Altered temporal pattern of evoked afferent activity in a rat model of vincristine-induced painful peripheral neuropathy.

It is known that the level of activity in nociceptive primary afferent nerve fibers increases in neuropathic conditions that produce pain, but changes in the temporal patterning of action potentials have not been analyzed in any detail. Because the patterning of action potentials in sensory nerve fibers might play a role in the development of pathological pain states, we studied patterning of mechanical stimulus-evoked action potential trains in nociceptive primary afferents in a rat model of vincristine-induced painful peripheral neuropathy. Systemic administration of vincristine (100 microg/kg) caused approximately half the C-fiber nociceptors to become markedly hyperresponsive to mechanical stimulation. Instantaneous frequency plots showed that vincristine induced an irregular pattern of action-potential firing in hyperresponsive C-fibers, characterized by interspersed occurrences of high- and low-frequency firing. This pattern was associated with an increase in the percentage of interspike intervals 100-199 ms in duration compared with that in C-fibers from control rats and vincristine-treated C-fibers that did not become hyperresponsive. Variability in the temporal pattern of action potential firing was quantified by determining the coefficient of variability (CV2) for adjacent interspike intervals. This analysis revealed that vincristine altered the pattern of action-potential timing, so that combinations of higher firing frequency and higher variability occurred that were not observed in control fibers. The abnormal temporal structure of nociceptor responses induced by vincristine in some C-fiber nociceptors could contribute to the pathogenesis of chemotherapy-induced neuropathic pain, perhaps by inducing activity-dependent post-synaptic effects in sensory pathways.

Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine-induced painful peripheral neuropathy in the rat.

Vincristine, along with other antimitotic chemotherapeutic drugs, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias, dysesthesias, and occasionally hypoesthesia, and by hyporeflexia (Holland et al. [1973] Cancer Res. 33:1258-1264; McLeod and Penny [1969] J Neurol Neurosurg Psychiatry 32:297-304; Postma et al. [1993] J Neurooncol. 15:23-27; Sandler et al. [1969] Neurology 19:367-374). Systemic administration of vincristine causes swelling of unmyelinated axons and disorientation of axonal microtubules (Tanner et al. [1998a1998a] J Comp Neurol. 395:481-492) at a time when it also produces allodynia and mechanical hyperalgesia (Aley et al. [1996] Neuroscience 73:259-265; Authier et al. [1999] Neuroreport 10:965-968) and enhanced responsiveness in C-fibers in the rat (Tanner et al. [1998b] J Neurosci. 18:6480-6491). Because slowing of A-fiber conduction velocities had also been demonstrated (Tanner et al. [1998b] J Neurosci. 18:6480-6491), and mechanical hyperalgesia can occur secondary to damage to large diameter sensory afferents (Basbaum et al. [1991] Can J Physiol Pharmacol. 69:647-651; Coggeshall et al. [1993] Pain 52:233-242; Woolf and Mannion [1999] Lancet 353:1959-1964), we sought to determine whether vincristine also induced ultrastructural changes in myelinated A-fibers. Moreover, since systemic treatment with vincristine did not cause profound microtubule depolymerization in the unmyelinated axons of the peripheral nerve, we hypothesized that the drug's effects may be more extensive in the cell body, because in the spinal ganglion, the blood-nerve barrier is less restrictive. We used quantitative ultrastructural methods to analyze the microtubule cytoskeleton in myelinated axons in the mid-shaft of the saphenous nerve and in the sensory ganglion cells. Vincristine induced swelling of the whole nerve and an increase in the cross-sectional areas of myelinated axons but no loss of myelinated axons. There was a significant decrease in axonal microtubules, as well as microtubule disorganization, in myelinated fibers from vincristine-treated rats. In the spinal ganglion, vincristine induced swelling of large diameter sensory neurons and a build-up of neurofilaments in the cell bodies and proximal axons, suggestive of impaired anterograde axonal transport.

Nociceptor hyper-responsiveness during vincristine-induced painful peripheral neuropathy in the rat.

Neuropathic pain accompanies peripheral nerve injury after a wide variety of insults including metabolic disorders, traumatic nerve injury, and neurotoxic drugs. Chemotherapy-induced neuropathic pain, caused by drugs such as vincristine and taxol, occurs in cancer patients who receive these drugs as antineoplastic agents. Although a variety of remediations have been attempted, the absence of knowledge concerning mechanisms of chemotherapy-induced neuropathic pain has hindered the development of treatment strategies. Vincristine, a widely used chemotherapeutic agent, produces painful peripheral neuropathy in humans and mechanical hyperalgesia in rats. To test the hypothesis that alterations in C-fiber nociceptor function occur during vincristine-induced painful peripheral neuropathy, we performed in vivo extracellular recordings of single neurons from the saphenous nerve of vincristine-treated rats. Forty-one percent of C-fiber nociceptors were significantly hyper-responsive to suprathreshold mechanical stimulation. As a population, these mechanically hyper-responsive nociceptors also had significantly greater responses to suprathreshold heat stimulation; however, heat hyper-responsiveness was found only in a subset of these nociceptors and was never detected in the absence of mechanical hyper-responsiveness. In addition, mean conduction velocities of A-fibers and C-fibers in vincristine-treated rats were significantly slowed. Mean heat and mechanical activation thresholds of C-fiber nociceptors, their distribution among subclasses, and the percentage of spontaneously active neurons in vincristine-treated rats were not statistically different from controls. Vincristine does not, therefore, cause generalized impairment of C-fiber nociceptor function but rather specifically interferes with mechanisms underlying responsiveness to suprathreshold stimuli. Furthermore, vincristine-induced nociceptor hyper-responsiveness may involve alterations specifically in mechanotransduction in some nociceptors and alterations in general cellular adaptation mechanisms in others.

Microtubule disorientation and axonal swelling in unmyelinated sensory axons during vincristine-induced painful neuropathy in rat.

Neuropathic pain accompanies peripheral nerve injury following a variety of insults including metabolic disorders, traumatic injury, and exposure to neurotoxins such as vincristine and taxol. Vincristine, a microtubule depolymerizing drug, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias and dysesthesias (Sandler et al., [1969] Neurology 19:367-374; Holland et al. [1973] Cancer Res. 33:1258-1264). The recent development of an animal model of vincristine-induced neuropathy provides an opportunity to investigate mechanisms underlying this form of neuropathic pain. Systemic vincristine (100 microg/kg) produces hyperalgesia to mechanical stimuli during the second week of administration, which persists for more than a week (Aley et al. [1996] Neuroscience 73:259-265). To test the hypothesis that changes in microtubule structure in nociceptive sensory neurons accompany vincristine-induced hyperalgesia, we analyzed unmyelinated axons in saphenous nerves of vincristine-treated rats. This study constitutes the first quantitative ultrastructural analysis of the cytoskeleton of unmyelinated axons in peripheral nerve during neuropathic hyperalgesia. There was no evidence of unmyelinated fiber loss or a decrease in the number of microtubules per axons. There was, however, a significant decrease in microtubule density in unmyelinated axons from vincristine-treated rats. This decrease in microtubule density was due to a significant increase in the cross-sectional area of unmyelinated axons, suggesting swelling of axons. In addition, vincristine-treated axons had significantly fewer microtubules cut in cross-section and significantly more tangentially oriented microtubules per axon compared to controls. These results suggest that vincristine causes disorganization of the axonal microtubule cytoskeleton, as well as an increase in the caliber of unmyelinated sensory axons.

Formalin induces biphasic activity in C-fibers in the rat.

While the formalin test is a widely used behavioral model of tonic chemogenic pain, little is known about the responses of primary afferent nociceptors to formalin. Formalin (2.5%, 50 microliters) was injected either directly in or adjacent to the mechanical receptive fields of single C-fibers isolated from the saphenous nerve of pentobarbital-anesthetized rats. The average formalin-evoked response in C-fibers (n = 29) over time was biphasic. This biphasic time course of the C-fiber response to formalin is similar to that of the behavioral response in the awake animal and is compatible with the hypothesis that increased C-fiber activity contributes to the behavioral response in phase 2, as well as in phase 1 of the formalin test.

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.