Adelta and C Primary Afferents Convey Dorsal Root Reflexes After Intradermal Injection of Capsaicin in Rats

Qing Lin, Xiaoju Zou, and William D. Willis

Department of Anatomy and Neuroscience, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069


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INTRODUCTION
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Lin, Qing, Xiaoju Zou, and William D. Willis. Adelta and C Primary Afferents Convey Dorsal Root Reflexes After Intradermal Injection of Capsaicin in Rats. J. Neurophysiol. 84: 2695-2698, 2000. Antidromic activity was recorded in anesthetized rats from single afferent fibers in the proximal ends of cut dorsal root filaments at the L4-6 level and tested for responses to acute cutaneous inflammation produced by intradermal injection of capsaicin. This antidromic activity included low-frequency spontaneous firing and dorsal root reflex (DRR) discharges evoked by applying von Frey hairs to the skin of the foot. DRRs could be recorded from both small myelinated (Adelta ) and unmyelinated (C) afferent fibers, as well as from large myelinated (Abeta ) fibers. After capsaicin was injected intradermally into the plantar skin of the foot, a significant enhancement of DRR activity was seen in Adelta and C fibers but not in Abeta fibers, and this increase lasted for ~1 h. This study supports the hypothesis that centrally mediated antidromic activity in Adelta and C primary afferent fibers contributes to the development of neurogenic inflammation, presumably by release of inflammatory substances in the periphery.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intradermal injection of capsaicin (CAP) results in acute cutaneous neurogenic inflammation, characterized by a local increase in blood flow and the development of edema (Lin et al. 1999). The inflammation is accompanied by an enhancement in dorsal root reflexes (DRRs), recorded as multi-unit activity from the cut central ends of dorsal root filaments (Lin et al. 1999). Antidromic activity in primary afferent fibers is believed to be an important mechanism by which neurogenic inflammation is induced. Peripheral events include release of neuropeptides, particularly substance P (SP) and calcitonin gene-related peptide (CGRP), from the terminals of primary afferents (reviewed by Holzer 1988; Willis 1999). The primary afferents involved are mainly C fibers, although Adelta fibers also play a role (Lam and Ferrell 1993; Lewin et al. 1992). However, no evidence has been reported so far that individual C and Adelta fibers conduct DRRs when neurogenic inflammation develops in the skin.

In this study, we recorded DRRs from single Adelta and C units in teased dorsal root filaments and tested the effects of intradermal injection of CAP in the foot on these DRRs. This is an important extension of our previous work because peptidergic afferents generally have small diameters, and we hypothesize that release of peptides from such fine afferent nociceptive fibers by DRRs underlies the induction of neurogenic inflammation. Preliminary results have been published (Lin et al. 2000; Willis et al. 2000).


    METHODS
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INTRODUCTION
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Male Sprague-Dawley rats (250-350 g) were used. The experiments were approved by the Animal Care and Use Committee and were consistent with the ethical guidelines of the National Institutes of Health and of the International Association for the Study of Pain.

Pentobarbital sodium was used to induce (50 mg/kg ip) and maintain (5-8 mg · kg-1 · h-1 iv) anesthesia. Once a stable level of anesthesia was reached, the animals were paralyzed with pancuronium (0.3-0.4 mg/h iv) and ventilated artificially. End-tidal CO2 was kept at 3.5-4.5%. Core body temperature was maintained at ~37°C using a thermostatically controlled heating blanket. Dorsal roots were exposed by laminectomy and a dorsal rootlet from the L4-L6 level was cut distally. The central stump was placed on a dental mirror. Fine filaments were dissected until a single unit could be isolated on the basis of its amplitude and waveform. A platinum unipolar hook electrode was used for extracellular recordings of single fiber discharges. A bipolar stimulating electrode was placed on the cut dorsal rootlet 15-25 mm proximal to the recording site. An evoked action potential with a fixed latency was recorded when the nerve was stimulated electrically using a suprathreshold stimulus. Conduction velocity was calculated by dividing the conduction distance by the latency of the action potential. The fiber type was then identified, based on conduction velocity, as an Abeta , Adelta , or C fiber. Recorded activity was amplified and led to a data analysis system (CED1401 plus with Spike-2 software) for off-line analysis. DRR activity was evoked by applying a series of calibrated von Frey filaments having graded bending forces to an empirically determined area on the foot. Since the threshold for evoking DRRs by mechanically applied stimuli varied, an appropriate set of von Frey filaments was chosen in each experiment. Spontaneous antidromic discharges were also seen.

The effects of intradermal injection of CAP (1%, 25 µl) on spontaneous antidromic activity and evoked DRRs were tested as in our previous paper (Lin et al. 1999). CAP was injected intradermally into the plantar skin of the foot near the site (~5 mm) to which the von Frey hairs were applied to evoke the DRRs. DRRs were recorded before and at 15, 30, and 60 min after CAP injection. In a separate group of animals, the vehicle (7% Tween 80 and 93% saline) used to dissolve CAP was injected into the foot in the same volume while DRRs were recorded. Discharge frequencies were compared before and after intradermal injection of CAP. Changes are expressed as a percentage of control. Statistical comparisons were performed with paired t-tests. P <=  0.05 was considered significant.


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To avoid giving an intradermal injection of CAP or vehicle twice in the same animal, only one nerve fiber was investigated in each rat. A total of 30 individual single fibers were recorded from 30 rats. Of these, 13 were Adelta fibers, 11 C fibers, and 6 Abeta fibers.

When the fiber type was identified, we had to consider the possibility that the action potentials evoked by stimulation of a dorsal rootlet might be either directly propagated or a DRR generated by activating spinal neuronal circuits by the orthodromically conducted volley. However, evoked DRRs do not have a fixed latency, since they are triggered by a multisynaptic pathway (Eccles et al. 1961). Figure 1 shows action potentials evoked in an Adelta and a C fiber by repeated stimuli. The action potentials occurred at an essentially fixed latency and were thus evoked directly. Slight variations in latency are expected because of changes in refractoriness due to spontaneous antidromic activity and because of alterations in the level of primary afferent depolarization (PAD) spreading to the stimulated dorsal root. The conduction velocities were calculated from the latency and conduction distance.



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Fig. 1. Action potentials recorded from an Adelta and a C dorsal root fiber and evoked by electrical stimulation of the same dorsal rootlets. Electrical stimuli were repeated at 0.5 Hz for 8 sequential traces. The evoked potentials had fixed latencies (with some minor jitter). up-arrow , the time that a stimulus was applied.

Spontaneous activity could be recorded from all Adelta and C fibers. The discharges were irregular and at low frequencies (0.05-3.74 Hz). In most units (22/24), DRRs could be evoked by applying a graded series of von Frey hairs of increasing bending force to the skin of the foot. The forces applied to evoke DRRs were in the noxious range, between 36 and 1,274 mN (Leem et al. 1993). Figures 2 and 3 show spontaneous antidromic activity and evoked DRRs recorded from an Adelta and a C fiber identified by conduction velocity (Figs. 2A and 3A). Both spontaneous and evoked activity increased following intradermal injection of CAP (Figs. 2B and 3B). The enhancement of the responses lasted for <= 1 h after CAP injection. Tests were done on 11 Adelta and 10 C fibers. A significant increase both in spontaneous and evoked DRRs was observed following CAP injection (Figs. 2C and 3C). The possible effects of intradermal CAP injection on DRRs were examined in recordings from six identified Abeta fibers. DRRs recorded from Abeta fibers were not significantly affected by CAP injection (Fig. 2C).



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Fig. 2. Changes in spontaneous antidromic activity and evoked dorsal root reflexs (DRRs) recorded from an Adelta dorsal root fiber before and after intradermal capsaicin (CAP) injection. A: antidromic action potential evoked by electrical stimulation used to determine conduction velocity (CV). B, top: baseline activity. Inset, left: the expanded spikes of DRRs. Middle: responses 15 min after CAP injection. Bottom: 60 min after CAP injection. Horizontal lines above histograms at the right indicate times of application of von Frey hairs. The bending forces are shown above the horizontal lines. C: summary of the effects of intradermal CAP injection on spontaneous antidromic activity (SPON.) and evoked DRRs (EVOKED) recorded from Abeta and Adelta fibers. Baseline antidromic activity was expressed as 100% and is indicated by dashed line. * and **, P < 0.05 and P < 0.01, respectively, compared with the values pre-CAP injection.



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Fig. 3. Changes in spontaneous antidromic activity and evoked DRRs recorded from a C dorsal root fiber before and after intradermal CAP injection. A and B: as in Fig. 2. C: summary of the effects of intradermal CAP injection on spontaneous antidromic discharges and evoked DRRs recorded from C fibers. - - -, baseline antidromic activity was expressed as 100%. **, P < 0.01 compared with the values pre-CAP injection.

Control experiments were done on two Adelta and one C fibers by intradermal injection of 25 µl of vehicle. No significant change was observed in either spontaneous antidromic activity or in evoked DRRs (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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In our previous study (Lin et al. 1999), we showed that multi-unit antidromic action potentials (DRRs) are enhanced following intradermal injection of CAP. The central mechanism of the DRRs involved GABAA, non-N-methyl-D-aspartate (non-NMDA), as well as NMDA receptors within dorsal horn circuits. Here we confirmed these results by recording DRRs from individual A and C fibers in the cut central ends of dorsal root filaments in the same intradermal CAP injection model.

DRRs triggered by PAD are known to occur in A fibers (Eccles et al. 1961; Toennies 1938). PAD has been shown indirectly in C fibers by excitability testing (Calvillo et al. 1982; Carstens et al. 1981; Fitzgerald and Woolf 1981; Hentall and Fields 1979). DRRs in unmyelinated articular fibers have been suggested by compound potential analysis in a model of acute arthritis (Sluka et al. 1995). However, so far as we are aware, DRRs have not previously been demonstrated in individual C fibers.

The present experiments provide direct evidence that enhanced DRRs after acute cutaneous inflammation induced by CAP injection can be evoked in both Adelta and C afferent fibers. In contrast, there was no significant increase in DRRs recorded from Abeta fibers after intradermal CAP injection. These results suggest strongly that the centrally mediated antidromic activity that contributes to neurogenic inflammation is conducted by small myelinated and/or unmyelinated primary afferent fibers. Peptides, such as SP and CGRP, are released from the peripheral terminals of fine primary afferent fibers when these fibers are antidromically stimulated (Kress et al. 1999; reviewed by Holzer 1988) and these peptides cause vasodilation and neurogenic edema (reviewed by Holzer 1988; Willis 1999).

In conclusion, this is the first electrophysiological evidence that DRRs can be recorded from individual C afferent fibers. An enhancement of the DRRs in Adelta and C afferents follows intradermal injection of CAP, supporting the hypothesis that neurogenic inflammation can be induced in part by DRRs in fine primary afferent fibers.


    ACKNOWLEDGMENTS

The authors thank G. Gonzales for assistance with the illustrations.

This work was supported by Recruitment Grant 2517-98 (Sealy Memorial Endowment Fund for Biomedical Research) and National Institute of Neurological Disorders and Stroke Grant NS-09743.


    FOOTNOTES

Address for reprint requests: W. D. Willis, Dept. of Anatomy and Neuroscience, Marine Biomedical Institute, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069 (E-mail: wdwillis{at}utmb.edu).

Received 22 May 2000; accepted in final form 31 July 2000.


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