Dorsal Root Reflexes and Cutaneous Neurogenic Inflammation After Intradermal Injection of Capsaicin in Rats

Qing Lin, Jing Wu, and William D. Willis

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lin, Qing, Jing Wu, and William D. Willis. Dorsal Root Reflexes and Cutaneous Neurogenic Inflammation After Intradermal Injection of Capsaicin in Rats. J. Neurophysiol. 82: 2602-2611, 1999. The role of dorsal root reflexes (DRRs) in acute cutaneous neurogenic inflammation induced by intradermal injection of capsaicin (CAP) was examined in anesthetized rats. Changes in cutaneous blood flow (flare) on the plantar surface of the foot were measured using a laser Doppler flowmeter, and neurogenic edema was examined by measurements of paw thickness. To implicate DRRs in neurogenic inflammation after CAP injection, the ipsilateral sciatic and femoral nerves were sectioned, dorsal rhizotomies were performed at L3--S1, and antagonists of GABA or excitatory amino acid receptors were administered intrathecally. Intradermal injection of CAP evoked a flare response that was largest at 15-20 mm from the injection site and that spread >30 mm. Acute transection of the sciatic and femoral nerves or dorsal rhizotomies nearly completely abolished the blood flow changes 15-20 mm from the CAP injection site, although there was only a minimal effect on blood flow near the injection site. These procedures also significantly reduced neurogenic edema. Intrathecal bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione, (CNQX) or D(-)-2-amino-7-phosphonoheptanoic acid (AP7), but not phaclofen, also reduced dramatically the increases in blood flow 15-20 mm from the CAP injection site, but had only a minimal effect on blood flow near the injection site. Neurogenic edema was reduced by the same agents that reduced blood flow. Multiunit DRRs recorded from the central stumps of cut dorsal rootlets in the lumbar spinal cord were enhanced after CAP injection. This enhanced DRR activity could be reduced significantly by posttreatment of the spinal cord with bicuculline, CNQX or AP7, but not phaclofen. It is concluded that peripheral cutaneous inflammation induced by intradermal injection of CAP involves central nervous mechanisms. DRRs play a major role in the development of neurogenic cutaneous inflammation, although a direct action of CAP on peripheral nerve terminals or the generation of axon reflexes also may contribute to changes in the skin near the injection site.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neurogenic inflammation forms part of the peripheral tissue response to injury. This inflammatory response is generally attributed to an effector function of primary afferent terminals (Jancsó et al. 1967, 1968; Lewis 1927) and is initiated by the release of vasoactive peptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP) (Brain and Williams 1985; Ferrell and Russell 1986). The primary afferents involved are thought to be mainly C fibers, although Adelta -fibers also play a role (Colpaert et al. 1983; Lam and Ferrell 1993; Lewin et al. 1992).

Although the effector action of primary afferent fibers is often attributed to axon reflexes (Barron and Matthews 1938; Carpenter and Lynn 1981; Lewis 1927), it also can be initiated in the spinal cord by primary afferent depolarization (PAD) large enough to trigger dorsal root reflexes (DRRs) (Eccles et al. 1961, 1962; Koketsu 1956; Willis 1999). Previous experiments by our group have shown that neural processing in the spinal cord dorsal horn contributes to the development of knee joint inflammation by the generation of DRRs (Rees et al. 1994, 1996). These nerve impulses are conducted antidromically in large and small myelinated and in unmyelinated axons, as shown by recordings of compound action potentials in primary afferent fibers from the central stumps of cut dorsal root filaments or the cut medial articular nerve after induction of acute arthritis (Sluka et al. 1995a). This antidromic activity could, in turn, result in the release of inflammatory mediators in the knee joint (Ferrell and Russell 1986; Yaksh 1988), setting up a positive feedback loop between the dorsal horn and periphery that tends to prolong the inflammatory state (Sluka et al. 1995b; Willis 1999; Willis et al. 1998).

The experiments described in this paper examined the effector role of primary cutaneous afferents in acute cutaneous inflammation and determined the possible involvement of DRRs in this process. Intradermal CAP injection is a simple means to evoke an acute neurogenic inflammation in rats (Jancsó et al. 1967, 1968). When applied to the skin, CAP causes vasodilation if the sensory innervation is intact (Bernstein et al. 1981; Carpenter and Lynn 1981). To evoke an acute cutaneous inflammation, CAP was injected intradermally into the plantar skin of the foot. The flare (a major characteristic of inflammation) was evaluated by measuring changes in cutaneous blood flow in the foot. Neurogenic edema was estimated by measurements of paw thickness. DRRs were evaluated by recording antidromic discharges from the central ends of cut dorsal rootlets in the lumbar spinal cord. DRRs also could be demonstrated in cutaneous afferents supplying the foot.

Some of the present results have been reported in abstract form (Lin et al. 1997b, 1999).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A total of 116 male Sprague-Dawley rats weighing 250-350 g were used for this study. All experimental protocols were approved by the Animal Care and Use Committee and were in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain.

Animals were initially anesthetized by pentobarbital sodium (50 mg/kg ip). The external jugular vein was cannulated, and anesthesia was then maintained by infusion of sodium pentobarbital (5-8 mg · kg-1 · h-1 ) in a saline solution. The level of anesthesia was monitored by frequent examination of pupillary size and responses to stimulation, absence of a flexion reflex, and stability of the level of end-tidal CO2. Once a stable level of surgical anesthesia was reached, the animals were paralyzed with pancuronium (0.3-0.4 mg/hr iv) and ventilated artificially. End-tidal CO2 was kept between 3.5 and 4.5% by adjusting the respiratory parameters. Rectal temperature was maintained near 37°C by a servo-controlled heating blanket.

Cutaneous blood flow measurement

Changes in cutaneous blood flow in the plantar skin of the foot were measured to reflect the local vasodilation (flare) that followed intradermal injection of CAP. The measurements were done by attaching laser Doppler flowmeter probes (Moor Instruments, UK) to the skin of the foot with adhesive tape. Blood flow, detected as blood cell flux by the flowmeter (laser wavelength 633 nm), was processed by a computer analysis system (CED1401 plus, with Spike2 software), and blood flow was shown as a voltage level. It has been reported that the depth of laser penetration can be <= 500-700 µm below the surface where the probe is placed (Silverman et al. 1994). Therefore the laser Doppler flow probe presumably picked up the blood flow signal mainly from the microvasculature in the dermis. A volume of 25 µl of CAP, dissolved in Tween 80 (7%) and saline (93%) to a concentration of 1%, was injected into the plantar surface of the foot. For control purposes, the vehicle for dissolving CAP was injected intradermally in the same volume as the CAP solution.

Experiments first were made to map the distribution of flare on the foot skin ipsilateral to CAP injection by placing three probes at sites ~5, 15-20, and 30 mm, respectively, from the spot where CAP was injected intradermally (Fig. 1A). Figure 1, B and C, shows that the maximal flare reaction was recorded from a site (Probe III) ~15-20 mm away from the CAP injection spot and that the blood flow changes were much less at a site (Probe II) ~30 mm away from the injection spot. Therefore blood flow responses at ipsilateral sites >20 mm from the CAP injection site were not studied further.



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Fig. 1. A: schematic diagram shows the sites where 3 laser Doppler probes were placed on the plantar surface of the rat foot to measure blood flow. , sites where 3 probes were placed at distances of ~5, 15, and 30 mm away from the capsaicin (CAP) injection spot. B: traces showing cutaneous blood flow recorded in a rat from these 3 areas on 1 foot (indicated by Probes I, II, and III in A) before and after ipsilateral injection of CAP. C: averaged peak percent blood flow changes after CAP injection (n = 4).

To determine if elimination of presumed DRRs would result in a reduction in the flare produced by CAP injection, the femoral and sciatic nerves were sectioned ipsilaterally or dorsal roots L3-S1 were cut on the day when experiments were performed. CAP was then injected to determine if the flare reaction could be evoked. Before peripheral nerves were sectioned, 2% lidocaine was applied to them at sites where these nerves were to be cut to minimize injury discharges. For control purposes, sham surgery was done in other animals.

In another series of animals, the tip of a catheter (PE10) was implanted into the spinal subarachnoid space at the T12-L1 vertebral level for intrathecal administration of drugs. The spinal cord was pretreated with a GABAA receptor antagonist (bicuculline, 5 µg), a GABAB receptor antagonist (phaclofen, 15 µg), a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist [6-cyano-7-nitroquinoxaline-2,3-dione, (CNQX) 0.1 µg] or an NMDA receptor antagonist [D(-)-2-amino-7-phosphonoheptanoic acid (AP7), 4 µg], to determine if blockade of spinal GABA or excitatory amino acid (EAA) receptors could reduce the flare. Drugs were dissolved in artificial cerebrospinal fluid (ACSF) in a volume of 15 µl and were given intrathecally 20 min before CAP injection. The same volume of ACSF was used in other animals for control purposes.

In four rats, the spinal cord was transected at the T12-L1 level >= 1 h before the blood flow measurements were performed. The effects of CAP injection on blood flow were evaluated in these spinalized rats.

The degree of cutaneous inflammation due to CAP injection was also assessed by paw-thickness measurements. This was done with a digital caliper, which was placed near the site where Probe 2 was placed. Care was taken to assure that the caliper was placed at the same site on the paw for each measurement. Paw thickness was measured before and at 2 h after CAP injection. Paw-thickness changes due to CAP injection also were observed in experiments in which the sciatic and femoral nerves were sectioned or dorsal roots were cut or in which the spinal cord was pretreated with GABA or EAA receptor antagonists. Additionally, paw thickness was measured after the same volume of vehicle was injected intradermally in some rats as controls.

DRR recordings

The lumbosacral spinal cord was exposed by laminectomy to provide access to dorsal roots for recordings of DRRs. The spinal cord was protected from drying and cooling by formation of a mineral oil pool between skin flaps. Heated water was circulated through metal tubes placed in the pool. A dorsal rootlet from the L4-L6 level was teased into several filaments and cut distally. Multiunit DRRs were recorded by placing the proximal stump of one dorsal root filament on a platinum unipolar hook electrode. DRRs were amplified and observed on analogue and digital storage oscilloscopes and discriminated from noise using a window discriminator. Digitized signals were processed by an interface (CED 1401) connected to a Pentium PC to construct peristimulus rate histograms for counting the firing rates. To assure that nerve impulses, instead of noise, were recorded throughout the experiment, analogue signals also were processed simultaneously with the use of Spike-2 wavemark software, which captured the original spikes after subtracting the noise level (see Fig. 5C). Data storage space is conserved by saving only the spikes and not the baseline. Different spikes can be distinguished on the basis of their waveforms. Spontaneous DRRs always were recorded, but these often had a low discharge rate. Additional DRR activity was evoked by applying a series of calibrated von Frey filaments having graded bending forces to a certain area on the foot. The sites from which DRRs could be evoked were considered the "receptive field" for the DRRs. Because the threshold for evoking DRRs by mechanically stimulating peripheral afferent terminals varied with each experiment, an appropriate set of von Frey filaments was chosen for each animal. The forces applied to the receptive fields to evoke DRRs were between 20 and 1,274 mN, which are in the noxious range of stimuli (Leem et al. 1993).

After control spontaneous antidromic activity and evoked DRRs were recorded, CAP was injected intradermally into the foot in the way described in the preceding section. DRRs then were recorded at 15 min after CAP injection. To verify that these antidromic discharges after CAP injection were influenced by the same drugs that reduced flare, the spinal dorsal horn was posttreated with the same GABA, non-NMDA or NMDA receptor antagonists. A catheter (PE10) was placed near the surface of the dorsal horn at the point where the dorsal root from which DRRs were recorded entered the spinal cord. Bicuculline, phaclofen, CNQX or AP7 was dissolved in 15 µl of artificial cerebrospinal fluid (ACSF) at the same concentrations as used for reducing flare. The drugs were administered topically onto the spinal cord at 30 min after CAP injection. For control purposes, the same volume of ACSF was administered onto the spinal cord. Each animal was posttreated with only one drug, and so the animals were grouped according to drug treatment. Recordings of DRRs were made 30 (immediately after drug administration), 60, 90, and 120 min after CAP injection.

In some animals separate from the preceding section, the vehicle (7% Tween 80 and 93% saline) that was used for dissolving CAP was injected into the foot in the same volume while DRRs were recorded.

In two animals, recordings of DRRs were made from the central end of a cut sural nerve. The effects of CAP on DRRs recorded from ipsilateral sural nerve were observed when CAP was injected intradermally into the area that was innervated by the tibial or saphenous nerves (Takahashi et al. 1994).

Data analysis

Baseline blood flow level was expressed as 100% and percentage changes after CAP injection were compared for different groups of animals. Recorded DRR activity was analyzed off-line from peristimulus time histograms. Spontaneous antidromic discharges were quantitated in terms of multiunit discharge rates. Evoked DRRs were evaluated by subtracting spontaneous discharges from all evoked responses and by adding all responses evoked by stimulation using five graded von Frey filaments to produce a total response value. The responses to CAP injection were expressed as a percentage of baseline, with baseline set at 100%. Statistical significance was tested using ANOVA with repeated measures and differences across time were assessed with post hoc paired t-tests. A grouped t-test was used to compare the difference in responses between groups having different treatments. A P < 0.05 was taken as significant. Values are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in cutaneous blood flow recorded from different areas of the ipsilateral foot after capsaicin injection and effects of nerve sectioning

A laser probe was placed on a spot near the CAP injection site (site of Probe I, Fig. 1A) and another 15-20 mm distally (site of Probe II, Fig. 1A) to observe simultaneously blood flow changes both near and distal to the CAP injection site. As shown in Fig. 1B, baseline cutaneous blood flow was recorded continuously for ~30-40 min before CAP was injected intradermally into one foot. The CAP effect then was observed for <= 2 h.

Experiments were initially done on rats with sham surgery without cutting the sciatic and femoral nerves (n = 7). An elevated blood flow was seen at both sites after CAP injection, but the enhanced responses recorded from Probe I (Fig. 2A) were less than the responses measured from the distal site (Probe II, Fig. 2B). Peak increases were 294 ± 53% (P = 0.01, compared with baseline level) recorded from Probe I and 643 ± 131% (P = 0.0067) recorded from Probe II. After the sciatic and femoral nerves were cut (in separate experiments, n = 6), the enhanced blood flow recorded from both sites (Fig. 2, A and B) was less than in rats with sham surgery, but the magnitude of the reduction in the response at the site distal to CAP injection site was much larger than that at the site near the CAP injection spot (Fig. 2, B vs. A). Peak increases were 137 ± 9% (P = 0.027) recorded from the Probe II and 185 ± 26% (P = 0.027) recorded from Probe I. The peak increase recorded from Probe II in the nerve-cut group became much smaller than that in the sham-operated group (P = 0.002, Table 1). However, there was no statistical difference in the peak increases measured by the Probe I between the nerve-cut and the sham-operated groups (P = 0.11, Table 1).



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Fig. 2. Effects of peripheral nerve section or dorsal rhizotomy on ipsilateral blood flow changes recorded from sites near (A) and distal (B) to the site of CAP injection. Data are from animals with the sciatic and femoral nerves cut (n = 6), with dorsal rhizotomies at L3-S1 (n = 6) or with sham surgery (n = 14). Two laser probes (I and II) were placed on the plantar skin of the foot on the side of CAP injection, as indicated in Fig. 1A, to measure blood flow from 2 sites simultaneously. Probe I was near the spot where CAP was injected and Probe II was 15-20 mm distal to the CAP injection spot.


                              
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Table 1. Effects of nerve transections (N cut) or dorsal rhizotomies (D Rz) on blood flow changes at sites of Probe I (PI) and Probe II (PII) following CAP injection

In six rats, a dorsal rhizotomy was performed at the L3-S1 levels before blood flow was measured. A result consistent with the nerve-cut study was obtained in animals that underwent dorsal rhizotomy (Fig. 2). The peak increases in blood flow after CAP injection were reduced to 295 ± 72% (P = 0.045) at Probe I (Fig. 2A) and 198 ± 24% (P = 0.009) at Probe II (Fig. 2B). In sham-surgery animals (n = 7), the peak increases were 383 ± 34% (P = 0.005) at Probe I (Fig. 2A) and 589 ± 42% (P = 0.0006) at Probe II (Fig. 2B). There was no statistically significant difference in the peak increases at Probe I between these two groups of animals (P = 0.97, Table 1). However, a significant attenuation of enhanced blood flow responses was seen when the peak increase from Probe II in dorsal rhizotomized rats was compared with that in sham-operated rats (P = 0.0001, Table 1).

Control experiments were done in six rats separate from those used for CAP injection. The same volume of vehicle (Tween 80 and saline) that did not contain CAP was injected intradermally into the foot. Blood flow was recorded from the site of Probe II and observations were made <= 40 min after vehicle injection. A slight increase in blood flow was seen (peak value 114 ± 5%) after vehicle injection, but this was without statistical significance.

To examine if supraspinal modulation affected the neurogenic inflammation after CAP injection, additional observations were made on the effects of CAP injection on blood flow in four spinalized rats. Peak increases recorded from Probes I and II were 148 ± 24% and 491 ± 39%, respectively. These values were slightly less than the changes seen in intact animals. However, the differences did not reach statistical significance (P = 0.16 and P = 0.41, respectively).

Effects of blockade of spinal GABA and excitatory amino acid receptors on blood flow responses after capsaicin injection

Pretreatment of the spinal cord by intrathecal infusion of the GABAA receptor antagonist, bicuculline, or the non-NMDA receptor antagonist, CNQX, reduced profoundly the CAP-induced blood flow responses at the site distal to CAP injection spot (Probe II, Fig. 3B). Peak increases were 222 ± 42% (P = 0.032) and 255 ± 53% (P = 0.018), respectively. These values were much smaller when compared with the peak increases in rats pretreated with ACSF (P = 0.0004 and P = 0.009, respectively, Table 2), in which CAP injection resulted in a 563 ± 78% peak increase (P = 0.002) at Probe II. The responses at the site (Probe I) near the CAP injection spot were also reduced slightly (Fig. 3A), but the changes did not reach statistical significance when compared with the peak increases in rats pretreated with ACSF (P = 0.22 and P = 0.11, respectively, Table 2).



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Fig. 3. Effects of intrathecal pretreatment of the spinal cord with GABAA, GABAB, non-N-methyl-D-aspartate (non-NMDA) and NMDA glutamate receptor antagonists on ipsilateral blood flow changes recorded from sites near (A) and distal (B) to the CAP injection spot after CAP injection. CAP was injected intradermally 20 min after intrathecal infusion of bicuculline (n = 6), phaclofen (n = 6), 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX; n = 6) or D(-)-2-amino-7-phosphonoheptanoic acid (AP7; n = 6). Infusion of artificial cerebrospinal fluid (ACSF; n = 6) was done in separate animals for control purposes. Sites for blood flow measurements were the same as in Fig. 2.


                              
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Table 2. Effects of drug treatment of the spinal cord on blood flow changes at sites of probe 1 and probe 2 and on DRRs after CAP injection

Interestingly, blood flow responses induced by CAP injection also were reduced by pretreatment with an NMDA receptor antagonist, AP7 (Fig. 3, A and B). The peak increases recorded from Probes I and II were 200 ± 27% (P = 0.031) and 257 ± 52% (P = 0.03), respectively. Compared with peak increases in rats pretreated with ACSF, the peak increase at Probe II was reduced significantly (P = 0.015, Table 2) with AP7 pretreatment. However, the reduction in the response recorded from Probe I was not significant when compared with the response at Probe I of ACSF-pretreated group (P = 0.15, Table 2).

In summary, the amplitudes of the blood flow responses at the site distal to CAP injection were reduced significantly compared with those of the blood flow responses at the site near the CAP injection site when the spinal cord was pretreated with GABAA, non-NMDA or NMDA receptor antagonists.

In contrast, a GABAB receptor antagonist, phaclofen, did not significantly affect blood flow responses (P = 0.83 for Probe I and P = 1.0 for Probe II compared with the responses of the ACSF-pretreated group, Table 2). Peak increases were 319 ± 63% (P = 0.016) at Probe I and 583 ± 125% (P = 0.007) at Probe II.

Changes in paw thickness after capsaicin injection and effects of nerve sectioning and blockade of spinal GABA and excitatory amino acid receptors

Both in sham-operated rats (n = 14) and in rats in which the sciatic and femoral nerves were cut (n = 6) or in which dorsal rhizotomies were performed (n = 6), the development of edema in the hindpaw on the side of CAP injection was observed. Within 2 h after CAP injection, paw thickness increased significantly in sham-operated rats (Fig. 4A). However, the increase in paw thickness was reduced significantly after section of sciatic and femoral nerves or after dorsal rhizotomy. (Fig. 4A).



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Fig. 4. Changes in paw thickness after intradermal CAP injection into the ipsilateral foot and the effects of nerve section, dorsal rhizotomy (A), and blockade of spinal GABA, non-NMDA and NMDA receptors (B). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with value before CAP injection in the same group. +P < 0.05, ++P < 0.01, and +++P < 0.001 compared with the increased response in rats pretreated with ACSF.

In four rats in which the same volume of vehicle was injected intradermally, the paw thickness was 5.4 ± 0.05 mm before vehicle injection. There was slight increase in paw thickness (5.5 ± 0.06 mm) after vehicle injection, but this was without statistical significance (P = 0.09).

Pretreatment of the spinal cord with ACSF did not obviously affect the increases in paw thickness (n = 6). The increase in paw thickness was similar to that in sham-nerve cut or sham-dorsal rhizotomized rats (Fig. 4B). However, when the spinal cord was pretreated with bicuculline (n = 6), the increase in paw thickness due to CAP injection was almost completely blocked (Fig. 4B). Pretreatment of the spinal cord with either CNQX (n = 6) or AP7 (n = 6) also prevented most of the increase in paw thickness due to CAP injection (Fig. 4B). However, blockade of spinal GABAB receptors with phaclofen did not affect significantly the enhanced paw thickness (n = 6).

Dorsal root reflexes recorded from the central ends of cut dorsal root filaments and changes after intradermal injection of capsaicin

Baseline multiunit spontaneous antidromic discharges were recorded from 42 animals. The discharges were irregular and had rates that varied over a wide range of frequencies (0.03-5.9 Hz). Usually, the activity had a very low rate. In most recorded units, additional DRR activity could be evoked by mechanically applying a series of von Frey hairs of increasing bending force to the skin of the foot. Figures 5, A and B, and 6 show examples of spontaneous and evoked DRRs recorded from four rats, respectively. Intradermal injection of CAP caused an increase in spontaneous and/or evoked activity (2nd rows of Figs. 5A and 6A), although enhanced responses could be predominantly in either spontaneous or in evoked activity in particular cases (see 2nd row of Fig. 6A). The enhancement of the responses lasted for <= 2 h after CAP injection.



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Fig. 5. A and B: rate histograms represent spontaneous multi-unit antidromic discharges recorded from dorsal root filaments after ipsilateral intradermal injection of CAP and the effects of topical applications of ACSF (A) or bicuculline (B). Top: baseline activity; 2nd row: spontaneous activity 15 min after CAP injection; 3rd row: 30 min after CAP injection with application of ACSF or bicuculline; 4th-6th rows: 60, 90, and 120 min after CAP injection, respectively, with ACSF or bicuculline still present. C: spontaneous multiunit antidromic discharges recorded from the cut proximal end of the sural nerve and the effects of intradermal injection of CAP into the ipsilateral foot. Spikes were wavemark files that were saved by the Spike 2 software program. The bottom record shows action potentials of 3 different units on an expanded sweep at 30 min after CAP injection.



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Fig. 6. Rate histograms represent dorsal root reflexes (DRRs) recorded from dorsal root filaments and evoked by applying von Frey hairs of increasing bending force to the skin of the foot before and after ipsilateral intradermal injection of CAP and the effects of topical application of ACSF (A) or bicuculline (B). Top: baseline activity; 2nd row: responses 15 min after CAP injection; 3rd row: 30 min after CAP injection with application of ACSF or bicuculline; 4th-6th rows: 60, 90, and 120 min after CAP injection, respectively, with ACSF or bicuculline still present. Horizontal lines above histograms indicate times of application of von Frey hairs. Bending forces are shown above the horizontal lines.

In contrast, no significant change was observed in spontaneous antidromic activity and evoked DRRs after the same volume of vehicle was injected intradermally into the foot in six rats. Spontaneous activity and evoked DRRs 15 min after vehicle injection increased slightly to 120 ± 24% and 102 ± 11%, respectively. However, the changes were not statistically significant when compared with the baseline (P = 0.15 and P = 0.11, respectively).

Enhanced DRRs could also be recorded from the cut central end of a peripheral sensory nerve after CAP injection. Experiments were done in two animals, and Fig. 5C shows the results in one rat. Recordings were made from the central end of the cut ipsilateral sural nerve. CAP injection into the area of the foot that is innervated by the tibial nerve resulted in an increase in spontaneous antidromic discharges. The activity recovered within 90 min after CAP injection.

Effects of posttreatment of the spinal cord with GABA, non-NMDA and NMDA receptor antagonists on enhanced DRRs after capsaicin injection

CAP injection produced a long-lasting increase both in spontaneous antidromic discharges and evoked DRRs. Posttreatment of the spinal cord with ACSF at 30 min (3rd rows of Figs. 5A and 6A) after CAP injection did not interfere with this enhanced activity. There was a significant increase in DRRs for >= 90 min after CAP injection with ACSF posttreatment (n = 8, P = 0.04, Fig. 7, A and B). However, increased responses were reversed nearly completely after the spinal cord was posttreated with bicuculline (3rd rows of Figs. 5B and 6B). The responses of eight animals with bicuculline posttreatment are summarized in Fig. 7, A and B, and Table 2. The CAP-induced responses were significantly reduced after posttreatment with bicuculline when compared with the values at the same time points after CAP injection in the ACSF group (P = 0.028 and P = 0.0009, respectively).



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Fig. 7. Summary of spontaneous antidromic activity (A) and evoked DRRs (B) after ipsilateral intradermal injection of CAP and effects of topical applications of GABAA (bicuculline, Bic, n = 8), GABAB (phaclofen, Pha, n = 7), non-NMDA (CNQX, n = 7), and NMDA (AP7, n = 8) receptor antagonists to the spinal cord at 30 min post CAP injection. * P < 0.05; ** P < 0.01; and *** P < 0.001 compared with the baseline level of the same group. +P < 0.05; ++P < 0.01; and +++P < 0.001 compared with the value at the same time point after CAP injection in the ACSF group.

To establish if the enhanced DRR responses after CAP injection were selectively reversed by the GABAA receptor antagonist, bicuculline, similar experiments (n = 7) were done using the GABAB antagonist, phaclofen. Posttreatment with phaclofen failed to reverse the increase in DRRs after CAP injection (P = 0.463 and P = 0.301, respectively, Fig. 7, A and B, Table 2).

A reversal of the enhanced DRRs was seen when the non-NMDA (n = 7) or NMDA (n = 8) receptor antagonists, CNQX or AP7, were applied to the spinal dorsal horn, using the same procedure as for bicuculline administration. The reversal of the enhanced DRRs is shown in Fig. 7, A and B, and Table 2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main finding of this study was that intradermal injection of CAP into the rat foot evoked vasodilation in that foot. This response occurred both near the CAP injection site and in a surrounding area that extended >30 mm away from the injection site. Flare is defined as a cutaneous vasodilation surrounding a site of damage (see Lewis 1927). Because the CAP-evoked spread of vasodilation found in this work is similar to the flare seen in human skin after CAP injection (LaMotte et al. 1991), we refer to it as flare. Flare was accompanied in these experiments by the development of edema as shown by an increase in paw thickness. Several lines of experimental evidence, including the data from DRR recordings provided by this study, indicate that much of the vasodilation and edema after intradermal injection of CAP is mediated by way of the dorsal horn of the spinal cord via an effector action of primary afferent fibers. We propose that the mechanism of these components of neurogenic cutaneous inflammation depend on antidromic discharges traveling from the dorsal horn along primary afferent fibers, that is on DRRs (Fig. 8).



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Fig. 8. Diagram illustrating a proposed model of the mechanism by which dorsal horn circuits produce primary afferent depolarization and generate DRRs. Two dorsal root ganglion cells are shown. One innervates the skin at the CAP injection site, and the other supplies the skin away from the injection site. CAP injection activates dorsal horn circuits that result in DRRs, which produce vasodilatation in the skin adjacent to the injection site.

It has been thought that inflammation-induced vasodilation around a cutaneous injury is initiated by axon reflexes (Barron and Matthews 1938; Carpenter and Lynn 1981; Lewis 1927). This term denotes a reflex that takes place entirely within a single neuron (Lewis 1927). It was proposed that sensory nerve fibers have branches that end in adjacent areas of skin. One branch forms a sensory ending for reception of the irritant stimulus, whereas another supplies a blood vessel. When the sensory ending is activated by inflammatory mediators released by tissue injury, such as after an intradermal injection of CAP, nerve impulses travel not only centrally but at the branch point also pass antidromically to the vicinity of the blood vessel to cause vasodilation by releasing vasoactive substances. This hypothesis is based on two lines of experimental evidence: 1) reflex flare depends on intact sensory innervation because it does not take place in denervated skin (Bernstein et al. 1981; Carpenter and Lynn 1981; Jancsó et al. 1968; Lewis 1927); and 2) the spread of flare is prevented by administration of a local anesthetic (Dux et al. 1996; Jancsó et al. 1968; Lewis 1927). Additionally, there is morphological evidence for an axon reflex arrangement because there are extensive arborizations of sensory nerve processes and some branches have been described near blood vessels (Chapman and Goodell 1964; Helme and McKernan 1985).

It recently was demonstrated in rats that antidromic stimulation of identified cutaneous C fibers produces an increase in blood flow in a localized area, the size of which coincides well with the afferent receptive field. (Gee et al. 1997). Therefore it is difficult to explain the flare that we observed simply by a local axon reflex because the area of flare extends greatly beyond the receptive fields of rat nociceptive primary afferent neurons. From our observations, an enhanced blood flow occurs both near the CAP injection site and up to >= 30 mm away. Although the part of the vasodilation near the injection site could be mediated by local axon reflexes or by a direct action of CAP, it seems unlikely that this mechanism could account for increased blood flow more than a few millimeters from the CAP injection site because nociceptors in the rat foot have receptive fields <6 mm2 in area (Bharali and Lisney 1992). Other arguments from our study that are also against the view that neurogenic inflammation is mediated only by a local axon reflex include 1) the blood flow that could be measured 15-20 mm from the CAP injection spot was increased more than that recorded at a site within 5 mm of the CAP injection spot; 2) proximal sectioning of the sciatic and femoral nerves (any sensory nerve bifurcations in the periphery remained intact) or dorsal rhizotomy abolished nearly completely the CAP-evoked increase in blood flow at the distal site, but the change in blood flow near the injection site was only slightly affected; and 3) more importantly, blockade of spinal GABAA, non-NMDA or NMDA receptors reduced dramatically the CAP-evoked increase in blood flow at the distal site, although the enhanced response near the injection site was only slightly decreased. The vasodilation near the site of injection could result mainly from local axon reflexes or from a direct action of CAP on sensory terminals (Szolcsányi 1996). The fact that there was a smaller vasodilation at a distance of 5 mm than at 15-20 mm from the CAP injection site could be the result of a blockade by CAP of nerve conduction in CAP-sensitive afferent nerve fibers and thus of DRRs conducted in these fibers. The widespread vasodilation or flare in the skin distal to the injection site appears to involve a spinally mediated mechanism (PAD that triggers DRRs) with an action on large numbers of primary afferent fibers (Willis 1999; Willis et al. 1998).

However, one possible concern is that sympathetic efferents might be involved in the mediation of the acute peripheral inflammatory response by interaction with primary afferent terminals (Jänig et al. 1996). The effects of sectioning the sciatic and femoral nerves and dorsal rhizotomy on the CAP-evoked changes in blood flow may indicate a central control of neurogenic inflammation by efferent activity of sensory neurons, but cannot rule out the possible involvement of sympathetic nerves. The role of sympathetic nerves in neurogenic peripheral inflammation still remains obscure. It has been shown that the development of peripheral inflammation depends on sympathetic postganglionic neurons (Green et al. 1993, 1997; Miao et al. 1996). However, observations in previous studies by our group and others showed that acute inflammation of the knee joint was unaffected by chemical or surgical sympathectomy (Lam and Ferrell 1991; Rees et al. 1994; Sluka et al. 1994b). In the present study, blockade of spinal GABAA, non-NMDA or NMDA receptors reduced profoundly the enhanced blood flow response after CAP injection. These observations provide evidence consistent with the idea that spinal cord modulation of neurogenic inflammation involves DRRs.

To obtain direct evidence that DRRs are involved in cutaneous neurogenic inflammation, we have used the same model as was used in experiments for blood flow measurements to determine if DRRs recorded from the cut central end of dorsal roots at the L4-6 levels become enhanced during acute cutaneous inflammation of the foot after CAP injection. Our results reveal that DRRs originating from the spinal cord are enhanced after intradermal injection of CAP. These antidromic impulses were blocked pharmalogically by posttreatment of the spinal cord with GABA and EAA receptor antagonists. The results are consistent with the data on neurogenic flare and edema. It thus is suggested that DRRs are likely to play a significant role in cutaneous neurogenic inflammation. The main reason why we recorded DRRs chiefly from dorsal root filaments instead of from peripheral cutaneous sensory nerves was based on the concern that the peripheral nerves contain sympathetic fibers, which needed to be excluded carefully in this experiment. The role of sympathetic nerves in neurogenic peripheral inflammation still needs to be considered (Green et al. 1993, 1997; Miao et al. 1996). DRRs also were recorded from the cut central ends of the sural nerve in separate experiments, and DRRs in this peripheral nerve responded to CAP injection in a manner similar to DRRs recorded from dorsal root filaments, indicating that DRRs generated in the spinal cord do travel along cutaneous afferents to the periphery where some presumably release inflammatory agents.

Therefore we conclude that the DRRs recorded from either dorsal root filaments or peripheral nerves are enhanced by activation of neighboring intact afferent fibers after CAP injection via central circuits within the spinal dorsal horn. This is supported by the observation that DRRs often could be evoked by mechanical stimulation applied to peripheral receptive fields.

There are several mechanisms by which inflammation might increase the likelihood for DRRs to be triggered by peripheral stimulation. Inflammation causes an upregulation of GABAergic mechanisms in the dorsal horn (Castro-Lopes et al. 1994a,b; Nahin and Hylden 1991). An increased release of GABA from GABAergic interneurons of the dorsal horn could result in more PAD and secondarily trigger DRRs (Willis 1999). PAD also is found to be mediated by spinal non-NMDA receptors (Evans and Long 1989; Hackam and Davidoff 1991). The most likely explanation of this is that primary afferent fibers release EAAs, which then activate non-NMDA receptors on GABAergic interneurons, causing them to release GABA at axoaxonic or dendroaxonic synapses on primary afferent terminals (Carlton and Hayes 1990; Sluka et al. 1995b). A significant alleviation of inflammatory responses, such as swelling, an increase in the temperature of the knee joint and hyperalgesia, was accompanied by an inhibition of DRRs when spinal GABAA or non-NMDA receptors were blocked (Sluka and Westlund 1993; Sluka et al. 1993, 1994a). Another mechanism could be an increased activity of the Na+-K+-Cl- cotransporter in primary afferent neurons (Willis 1999). This cotransporter is regulated by second-messenger systems that are affected by a variety of neurotransmitters, hormones, and growth factors (Alvarez-Leefmans et al. 1998). Second-messenger systems may control the cotransporter by phosphorylation and dephosphorylation. Central sensitization of spinothalamic tract neurons after intradermal injection of CAP is associated with the activation of protein kinase pathways (Lin et al. 1996, 1997a). Thus it is plausible to suggest that the Na+-K+-Cl- cotransporter could be phosphorylated by protein kinases that are activated after CAP injection.

The results of this and our preceding paper support previous studies done in models of arthritis (Rees et al. 1994-1996; Sluka et al. 1993, 1994a, 1995a) and suggest that excessive PAD leading to the generation of DRRs may be an important factor in acute cutaneous neurogenic inflammation. The neurotransmitter receptors that mediate DRR activity in arthritis include GABAA and non-NMDA glutamate receptors. However, the present study indicates that spinal NMDA receptors also are involved in the generation of DRRs in an inflammatory model involving the skin, suggesting that the mechanisms by which DRRs are generated in the development of cutaneous inflammation differ somewhat from those in joint inflammation. Thus the dorsal horn circuits that produce DRRs in cutaneous nerve fibers presumably involve GABAA, non-NMDA and NMDA receptors (Fig. 8). The primary afferent fibers activate GABAergic interneurons by release of EAAs (Evans and Long 1989), and PAD then is initiated by the action of GABA on GABAA receptors on primary afferent terminals (Curtis and Lodge 1982; Eccles et al. 1963). The PAD may become excessive and trigger DRRs, which in turn propagate peripherally and release inflammatory agents in peripheral tissue, including the skin (Fig. 8). The result is the induction of the initial stages of neurogenic inflammation.


    ACKNOWLEDGMENTS

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

This work was supported by Recruitment Grant 2517-98 from the 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.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 March 1999; accepted in final form 2 August 1999.


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