transmitter release, and this may contribute to the symptoms seen in neuropathy.     

Sciatic nerve transection triggers release and intercellular transfer of a genetically expressed macromolecular tracer in dorsal root ganglia

We recently developed a genetic transneuronal tracing approach that allows for the study of circuits that are altered by nerve injury. We generated transgenic (ZW-X) mice in which expression of a transneuronal tracer, wheat germ agglutinin (WGA), is induced in primary sensory neurons, but only after transection of their peripheral axon. By following the transneuronal transport of the tracer into the central nervous system (CNS) we can label the circuits that are engaged by the WGA-expressing damaged neurons. Here we used the ZW-X mouse line to analyze dorsal root ganglia (DRG) for intraganglionic connections between injured sensory neurons and their neighboring "intact" neurons. Because neuropeptide Y (NPY) expression is strongly induced in DRG neurons after peripheral axotomy, we crossed the ZW-X mouse line with a mouse that expresses Cre recombinase under the influence of the NPY promoter. As expected, sciatic nerve transection triggered WGA expression in NPY-positive DRG neurons, most of which are of large diameter. As expected, double labeling for ATF-3, a marker of cell bodies with damaged axons, showed that the tracer predominated in injured (i.e., axotomized) neurons. However, we also found the WGA tracer in DRG cell bodies of uninjured sensory neurons. Importantly, in the absence of nerve injury there was no intraganglionic transfer of WGA. Our results demonstrate that intraganglionic, cell-to-cell communication, via transfer of large molecules, occurs between the cell bodies of injured and neighboring noninjured primary afferent neurons.     

Paclitaxel inhibits the activity and membrane localization of PKCα and PKCβI/II to elicit a decrease in stimulated calcitonin gene-related peptide release from cultured sensory neurons

Peripheral neuropathy is a dose-limiting and debilitating side effect of the chemotherapeutic drug, paclitaxel. Consequently, elucidating the mechanisms by which this drug alters sensory neuronal function is essential for the development of successful therapeutics for peripheral neuropathy. We previously demonstrated that chronic treatment with paclitaxel (3–5 days) reduces neuropeptide release stimulated by agonists of TRPV1. Because the activity of TRPV1 channels is modulated by conventional and novel PKC isozymes (c/nPKC), we investigated whether c/nPKC mediate the loss of neuropeptide release following chronic treatment with paclitaxel (300 nM; 3 and 5 days). Release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Following paclitaxel treatment, cultured dorsal root ganglia sensory neurons were stimulated with a c/nPKC activator, phorbol 12,13-dibutyrate (PDBu), or a TRPV1 agonist, capsaicin, in the absence and presence of selective inhibitors of conventional PKCα and PKCβI/II isozymes (cPKC). Paclitaxel (300 nM; 3 days and 5 days) attenuated both PDBu- and capsaicin-stimulated release in a cPKC-dependent manner. Under basal conditions, there were no changes in the protein expression, phosphorylation or membrane localization of PKC α, βI or βII, however, paclitaxel decreased cPKC activity as indicated by a reduction in the phosphorylation of cPKC substrates. Under stimulatory conditions, paclitaxel attenuated the membrane translocation of phosphorylated PKC α, βI and βII, providing a rationale for the attenuation in PDBu- and capsaicin-stimulated release. Our findings suggest that a decrease in cPKC activity and membrane localization are responsible for the reduction in stimulated peptide release following chronic treatment with paclitaxel in sensory neurons.     

Ultrastructural features of functionally identified primary afferent neurons with C (unmyelinated) fibers of the guinea pig: classification of dorsal root ganglion cell type with reference to sensory modality

Intracellular labeling of neurons permitted a direct correlation of neuronal profiles with sensory modality of cutaneous receptors. In the guinea pig, 47 neurons in the dorsal root ganglion (displaying C-fiber conduction velocities) were labeled with horseradish peroxidase (HRP) by iontophoresis after determining the sensory modality. Receptive field were explored with systematic "natural" stimuli. Cell areas of all C-fiber units were measured by tracing the cellular contour in light microscopy. The mean cellular diameter calculated from cell areas was 21.8 microns in the second cervical ganglion of the guinea pig. Mean cell diameter for high-threshold mechanoreceptors was 20.9 microns, 24.6 microns for polymodal nociceptors, and 25.7 microns for mechanical-cold nociceptors. Electron microscopic observations showed that all labeled neurons of C-fiber units had profiles of small, dark type-B neurons. Neurons representative of each sensory modality exhibited different cell features, each belonging to a distinct subtype of small B neurons. High-threshold mechanoreceptor and mechanical-cold nociceptor displayed a peripheral lamellar arrangement of cisternae of endoplasmic reticulum (ER), corresponding to the B1 subtype. Polymodal nociceptor units were characteristically of the B2 subtype, in which stacks of long and short cisternae of ER were distributed randomly throughout the cytoplasm, and the arrangement of Golgi bodies varied among these cells. Cooling receptors displayed poorly developed, flattened cisternae of ER and numerous vesicles, typical of the B3 subtype. These results imply that all C-fiber cells belong to the small B-type cell category and that the ultrastructural features of the neuron in the dorsal root ganglion may reflect the sensory modality of the receptive field.     

Paraneoplastic encephalomyelitis/sensory motor peripheral neuropathy - an autopsy case study

Paraneoplastic neurological anti-Hu syndrome is one of the most frequent remote effects of cancer and usually manifests as encephalomyelitis combined with peripheral neuropathy. Subacute sensory neuronopathy, which results from the inflammatory destruction of sensory neurone cell bodies in the dorsal root ganglia, is thought to be the principal presentation of peripheral neuropathy. In addition to sensory involvement, evidence of motor nerve involvement is frequently found. The mechanisms of motor involvement remain largely unclear and there have been only a limited number of pathological studies. We present an autopsy case study of anti-Hu paraneoplastic encephalomyelitis/sensory-motor neuropathy, which confirms an inflammatory paraneoplastic destruction of sensory neuron cell bodies in the dorsal root ganglia and lower motor neurons in the spinal cord, as a cause of clinically rapidly progressive peripheral sensory-motor neuropathy.     

Identification of fluocinolone acetonide to prevent paclitaxel‐induced peripheral neuropathy

Paclitaxel (PTX) is among the most commonly used cancer drugs that cause chemotherapy‐induced peripheral neuropathy (CIPN), a debilitating and serious dose‐limiting side effect. Currently, no drugs exist to prevent CIPN, and symptomatic therapy is often ineffective. In order to identify therapeutic candidates to prevent axonal degeneration induced by PTX, we carried out a phenotypic drug screening using primary rodent dorsal root ganglion sensory neurons. We identified fluocinolone acetonide as a neuroprotective compound and verified it through secondary screens. Furthermore, we showed its efficacy in a mouse model of PTX‐induced peripheral neuropathy and confirmed with four different cancer cell lines that fluocinolone acetonide does not interfere with PTX's antitumor activity. Our study identifies fluocinolone acetonide as a potential therapy to prevent CIPN caused by PTX.     

In vivo gene therapy for pyridoxine-induced neuropathy by herpes simplex virus-mediated gene transfer of neurotrophin-3.

Neurotrophic factors have been demonstrated to prevent the development of peripheral neuropathy in animal models, but the therapeutic use of these factors in human disease has been limited by the short serum half-life and dose-limiting side effects of these potent peptides. We used peripheral subcutaneous inoculation with a replication-incompetent, genomic herpes simplex virus-based vector containing the coding sequence for neurotrophin-3 to transduce sensory neurons of the rat dorsal root ganglion in vivo, and found that expression of neurotrophin-3 from the vector protected peripheral sensory axons from neuropathy induced by intoxication with pyridoxine assessed by electrophysiological (foot sensory response amplitude, and conduction velocity, and H-wave), histological (nerve morphology and morphometry), and behavioral measures of proprioceptive function. In vivo gene transfer using herpes simplex virus vectors provides a unique option for treatment of diseases of the sensory peripheral nervous system.     

Schedule dependency of paclitaxel-induced neuropathy in mice: a morphological study

Paclitaxel, which is known to induce peripheral neuropathy in humans, was administered to BDF1 mice, and a neuropathological examination was then conducted. Paclitaxel was administered at a dose of 30 mg/kg once or several times at different intervals, 3 times, every 3 hours (q3hx3), every day (q1dx3), every 2 days (q2dx3) and 4 times, once a week (q7dx4). The spinal cord, spinal ganglion and peripheral nerves (sciatic and tibial) were then processed for neuropathological examination following perfusion of the anesthetized mice. The effect on the peripheral nerves was evaluated by counting the number of abnormal nerve fibers in the preparations of teased nerve fibers and in the epoxy resin 1-microm-thick sections. The drug-induced degeneration, such as axonal and myelin fragmentations and phagocytosis in the nerve fibers, was detected in the dorsal funiculus, the dorsal spinal roots and the peripheral nerves (sciatic and tibial). No drug-related degeneration was observed in the motor neurons in the spinal cord nor in the ventral spinal roots. The single treatment did not induce any changes. The severity of the degeneration was as follows: q2dx3>q1dx3>q3hx3 and q7dx4. The degeneration in the mice treated on q3hx3 and q7dx4 was very slight, but it was clear that paclitaxel also induced degeneration on these schedules. These results suggest that paclitaxel induces a predominantly sensory neuropathy in mice and the severity is obviously dependent on the treatment schedule.     

Neuronal projections to the guinea pig stellate ganglion investigated by retrograde tracing

Previous electrophysiological studies have revealed a peripheral sensory input to the stellate ganglion which does not originate from the dorsal root ganglia. The present retrograde tracing study aimed at evaluating whether the parent cell bodies are located in the periphery, i.e. in mediastinal ganglia. Following injection of Fast blue or wheat germ agglutinin-horseradish peroxidase into the right stellate ganglion of the guinea pig, retrogradely labelled cell bodies were observed in the intermediolateral and intercalated nuclei of the spinal cord as well as in dorsal root ganglia at segmental levels C8 to T6. In another case, the stellate ganglion was resected and replaced by a sponge soaked with 10 μl of Fast blue. Labelling of preganglionic and sensory neurons parallelled that obtained by tracer injections. In neither case, however, were retrogradely labelled neurons found within or around the thoracic viscera (thymus, trachea, bronchi, esophagus, heart, great vessels of upper mediastinum) when these were cut serially en bloc. Controls performed by injection of Fast blue into the inferior mesenteric ganglion and investigation of the distal colon showed that our experimental protocol was able to visualize a peripheral projection towards a sympathetic ganglion — in this case from myenteric ganglia to the inferior mesenteric ganglion. We conclude that, in contrast to the circuitry connecting prevertebral sympathetic ganglia with the gut, the neuronal cell bodies providing peripheral sensory input from thoracic viscera to the right stellate ganglion most likely are not located within the mediastinal ganglia. Instead, they may reside within the stellate ganglion itself.     

Protective effect of HSV-mediated gene transfer of nerve growth factor in pyridoxine neuropathy demonstrates functional activity of trkA receptors in large sensory neurons of adult animals

The distinct distribution of trkA receptors on small neurons and trkC receptors on large neurons in the dorsal root ganglion correlates with the dependence of these two classes of neurons on nerve growth factor and neurotrophin-3, respectively, for survival during development. In adult animals, the distribution of high affinity neurotrophin (trk) is complex and overlapping; neurotrophins are not required for cell survival, but may influence cell phenotype and the response to injury. In order to test the functional activity of trkA receptors in the sensory ganglia of adult animals in vivo, we examined the ability of a nerve growth factor-expressing recombinant replication-defective herpes simplex virus-based vector to prevent the selective degeneration of large sensory fibres caused by intoxication with pyridoxine. Transduction of dorsal root ganglion neurons in vivo by subcutaneous inoculation of the nerve growth factor-expressing vector prevented the development of pyridoxine-induced neuropathy measured by electrophysiological, morphological and behavioural measures. These results demonstrate a functional activity of trkA receptors expressed on large neurons in the dorsal root ganglion in mature animals; this observation has important implications for the choice of neurotrophic factors for treatment of peripheral nerve disease.     

The role of p-c-Jun in survival and outgrowth of developing sensory neurons

c-Jun activation has been implicated not only in neuronal apoptosis, but also in survival and regeneration. This Janus facet of c-Jun activation could be related to neuronal cell type or to the developmental stage of the neuron. We investigated c-Jun activation in E18 sensory neurons. Cultures of rat dorsal root ganglia neurons were maintained with or without the addition of nerve growth factor or the c-Jun N-terminal kinase inhibitor, (D)-JNKI1. Few dorsal root ganglia neurons survived nerve growth factor deprivation, whereas neurons supplied with nerve growth factor survived and exhibited extensive axonal outgrowth. Activated c-Jun was present in the nuclei of neurons with regenerating axons, but not in apoptotic neurons. c-Jun N-terminal kinase inhibition reduced the number of p-c-Jun immunoreactive and regenerating neurons, and increased cell death. Thus, activation of c-Jun seems to be required for survival and regeneration of developing sensory neurons.     

Preferential loss of large lumbar primary sensory neurons in carcinomatous sensory neuropathy

Morphometric studies at autopsy of a patient with sensory neuropathy associated with small-cell lung carcinoma showed preferential loss of large-diameter sensory nerve cell bodies (fifth lumbar dorsal root ganglion), marked decrease of large myelinated fibers in the dorsal root and sural nerve, and almost total loss of myelinated fibers in the fasciculus gracilis.     

The Janus role of c-Jun: cell death versus survival and regeneration of neonatal sympathetic and sensory neurons

We investigated the functional outcome of c-Jun activation in sympathetic and sensory neurons of neonatal rat superior cervical ganglion (SCG) and dorsal root ganglion (DRG), respectively. Distinctly different roles of c-Jun activation have been suggested for these two types of neurons. In dissociated sympathetic neurons, c-Jun has been demonstrated to promote apoptosis, whereas in sensory neurons it stimulates axonal outgrowth. In organ-cultured ganglia, we found that c-Jun was activated within 24 h of explantation in both types of neurons, and that the JNK inhibitor SP600125 could mitigate this response. In both types of neurons, c-Jun activation was also reduced by NGF treatment. Inhibition of c-Jun activation did not affect the viability of sympathetic neurons, whereas the number of apoptotic sensory neurons increased. Furthermore, inhibition of c-Jun reduced axonal outgrowth from both SCG and DRG. Thus, in organ culture, c-Jun activation may be required for axonal outgrowth and, at least in sensory neurons, it promotes survival. The role of ATF3, a neuronal marker of injury and a c-Jun dimerization partner, was also examined. We found an ATF3 induction in both SCG and DRG neurons, a response, which was reduced by JNK inhibition. The reduction of ATF3 upon JNK inhibition was much larger in DRG than in SCG, a result which might account for the higher number of apoptotic neurons in JNK inhibitor exposed DRG. Taken together, and contrary to our expectations, neonatal sympathetic and sensory neurons seem to respond to axonal injury similarly with respect to c-Jun activation, and in no case was this activation pro-apoptotic.     

Pyridoxine-induced toxicity in rats: a stereological quantification of the sensory neuropathy

Excess ingestion of pyridoxine (vitamin B6) causes a severe sensory neuropathy in humans. The mechanism of action has not been fully elucidated, and studies of pyridoxine neuropathy in experimental animals have yielded disparate results. Pyridoxine intoxication appears to produce a neuropathy characterized by necrosis of dorsal root ganglion (DRG) sensory neurons and degeneration of peripheral and central sensory projections, with large diameter neurons being particularly affected. The major determinants affecting the severity of the pyridoxine neuropathy appear to be duration and dose of pyridoxine administration, differential neuronal vulnerability, and species susceptibility. The present study used design-based stereological techniques in conjunction with electrophysiological measures to quantify the morphological and physiological changes that occur in the DRG and the distal myelinated axons of the sciatic nerve following pyridoxine intoxication. This combined stereological and electrophysiological method demonstrates a general approach that could be used for assessing the correlation between pathophysiological and functional parameters in animal models of toxic neuropathy.     

Calbindin-immunoreactive sensory neurons of dorsal root ganglion project to skeletal muscle in the chick

In the chicken dorsal root ganglia, two neuronal subpopulations referred to as A1 and B1 share in common an immunoreactivity to antisera raised to calbindin D-28k but are distinguished by their cytological and ultrastructural characteristics. To determine the peripheral targets innervated by calbindin-immunoreactive neurons in lumbosacral dorsal root ganglia, cryostat sections of various hindlimb tissues were treated with anticalbindin antisera. Calbindin-immunostained axons were clearly detected in skeletal muscle. Large myelinated nerve fibres and afferent axon terminals in neuromuscular spindles were calbindin-immunoreactive; thin unmyelinated nerve fibres were also immunostained in nerve bundles of the perimysium. Since motoneurons and neurons of the autonomic nervous system were devoid of calbindin immunostaining, it was suggested that the immunoreactive axons found in skeletal muscle originate from sensory neurons expressing a calbindin immunoreaction in the dorsal root ganglia. This hypothesis was corroborated after introduction of wheat germ agglutinin coupled with horseradish peroxidase or colloidal gold particles into the sartorius muscle. The retrogradely transported tracer was collected only in ganglion cell bodies which displayed the ultrastructural characteristics of A1 and B1 sensory neurons. On the basis of calbindin immunoreaction and of tracer retrograde transport, it is concluded that ganglion cells of subclasses A1 and B1 contribute to the sensory innervation of skeletal muscle in the chicken.     

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.