Department of Anatomy and Neuroscience, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069
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ABSTRACT |
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Lin, Qing,
Xiaoju Zou, and
William D. Willis.
A 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 (A
) and unmyelinated (C) afferent fibers, as well as from
large myelinated (A
) fibers. After capsaicin was injected
intradermally into the plantar skin of the foot, a significant
enhancement of DRR activity was seen in A
and C fibers but not in
A
fibers, and this increase lasted for ~1 h. This study supports
the hypothesis that centrally mediated antidromic activity in A
and
C primary afferent fibers contributes to the development of neurogenic
inflammation, presumably by release of inflammatory substances in the periphery.
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INTRODUCTION |
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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 A
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 A
fibers conduct DRRs when
neurogenic inflammation develops in the skin.
In this study, we recorded DRRs from single A 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
).
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METHODS |
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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 · kg1 · 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 A
, A
, 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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RESULTS |
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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 A fibers, 11 C fibers, and 6 A
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 A
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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Spontaneous activity could be recorded from all A 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
A
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 A
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 A
fibers. DRRs
recorded from A
fibers were not significantly affected by CAP
injection (Fig. 2C).
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Control experiments were done on two A 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).
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DISCUSSION |
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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 A and C afferent fibers. In contrast, there was no
significant increase in DRRs recorded from A
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 A 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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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