Blood flow in the sciatic nerve is regulated by vasoconstrictive and vasodilative nerve fibers originating from the ventral and dorsal roots of the spinal nerves

NEUROSCIENCE RESEARCH ELSEVIER

Neuroscience Research 21 (1994) 125-133

Blood flow in the sciatic nerve is regulated by vasoconstrictive and vasodilative nerve fibers originating from the ventral and dorsal roots of the spinal nerves Akio Sato *a, Yuko Sato b, Sae Uchida a aDepartment of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, Itabashi-Ku, Tokyo 173, Japan bLaboratory of Physiology, Tsukuba College of Technology, Tsukuba 305, Japan Received 31 August 1994; accepted 20 September 1994

Abstract Anesthetized rats were subjected to repetitive electrical stimulation of either the ventral or dorsal root of the spinal nerves between the 11th thoracic and 2nd sacral spinal segments. The response of nerve blood flow (NBF) in the sciatic nerve was examined using laser Doppler flowmetry. For all nerve fibers stimulation was for a 10-30-s period at a supramaximal intensity. (1) Stimulation of the T11-LI ventral roots produced an increase in mean arterial pressure (MAP) and a biphasic NBF response was comprised of an initial increase and a subsequent decrease. The initial increase was a passive vasodilation due to the increase in MAP, while the following decrease in NBF resulted from active vasoconstriction of the vasa nervorum due to the activation of sympathetic nerves innervating the sciatic vasa nervorum. (2) Stimulation of the ventral root of the L6 segment produced an increase in NBF, even though MAP decreased. This increase in NBF was apparently mediated by activation of parasympathetic cholinergic vasodilators, because the response was abolished by i.v. injection of atropine, a muscarinic cholinergic receptor antagonist. (3) Stimulation of the dorsal roots between the L3 and S1 segments produced an increase in NBF, independent of changes in MAP. This increase in NBF appeared to be mediated by activation ofa calcitonin gene-related peptide (CGRP) containing afferent fibers innervating the vasa nervorum, because the response was abolished by topical application of hCGRP (8-37), a CGRP receptor antagonist. In conclusion, NBF in the sciatic nerve is regulated by: (1) sympathetic vasoconstrictors exiting the ventral roots of the spinal cord via the T11-LI segments; (2) parasympathetic vasodilators exiting the ventral roots of the spinal cord via the L6 segment; and (3) afferent CGRP containing vasodilators entering the dorsal roots of the spinal cord via the L3-SI segments. Keywords: Vasa nervorum; Spinal root; Vasoconstriction; Vasodilation; Nerve blood flow; Acetylcholine; Calcitonin gene-related peptide; Rat

1. Introduction Peripheral nerves receive their oxygen and nourishment from blood flowing in the vasa nervorum. Histochemical studies have demonstrated that the nerve fibers innervating the vasa n e r v o r u m contain noradrenaline, acetylcholine, serotonin and various polypeptides, such as vasoactive intestinal polypeptide, substance P (SP), neuropeptide Y, and calcitonin gene-related peptide * Corresponding author, Tel.: +81 3 3964 3241 (Ext. 3087); Fax: +81 3 3964 1415.

( C G R P ) (Amenta et al., 1983; Appenzeller et al., 1984; Dhital and Appenzeller, 1988; Milner et al., 1992) and appear to have specific functions in the neural regulation o f nerve blood flow (NBF). Sympathetic noradrenergic innervation o f the vasa nervorum, appears to have a vasoconstrictive function. Chemical sympathectomy o f the rat causes complete disappearance o f catecholamines from the vasa nervorum o f the sciatic nerve as determined by histofluorescence methods (Amenta et al., 1983). Repetitive electrical stimulation o f the lumbar sympathetic nerve trunk in anesthetized rats decreases N B F in the sciatic

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126

A. Sato et al./Neurosci, Res. 21 (1994) 125-133

nerve via activation of ot-adrenergic receptors (Hotta et al., 1991). These data indicate the existence of a vasoconstrictive sympathetic nerve fiber innervating the vasa nervorum in the sciatic nerve. This vasoconstrictive sympathetic nerve fiber may exit the spinal cord via the spinal ventral roots, however, it is not known which spinal levels possess this putative innervation. NBF in the sciatic nerve increased following repetitive electrical stimulation of the sciatic nerve trunk suggesting the existence of vasodilatory nerve fibers innervating the vasa nervorum (Zochodne and Ho, 1991b). Cholinergic vasodilative innervation of the vasa nervorum, has not yet been demonstrated. The evidence that acetylcholinesterase positive nerve fibers in the vasa nervorum are not altered by chemical sympathectomy (Amenta et al., 1983) is consistent with the theory that cholinergic nerve fibers in the vasa nervorum are of parasympathetic origin. Although the existence of muscarinic cholinergic receptors localized in the wall of the vasa nervorum of the rat sciatic nerve (Zaccheo et al., 1991) suggests the existence of cholinergic innervation of the vasa nervorum, it is not known what role these cholinergic parasympathetic fibers of the vasa nervorum have in the regulation of NBF. It is also not known at what spinal levels these nerve fibers exit the spinal cord. NBF in the sciatic nerve is also suggested to be regulated by peptidergic afferent nerve fibers in the vasa nervorum (Zochodne and Ho, 1991a). NBF in the sciatic nerve is increased after topical epineurial application of capsaicin (Zochodne and Ho, 1991a), or after sciatic nerve injury (Zochodne and Ho, 1992) and these vasodilative NBF responses are abolished by topical epineurial application of a CGRP receptor antagonist. However, to date the nature of the peptidergic vasodilative afferent innervation of the vasa nervorum of sciatic nerve is obscure as no systematic investigation has been performed. The present study aims to investigate the role of the sympathetic vasoconstrictive, cholinergic parasympathetic vasodilative, and peptidergic afferent vasodilative fibers in the regulation of NBF in the sciatic nerve. We used repetitive electrical stimulation of either the ventral or dorsal roots of the spinal nerves between the T11 and $2 segments and measured the response of NBF in the sciatic nerve using laser Doppler flowmetry. 2. Materials and methods

Twenty-four male Fischer-344 rats, 6-10 months old (330-430 g body wt), were used for the present experiments. Animals were anesthetized with urethane (1.1 g/kg i.p.). Additional urethane was administered (0.05-0.1 g/kg, i.v.) to maintain the depth of anesthesia,

if systemic arterial blood pressure was observed to fluctuate. Respiration was maintained using an artificial respirator (model 683, Harvard, USA) and end-tidal CO 2, monitored using a gas monitor (1H26, NEC Sanei, Tokyo), was kept at 3.0-4.0% by changing the tidal volume and frequency of the respirator. The rectal temperature of the rat was maintained at 37.5 ± 0.5°C using a thermostatically-regulated heating pad and tamp (ATB 1100, Nihon Kohden, Tokyo). Mean arterial pressure (MAP) was recorded continuously through a cannula in a common carotid artery. The jugular vein was cannulated for the intravenous administration of drugs. Pancronium bromide (Mioblock, Organon, Netherlands) (0.2 mg/h, i.v.) was used for immobilization during the experiments. The animal was placed in the prone position and laminectomy was performed between the spinal levels of T ll and $2. After opening the dura mater and arachnoidea, the ventral and dorsal roots of the spinal nerves between the T11 and $2 levels were separated and cut close to the spinal cord. These nerves were covered with warm liquid paraffin and the cut peripheral segments were placed on bipolar platinum-iridium wire stimulating electrodes. Repetitive electrical square pulse stimuli of 0.5 ms width, with varying frequencies, intensities and periods, were applied. At the end of some experiments the threshold intensities were determined by recording evoked volleys from the stimulated L6 ventral or dorsal root about 5-10 mm distal from the stimulation site. The sciatic nerve was exposed 2.5-3 cm above the knee joint and NBF was measured using laser Doppler flowmeter (ALF 2100, Advance Co., Ltd., Tokyo). The probe of the flowmeter (diameter 0.8 mm) was gently placed in contact with the surface of the sciatic nerve and care was taken to avoid compressing the nerve. A black vinyl sheet (4 mm square) was placed under the sciatic nerve to prevent the measurement of blood flow in the underlying skeletal muscles. The exposed sciatic nerve was covered with warm liquid paraffin. The following drugs, dissolved in saline, were used; (1) atropine sulfate (Sigma Chemical Co., USA), a muscarinic cholinergic receptor antagonist; (2) hCGRP (8-37) (Peptide Institute, Osaka), a CGRP receptor antagonist; and (3) CP-96345 (Pfizer, USA), an SP receptor antagonist. Atropine and CP-96345 were administered intravenously, hCGRP (8-37) was applied topically to the sciatic nerve using about a 0.2-ml volume at concentrations of 10 -4 M or 10 -5 M according to the method of Zochodne and Ho (1991a). Data were expressed as mean ± S.E.M. The statistical significance was determined by analysis of variance (ANOVA) followed by Fisher's least significant difference test.

A. Sato et al./ Neurosci. Res. 21 (1994) 125-133 3. R e s u l t s

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3.1. The effects o f repetitive electrical stimulation of the ventral roots on N B F in the sciatic nerve

127

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Repetitive electrical stimulation with parameters of 10 V, 10 Hz for 30 s of the ventral roots between the T11 and $2 spinal segments induced various effects on MAP and NBF in the sciatic nerve depending on the level of ventral roots stimulated. The results were classified into two groups according to the level of spinal segments. Group 1 consisted of responses of the T1 l - L 1 segments. Group 2 was limited to responses of the L6 ventral root segment. 3.1.1. Stimulation o f the T l l - L 1 ventral roots Fig. 1 (Rat A) shows examples of NBF and MAP responses to stimulation of the ipsilateral T1 l - L 2 ventral roots in one rat. Stimulation of the T l l - L I segments produced biphasic NBF responses, composed of an initial increase and a subsequent decrease in NBF, both accompanied by a pressor response. This biphasic response was similar to the biphasic response elicited by electrical stimulation of the lumbar sympathetic trunk demonstrated in our previous study (Hotta et al., 1991). In contrast, stimulation of the ipsilateral and the contralateral L2 ventral root induced simultaneous increases in both NBF and MAP.

Rat A

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t

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Fig. 2. The effects of stimulation of the unilateral L6 ventral root (10 V, 10 Hz, 30 s, shown by horizontal bars) on NBF in the sciatic nerve ipsilateral (A) and contralateral (B) to the stimulation in one rat.

3.1.2. Stimulation o f the L6 ventral root Fig. 1 (Rat B) shows examples of NBF and MAP responses to stimulation of the ipsilateral L 4 - $ 2 ventral roots in one rat. The stimulation of L 4 - L 5 and S1-$2 had no effect on either NBF or MAP, whereas stimulation of the L6 ventral root produced changes in both NBF and MAP. Stimulation of the L6 ventral root induced a biphasic response in NBF composed of an initial slight decrease and a subsequent large increase during which time MAP decreased slightly. Fig. 2 shows responses of NBF and MAP elicited by ipsilateral (A) and contralateral (B) stimulation of the L6 ventral root. When the contralateral ventral root was stimulated with the same stimulus parameters used for stimulation of the ip-

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Fig. 1. The effectsof stimulation of the unilateral ventral roots between TI l-L2 (from Rat A) and L4-$2 (from Rat B) on blood flow in the sciatic nerve ipsilateral to the stimulation. Sample recordingsof the nerve blood flow (NBF) and mean arterial pressure (MAP) after electrical stimulation of ventral root with parameters of 0.5 ms, 10 V, 10 Hz, for 30 s, indicated by horizontal bars.

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A. Sato et aL /Neurosci. Res. 21 (1994) 125-133

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Fig. 3. Responses of NBF and MAP to stimulation of the L6 ventral root (10 V, 10 Hz, 30 s) in 4 rats before (closed circles) and 15 min after (open squares) administration of atropine (1-2.5 mg/kg, i.v.). Changes in NBF (A) and MAP (B) were calculated every 10 s, and are expressed as percentages of the prestimulus values just before the stimulation (ordinates). The horizontal bar between the dashed vertical lines indicates the time during which the L6 ventral root was stimulated. Each point with a vertical bar represents a mean ± S.E.M. Onset of electrical stimulation of the L6 ventral root is expressed as zero (abscissa). *P < 0.05, **P < 0.01; significantly different from prestimulus control values, ap < 0.05, bp < 0.01; significantly different from respective values before administration of atropine.

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stim. DR (10V,10Hz,10s) Fig. 5. The effects of stimulation of the dorsal roots between L4 and $2 on MAP and NBF in the sciatic nerve ipsilateral to the stimulation. Electrical stimulation of the dorsal roots with parameters of 10 V, 10 Hz, for 10 s is indicated by horizontal bars.

tion. The NBF reached its maximum around 10 s after the end of stimulation, at about 120% of the prestimulus control level of NBF. NBF gradually returned to the control level within 80 s after the end of stimulation. The summarized data of simultaneously recorded MAP are plotted in Fig. 3B (closed circles). The MAP showed a slight decrease during stimulation of the L6 ventral root. The decrease was maximal at the end of stimulation dropping to about 95% of the prestimulus control level

silateral ventral root, NBF showed a slight decrease, accompanied by a simultaneous decrease in MAP (Fig.

2B). The responses of ipsilateral NBF following repetitive electrical stimulation (10 V, 10 Hz, 30 s) of the L6 ventral root in four rats are summarized in Fig. 3A (dosed circles). Following an initial slight decrease, the NBF started to increase within 20 s after the onset of stimula-

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Fig. 4. Relationships between intensities (A) and frequencies (B) of electrical stimulation of L6 ventral root and magnitudes of NBF responses. Maximum NBF within l rain after onset of stimulation is expressed as % of the maximum NBF within 30 s before stimulation (ordinate). (A) Stimulation with 10 Hz, for 30 s, with various intensities (abscissa) in one rat. Threshold intensity for C fibers is indicated by an arrow (Tc). (B) Stimulation with l0 V, for 30 s, with various frequencies (abscissa) in another rat.

A. Sato et al./Neurosci. Res. 21 (1994) 125-133 A

ipsilateral

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stimulus frequencies, the response of NBF was dependent on stimulus frequency as shown in Fig. 4B. A frequency of 1 Hz was subthreshold and failed to produce any significant change in NBF. The increase in NBF was frequency dependent between 2 and 10 Hz. The response was saturated at 10 Hz. This tendency was confirmed in another rat. The effect of a muscarinic cholinergic receptor antagonist on the NBF response. To examine whether the increase in NBF induced by repetitive electrical stimulation of the L6 ventral root (with stimulus parameters of 10 V, 10 Hz and 30-s period) was produced by a release of acetylcholine from the cholinergic efferent nerve terminals and activation of the muscarinic cholinergic receptors in the vasa nervorum of the sciatic nerve, atropine, a cholinergic muscarinic receptor antagonist, was administered intravenously. As summarized in Fig. 3A, the response of NBF following repetitive electrical stimulation of the L6 ventral root was abolished by i.v. injection of atropine (1-2.5 mg/kg) (n = 4 rats). The attenuating effect of atropine started to appear within 10 min after the injection and lasted for more than 30 min. The i.v. administration of atropine did not modify the response of MAP as shown in Fig. 3B.

mV 450

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Fig. 6. The effects of stimulation of the unilateral L6 dorsal root (10 V, 10 Hz, 10 s, shown by horizontal bars) on NBF in the sciatic nerve ipsilateral (A) and contralateral (B) to the stimulation in one rat.

of MAP. After the end of stimulation the MAP recovered within 30 s. Stimulus intensity. Threshold intensity of C fibers determined by recording evoked volley from the stimulated L6 ventral root was 0.7 V (n = 2 rats). No response of ipsilateral NBF to electrical stimulation of the L6 ventral root was obtained with stimulus intensities below 0.5 V, which was subthreshold for C fibers. An increase in NBF was obtained with stimulus intensities above 1 V, which is above the threshold for unmyelinated C fibers in the ventral root (Fig. 4A). The response was elicited in a stimulus intensity-dependent manner between 1 and 5 V and saturated at 5 V. This tendency was confirmed in another rat. This indicates that activation of C efferent fibers is important in eliciting the response, and also that myelinated efferent fibers cannot produce the response. Stimulus frequency. When the L6 ventral root was electrically stimulated with 10 V intensity at various

A

129

3.2. The effects o f repetitive electrical stimulation o f the dorsal roots on N B F in the sciatic nerve N B F in t h e sciatic n e r v e w a s m e a s u r e d f o l l o w i n g r e p e t i t i v e e l e c t r i c a l s t i m u l a t i o n o f t h e d o r s a l r o o t bet w e e n t h e T11 a n d $2 s e g m e n t s . N B F in t h e sciatic n e r v e increased w h e n the ipsilateral dorsal roots o f the L 3 - S 1

stim. L 6 D R (10V, 10Hz, 10s)

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Fig. 7. Responses of NBF and MAP to stimulation of the L6 dorsal root (10 V, 10 Hz, 10 s) in 4 rats before (closed circles) and 25 min after (open squares) topical application of hCGRP (8-37) (10 -4 M). Changes in NBF (A) and MAP (B) were calculated every 10 or 20 s, and are expressed as percentages of the prestimulus values just before the stimulation (ordinates). The horizontal bar between the dashed vertical lines indicates the time during which the L6 dorsal root was stimulated. Each point with a vertical bar represents a mean ± S.E.M. Onset of electrical stimulation of the L6 dorsal root is expressed as zero (abscissa). *P < 0.05, **P < 0.01; significantly different from prestimulus control values, ap < 0.05, bp < 0.01; significantly different from respective values before application of hCGRP (8-37).

130

A. Sato et al./Neurosci. Res. 21 (1994) 125-133

segments were stimulated with parameters of 10 V, 10 Hz and 10-s period. The increase in NBF was observed in about 20% of trials when the L3 or S1 dorsal roots were stimulated, and in about 70% of trials when the L4, L5 or L6 dorsal roots were stimulated. Electrical stimulation of the T11, TI2, T13, L1, L2 and $2 segments did not produce any significant responses in NBF. Fig. 5 demonstrates examples of the responses of NBF ipsilateral to the stimulation of the L 4 - $ 2 dorsal roots with stimulus parameters of 10 V, 10 Hz, and 10-s period. When each of the L 4 - S 1 dorsal roots was stimulated, the NBF started to increase 10-30 s after the end of stimulation, reached the maximum after approximately 60 s and remained at the increased level for several minutes. MAP was slightly decreased or not influenced by these stimuli. The most marked increase in NBF, with regard to amplitude and duration of the response, was obtained when the L6 dorsal root was stimulated. Thus stimulation of the L6 dorsal root was used in the analysis of the neural mechanisms regulating NBF. The stimulation of the L6 dorsal root did not influence NBF in the contralateral sciatic nerve, as shown in Fig. 6B (n = 3 rats). The responses in four rats of ipsilateral NBF following electrical stimulation of the L6 dorsal root with parameters of 10 V, 10 Hz for 10 s are summarized in Fig. 7A (closed circles). The NBF started to increase after the end of stimulation, and was maximal between 1 and 2 rain, poststimulation, reaching about 150% of the control NBF. NBF gradually returned to prestimulation levels after more than 6 min. The stimulation of the L6 dorsal root did not significantly affect MAP (Fig. 7B, closed circles). Stimulus intensity. Threshold intensity of C fibers determined by recording evoked volley from the stimu-

lated L6 dorsal root was above 0.9 V (n = 3 rats). The ipsilateral NBF response following stimulation of the L6 dorsal root was obtained only with stimulus intensities above the threshold of intensity for unmyelinated C fibers in the dorsal root, as shown in Fig. 8A. The response elicited was stimulus intensity-dependent, and saturated at 10 V (n = 4 rats). Stimulus frequency. When the L6 dorsal root was stimulated with 10 V, the elicited NBF response was stimulus frequency-dependent (above 2 Hz) and saturated at 10 Hz as shown in Fig. 8B. One and 2 Hz stimulation did not produce any significant changes in NBF (n = 3 rats). The effect of a CGRP and an S P receptor antagonist on the N B F response. To examine whether the increase

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Fig. 8. Relationships between intensities (A) and frequencies (B) of electrical stimulation of L6 dorsal root and magnitudes of NBF responses in one rat. Maximum NBF within 2 min after onset of stimulation is exprt~,sed as % of the maximum NBF within 30 s before stimulation (ordinate). (A): Stimulation with 10 Hz, for 10 s, with various intensities (abscissa). Threshold intensity for C fibers is indicated by an arrow (Tc). (13): Stimulation with 10 V, for 10 s, with various frequencies (abscissa).

m

va$OCull=u i~uu=

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Fig. 9. Schematic representation of proposed neural mechanisms for regulating sciatic NBF in the rat. Excitation of the unmyelinated efferent fibers in the T11-LI ventral roots will excite vasoconstrictor fibers in the lumbar sympathetic trunk to release noradrenaline (NA), activating ~-adrencrgi¢ receptors, resulting in vasoconstriction of the vasa nervorum in the sciatic nerve. Excitation of the unmyelinated efferent fibers in the L6 ventral root will release acctylcholin¢ (ACh), activating muscarinic cholinergic receptors, resulting in vasodilation of the vasa nervorum. In both cases mentioned above, synaptic transmissions at the autonomic ganglia arc deleted for simplification of the diagram. Antidromic activation of the unmydinated afferent fibers in the L3-S! dorsal roots will release CGRP, activating CGRP receptors, and produce vasodilation of the vasa nervorum in the sciatic nerve.

A. Sato et al./ Neurosci. Res. 21 (1994) 125-133

in sciatic NBF induced by stimulation of the L6 dorsal root was produced by release of ACh, CGRP, and/or SP from the nerve terminals in the vasa nervorum, antagonists of the muscarinic, C G R P and SP receptors were used. The increase in NBF following stimulation of the L6 dorsal root was totally abolished after topical application of 10 -4 M h C G R P (8-37), a C G R P antagonist, as shown in Fig. 7A (n = 4 rats). This effect of hCGRP (8-37) appeared within 5 min of topical application and lasted for more than 45 min. MAP was not affected by this topical application of the C G R P antagonist (Fig. 7B). Topical application of 10 -5 M h C G R P (8-37) attenuated, but did not abolish the NBF response (n = 3 rats). Topical application of saline did not affect the NBF response (n = 3 rats). The increase of NBF induced by stimulation of the L6 dorsal root was not influenced by intravenous injections of CP-96345 (3 /~mol/kg; this dose was adopted from Lembeck et al., 1992), the SP receptor antagonist, or atropine (2.5 mg/kg), a muscarinic receptor antagonist. 4. Discussion The present study is the first to demonstrate that electrical stimulation between lower thoracic and upper sacral ventral or dorsal roots produces a decrease or increase in NBF of the ipsilateral sciatic nerve in rats. The results will be discussed in three sections. 4.1. Vasoconstriction o f the vasa nervorum o f the sciatic nerve following T 1 1 - L 1 ventral root stimulation

Generally speaking, cell bodies of sympathetic preganglionic neurons are located in the C 8 - L 3 segments of the spinal cord (Strack et al., 1988). The sympathetic vasoconstrictor nerves innervating blood vessels of the hindlimbs leave the spinal cord via the ventral roots of the spinal nerves mainly at the T 1 2 - L I segments in humans (Mitchell, 1956) and dogs (Donald and Ferguson, 1970), and at the L 1 - L 3 segments in cats (Sonnenschein and Weissman, 1978) and monkeys (Sheehan and Marrazzi, 1941). In rats, anatomical evidence that the sympathetic control of blood vessels in a single hindlimb muscle (gastrocnemius muscle) originates from five spinal segments (T1 l - L 2 ) with T13 being the major source (Rotto-Percelay et al., 1992). The biphasic response of sciatic NBF elicited in this study by repetitive electrical stimulation of the T1 l - L 1 ventral roots was similar to that elicited by repetitive electrical stimulation of the lumbar sympathetic trunk in rats reported by Hotta et al. (1991). They reported that the biphasic response of sciatic NBF only elicited by ipsilateral lumbar sympathetic trunk. Furthermore, stimulation of the contralateral L2 ventral root produced an increase in NBF accompanied by an increase in MAP. It is unlikely that the vasa nervorum of the sciatic nerve

131

are innervated by sympathetic nerves passing through the contralateral L2 ventral root. The biphasic response of NBF to stimulation is the summation of two opposing mechanisms. The initial increase in NBF is a passive response resulting from the associated increase in systemic arterial blood pressure, while the following decrease in NBF is most likely the result of activation of sympathetic vasoconstrictive fibers innervating the sciatic vasa nervorum via activation of a-adrenergic receptors as reported by Hotta et al. (1991). It is evident that all or at least, some, of the sympathetic vasoconstrictive fibers contained in the T1 l - L 1 ventral roots pass through the lumbar sympathetic trunk on their way to the sciatic vasa nervorum. This information is summarized schematically in Fig. 9. 4.2. Vasodilation o f the vasa nervorum o f the sciatic nerve following L6 ventral root stimulation

The increase in sciatic NBF elicited by stimulation of the ipsilateral L6 ventral root was accompanied by a decrease in MAP. On the other hand, stimulation of the contralateral L6 ventral root produced a decrease in sciatic NBF accompanied by a MAP depressor response. In the latter case, the decrease in NBF can be explained as a passive response to the decrease in MAP (Sundqvist et al., 1985). In contrast, the increase in NBF following the stimulation of the ipsilateral L6 ventral root is due to vasodilative mechanisms of the vasa nervorum which are independent of MAP. This vasodilatory response may result from either metabolite-induced vasodilation or directly in response to neural activation. A sciatic nerve contains both myelinated and unmyelinated fibers. The myelinated motor fibers in the sciatic nerve pass through the ventral roots between the L4 and L6 segments in rats (LaMotte et al., 1991). In the present study, the increase in sciatic NBF was elicited only by stimulation of unmyelinated fibers in the L6, but not in the L4 or L5 ventral roots. This precludes the possibility that vasodilation of the sciatic vasa nervorum resulted from the local accumulation of metabolites. The abolition by atropine of the increase in sciatic NBF induced by stimulation of the ipsilateral L6 ventral root strongly suggests the involvement of cholinergic vasodilative nerve fibers in the response. Excitation of these cholinergic fibers may release acetylcholine from the nerve terminals on/or near the vasa nervorum of the sciatic nerve, activating muscarinic receptors in the vasa nervorum. This results in vasodilation of the vasa nervorum and an increase in blood flow (Fig. 9). In rats, parasympathetic preganglionic neurons are located in the spinal cord at the levels of the L6-S1 segments, and the great majority of preganglionic fibers are unmyelinated (Gabella, 1985). Thus, the vasodilative un-

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myelinated nerve fibers in the L6 ventral root may be parasympathetic preganglionic fibers. Parasympathetic vasodilatative neural regulation of sacral organs has been demonstrated in the colon (Hult6n, 1969; Fasth et al., 1980), bladder (Andersson et al., 1990), and penis (Andersson et al., 1987). Although not measured, vasodilation of these organs could be responsible for the decrease in MAP observed when the L6 ventral root was stimulated. It is interesting to note that although the increase in NBF was attenuated by cholinergic blockade the decrease in MAP was unaffected. This suggests that if the decrease in MAP was a consequence of neurally induced vasodilation in sacral organs, then a non-muscarinic cholinergic mechanism may be involved. 4.3. Vasodilation o f the vasa nervorum o f the sciatic nerve following dorsal roots stimulation

This study is the first to demonstrate that repetitive electrical stimulation of the dorsal roots at the levels of L3-S1 elicits an increase in sciatic NBF. This increase in sciatic NBF was independent of changes in MAP. This may indicate that these particular dorsal root segments do not have vasodilative innervations to other major sacral organs, which would have produced a decrease in MAP, but rather have innervations to restricted body areas, such as the hindlimbs of dogs (Bayliss, 1901) or the gastrocnemius muscles of cats (Hilton and Marshall, 1980). Dorsal root stimulation required a higher stimulus frequency than did the ventral root stimulation to induce an increase in NBF. Electrophysiological studies by Lundberg and H6kfelt have demonstrated that release of neuropeptides requires a higher frequency of stimulation of the nerve than that required for the release of the classical transmitters such as acetylcholine or noradrenaline (Lundberg and H6kfelt, 1983). This, coupled with the remarkably long lasting response of the NBF, reaching almost several minutes after the end of only 10 s of stimulation, is consistent with the theory that peptidergic vasodilative substances are released from the nerve fibers of the dorsal root of the spinal nerves. The local application of C G R P is known to produce long lasting vasodilatation in the coronary artery (Franco-Cereceda et al., 1987; Franco-Cereceda and Rudehill, 1989), the skin (Brain and Williams, 1989), and the knee joint (Lam and Ferrell, 1993) as well as in the sciatic nerve (Zochodne and Ho, 1991a). The response of increase in NBF was obtained with stimulus intensities above the threshold of intensity for exciting unmyelinated C fibers in the dorsal roots. Furthermore, the response was abolished by an antagonist of CGRP. This suggests that only or mainly vasodilative unmyelinated C G R P containing nerve fibers in the dorsal roots are responsible for the vasodilation of the

sciatic vasa nervorum in response to stimulation of the L3-S1 dorsal roots. Many afferent fibers have been shown to contain neuropeptides, such as SP and C G R P (Lundberg et al., 1985; Holzer, 1988; McCarthy and Lawson, 1990). However, the results of this study indicate an important function for C G R P rather than SP in regulation of sciatic NBF. The possibility cannot be denied that the L3-S1 dorsal roots contain C G R P and function as efferent vasodilative fibers to the sciatic vasa nervorum. However, at this moment, we propose an axonal reflex mechanism whereby antidromic activation of unmyelinated CGRP-containing afferent fibers in the L3-S1 dorsal roots leads to release of C G R P at their peripheral terminals in the sciatic vasa nervorum. This activates CGRP receptors in the vasa nervorum with resultant vasodilation of these vessels (Fig. 9). The increase in NBF induced when the afferent nerves are excited may prove to be a useful method to increase NBF by the axon reflex mechanisms when some inflammatory process occurs in the region of the vasa nervorum. In conclusion, the present study documents three major neural mechanisms of regulation of the sciatic vasa nervorum in rats: (1) sympathetic noradrenergic vasoconstrictive fibers emerging from the spinal cord via the T1 l - L 1 ventral roots; (2) parasympathetic cholinergic vasodilative fibers emerging from the spinal cord via the L6 ventral root; and (3) afferent CGRPergic vasodilative fibers entering the spinal cord via the L3-S 1 dorsal roots. The physiological relevance of these three neural mechanisms are currently being investigated.

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