Sensory fibers containing vanilloid receptor-1 (VR-1) mediate spinal cord stimulation-induced vasodilation

BACKGROUND AND AIMS: Spinal cord stimulation (SCS) is used to improve peripheral blood flow in selected populations of patients with ischemia of the extremities. Previous studies show that antidromic activation of sensory fibers is an important mechanism that contributes to SCS-induced vasodilation. However, the characteristics of sensory fibers involved in vasodilation are not fully known. This study investigated the contribution of vanilloid receptor type 1 (VR-1) containing fibers to SCS-induced vasodilation. METHODS: A unipolar ball electrode was placed on the left dorsal column at the lumbar 2-3 spinal cord segments (L2-L3) in sodium pentobarbital anesthetized, paralyzed and ventilated rats. Cutaneous blood flows from both ipsilateral (left) and contralateral (right) hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS (50 Hz; 0.2 ms) was applied through the ball electrode at 30%, 60%, 90% and 300% of motor threshold (MT). Resiniferatoxin (RTX), an ultra potent analog of capsaicin and VR-1 receptor agonist, was used to suppress the activities of VR-1 containing sensory fibers. RESULTS: SCS at 30%, 60%, 90% and also at 300% of MT significantly increased cutaneous blood flow in the ipsilateral foot pad compared to that in the contralateral side. RTX (2 microg/kg, i.v.) significantly attenuated SCS-induced vasodilation of the ipsilateral side (P<0.05, n=7) compared with responses prior to RTX administration. A pledget of cotton soaked with RTX (2 microg/ml) placed on L2-L3 spinal cord significantly decreased SCS-induced vasodilation of the ipsilateral side at 30%, 60%, 90% and 300% of MT (P<0.05, n=7) compared with responses prior to RTX administration. Additionally, topical application of a pledget of cotton soaked with RTX (2 microg/ml) on the sciatic nerve at the middle level of the thigh or on the tibial nerve at the lower level of the lower hindlimb also decreased SCS-induced vasodilation (n=5). CONCLUSION: SCS-induced vasodilation is predominantly mediated via VR-1 containing sensory fibers.     

Low intensity spinal cord stimulation may induce cutaneous vasodilation via CGRP release

This study examined whether spinal cord stimulation (SCS) at intensities below motor threshold (MT) produces cutaneous vasodilation through sympathetic inhibition and/or antidromic activation of sensory fibers. SCS was applied to anesthetized rats with stimulus parameters used clinically, i.e. 50 Hz, 0.2 ms and stimulus intensities at 30, 60 or 90% of MT. SCS-induced vasodilation was not attenuated by hexamethonium, an autonomic ganglion blocking agent, but was abolished by CGRP-(8-37), an antagonist of the calcitonin gene-related peptide (CGRP) receptor. We concluded that SCS-induced vasodilation under the conditions of this study was mediated by peripheral release of CGRP via antidromic activation of sensory fibers.     

Mechanisms of sustained cutaneous vasodilation induced by spinal cord stimulation

This study was performed to investigate whether spinal cord stimulation (SCS) at intensities below motor threshold prolongs cutaneous vasodilation and whether sustained vasodilation by SCS is mediated through sympathetic inhibition and/or antidromic activation of sensory fibers. SCS was applied to the dorsal surface of the L2-L3 spinal cord of anesthesized rats with stimulus parameters used clinically (i.e., 50 Hz, 0.2 ms duration, and stimulus intensity at 30%, 60%, or 90% of motor threshold). Peripheral vasodilation induced by 5-min SCS was not attenuated by hexamethonium, an autonomic ganglion-blocking agent, but was abolished by dorsal rhizotomy. SCS at < or = 60% of motor threshold increased cutaneous blood flow to the level similar to that obtained at 90% of motor threshold, but the vasodilation did not last for 5 min. SCS-induced vasodilation at 90% of motor threshold persisted for the entire stimulation period up to 30 min, and the vasodilation was not attenuated by hexamethonium. It is concluded that sustained vasodilation, which is induced by SCS at only 90% of motor threshold, in this study was mediated via antidromic activation of sensory fibers.     

Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat

The numbers of myelinated and unmyelinated fibers were counted in dorsal roots of adult rats treated neonatally with capsaicin in doses ranging from 5 to 100 mg/kg. Substance P and somatostatin levels in the spinal cord, dorsal roots, and sensory ganglia also were determined in control and treated animals. Capsaicin administration lead to the loss of both small myelinated and unmyelinated fibers from dorsal roots. However, whereas a near total loss, up to 94%, of unmyelinated fibers was achieved after high doses of capsaicin, the reduction of myelinated fibers, even of the smallest caliber, did not exceed 40%. The degree of fiber loss showed a clear dose dependency, with little detectable damage to myelinated fibers at doses of less than 50 mg/kg and with an ED50 for damage to unmyelinated fibers of 5 to 10 mg/kg. In all of the structures examined, particularly the dorsal roots, a roughly parallel decrease of substance P and somatostatin was found with capsaicin dose. The depletions of spinal cord substance P (55%) and somatostatin (20%) produced by neonatal capsaicin treatment were similar to those produced by dorsal rhizotomy. Capsaicin does not appear to be specific for primary afferents containing either substance P or somatostatin.     

A quantitative study of the effects of neonatal capsaicin treatment and of subsequent peripheral nerve transection in the adult rat

The effects of unilateral intercostal nerve transection have been studied in adult rats neonatally treated with capsaicin. Two rats were treated with capsaicin-free vehicle and served as controls. It is shown that capsaicin treatment leads to a dorsal root ganglion cell loss of about 43%, and about a 72% loss of unmyelinated dorsal root fibers. Myelinated dorsal root fibers remain unaffected. The cell size spectrum shows a 27% increase in median cell area in the capsaicin-treated rats, indicating a loss of preferentially smaller cells. A subsequent peripheral nerve transection resulted in a further cell loss of about 30% with no major change in cell size spectrum. The peripheral nerve transection also somewhat further reduces both the number of unmyelinated and myelinated dorsal root axons. The data indicate that neurons which die after a prripheral nerve transection belong to both the groups of cells, which are capsaicin-sensitive and capsaicin-resistant.     

Putative mechanisms behind effects of spinal cord stimulation on vascular diseases: a review of experimental studies

Spinal cord stimulation (SCS) is a widely used clinical technique to treat ischemic pain in peripheral, cardiac and cerebral vascular diseases. The use of this treatment advanced rapidly during the late 80's and 90's, particularly in Europe. Although the clinical benefits of SCS are clear and the success rate remains high, the mechanisms are not yet completely understood. SCS at lumbar spinal segments (L2-L3) produces vasodilation in the lower limbs and feet which is mediated by antidromic activation of sensory fibers and decreased sympathetic outflow. SCS at thoracic spinal segments (T1-T2) induces several benefits including pain relief, reduction in both frequency and severity of angina attacks, and reduced short-acting nitrate intake. The benefits to the heart are not likely due to an increase, or redistribution of local blood flow, rather, they are associated with SCS-induced myocardial protection and normalization of the intrinsic cardiac nervous system. At somewhat lower cervical levels (C3-C6), SCS induces increased blood flow in the upper extremities. SCS at the upper cervical spinal segments (C1-C2) increased cerebral blood flow, which is associated with a decrease in sympathetic activity, an increase in vasomotor center activity and a release of neurohumoral factors. This review will summarize the basic science studies that have contributed to our understanding about mechanisms through which SCS produces beneficial effects when used in the treatment of vascular diseases. Furthermore, this review will particularly focus on the antidromic mechanisms of SCS-induced vasodilation in the lower limbs and feet.     

Is Ross syndrome a dysautonomic disorder only? An electrophysiologic and histologic study.

OBJECTIVE: To define the involvement of peripheral nerve fibers in Ross syndrome. METHODS: Mechanical pain perception, tactile and thermal thresholds on hand, foot dorsum, thigh, median nerve orthodromic sensory conduction velocity (SCV) and motor conduction velocity (MCV), sural nerve antidromic SCV, peroneal nerve MCV, H-reflex, F-wave, median, tibial nerve somatosensory evoked potentials (SSEPs), perioral, hand CO(2) laser late (LEPs) and ultralate evoked potentials, sympathetic skin response (SSRs), cardiovascular, Minor sweat, silastic imprint, histamine, photopletysmographic and pupil pilocarpine tests, cutaneous innervation immunohistochemical techniques were studied in 3 patients with Ross syndrome. RESULTS: Quantitative sensory testing showed altered results in patients 1 and 2, and patient 3 had a slight impairment of mechanical pain perception. Nerve conduction, except for a median nerve distal reduction of sensory conduction in patient 1, F-wave and SSEP findings were normal; H-reflex was absent at rest in all patients. Hand LEPs were absent in patient 2, ultralate potentials were absent in patients 1 and 2. Skin biopsy showed a disease duration related reduction of unmyelinated and myelinated sensory fibers and a lack of unmyelinated autonomic fibers in all patients. CONCLUSIONS: Our data suggest that Ross syndrome is a degenerative disorder involving progressive sudomotor fibers, and then epidermal sensory unmyelinated and myelinated fibers.     

Axonal regeneration in an articular branch following rat sciatic nerve lesions

This study examines the outcome of axonal regeneration in the posterior articular nerve of the adult rat knee joint (PAN), after sciatic nerve lesions. Some animals had previously been subjected to chemical sympathectomy with guanethidine. In crushed cases the number of myelinated PAN axons was 50% above control level. The occurrence of C-fibers was doubled, mainly due to an increased number of sympathetic efferents. In neurotomy/suture cases the number of myelinated fibers was clearly elevated, but the number of C-fibers was close to normal. Most C-fibers were sensory. Similar, but less marked, post-regeneration abnormalities were seen in the nerve to the lateral gastrocnemius muscle. The sural nerve exhibited moderately increased numbers of myelinated and unmyelinated fibers in crushed cases. In neurotomy cases, the myelinated axons had increased and the C-fibers had decreased in number. The size distribution of myelinated PAN axons was less abnormal in crushed cases than after neurotomy, like in the other nerves. These results show that the outcome of axon regeneration in an articular branch of the lesioned rat sciatic nerve differs from that in non-articular branches, and suggest that joints may become hyperinnervated by C-fibers after nerve crush lesions.     

A quantitative ultrastructural study of the optic nerve of the chameleon.

The optic nerve of adult chameleons was investigated with an electron microscope. The total number of retinal ganglion cell axons, the proportion of myelinated axons, the frequency distributions of myelinated and unmyelinated axon diameters were estimated, together with the volume occupied by glial processes. These were distinguished from unmyelinated axons using an antibody directed against glial fibrillary acidic protein, in a post-embedding procedure. The total number of fibers was estimated to be 405,235 +/- 60,000 axons. The proportion of myelinated fibers varied with position between the eyeball and the chiasma; being 22-27% close to the eyeball, rising to 42-47% halfway along the optic nerve and to 56-62% close to the chiasma. Myelinated and unmyelinated fiber diameter distributions were unimodal and positively skewed, with modes of 0.7 microm and 0.2 microm, respectively. There was a significant regional variation in the size of optic nerve axons. Large myelinated axons were observed in the dorsal and ventral periphery, whereas smaller myelinated fibers and a high proportion of unmyelinated fibers were found in the center of the nerve.     

Unmyelinated nerve fiber degeneration in chronic inflammatory demyelinating polyneuropathy

To determine whether unmyelinated nerve fibers escape degeneration as one might expect in an immune response exclusively directed at myelin, we performed a morphometric examination of unmyelinated axons and myelinated nerve fibers in sural nerve biopsy specimens of 14 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP) and of 12 age-matched normal controls. The numbers of unmyelinated axons, myelinated nerve fibers, denervated Schwann cell units and collagen pockets were quantified and related to the clinical and electrophysiological data of the patients with CIDP. In 4 patients with a rapid onset of the neuropathy and a highly elevated CSF protein, the numbers of both unmyelinated axons and myelinated nerve fibers were decreased equally. In 8 patients we found that the unmyelinated axons were relatively spared compared with the loss of myelinated nerve fibers. In these patients, however, the presence of denervated Schwann cell units and of collagen pockets was increased. We conclude that unmyelinated nerve fibers are affected in patients with CIDP.     

Origin of small primary afferent substance P-immunoreactive nerve fibers in the guinea-pig heart

Primary afferent and substance P (SP)-immunoreactive nerve fibers of the guinea pig and rat heart were investigated by physiological and immunohistochemical methods. Immunohistochemistry revealed abundant SP-positive fibers in the guinea pig atria, with fewer in the ventricles. Only an occasional fiber was seen in the rat atrium or ventricle. Sectioning the vagus nerve did not noticeably influence the supply of SP-immunoreactive nerve fibers in the guinea pig heart. When the atria or ventricles were stimulated, afferent nerve fiber activity was recorded from the second and third thoracic dorsal roots. In guinea pig atria 3 types of fibers were identified on the basis of conduction velocities: A delta 1, A delta 2 and C fibers. Only A delta fibers were identified in the ventricle. By vagal recordings, A delta fibers were demonstrated but a C fiber response could not be shown in this nerve. SP-immunoreactivity in primary afferent fibers was depleted by the neurotoxin capsaicin. Capsaicin treatment also caused a reduction in the conduction velocity of small diameter myelinated A delta 2 (by 29%) and unmyelinated C fibers (by 46%). In the rat heart, evidence for A delta 2 or C fibers was not found. These results indicate that primary afferent and SP-immunoreactive fibers are numerous in guinea pig heart, but few in the rat. It is concluded that most of these fibers have their cell bodies of origin in the dorsal root ganglia.     

Unmyelinated axons in the ventral roots of the cat lumbosacral enlargement

The ventral roots L₇ and S₁ of the cat spinal cord were examined with the light and electron microscopes. Differences in the morphology of Schwann cells associated with large myelinated fibers and with small myelinated or unmyelinated fibers were observed. The blood vessels were largely encircled by pericytes. The most noteworthy finding was that 29% of the axons in these roots were unmyelinated. These unmyelinated axons were greatly reduced in number proximal but not distal to a ventral rhizotomy. Furthermore, they were reduced in number following dorsal root ganglionectomy, but not after dorsal rhizotomy, sympathectomy or peripheral nerve section. It is concluded that the ventral roots of the lumbosacral enlargement contain a large population of unmyelinated fibers originating from dorsal root ganglion cells.     

Age differences in the morphology of posterior tibial nerves of mice

Light microscopic examination of posterior tibial nerves of C57BL/10 female mice at 3, 8 and 26 months of age revealed an age-related decrease in the total number of myelinated fibers per nerve with a fairly uniform loss across all caliber of fibers present. However, the size of the individual nerves increased with age as did also mean axon diameter of individual fibers and the proportion of connective tissue between fibers in the older nerves. Ultrastructural comparisons revealed an increase in mean axone circumference of myelinated fibers between 3 and 8 months and, of unmyelinated fibers between 8 and 26 months. Also, there was a significant decrease in mitochondrial concentration in myelinated fibers between 8 and 26 months of age. There was no change in blood vessels, myelin sheath thickness, concentration of unmyelinated fibers per nerve, and in the concentration of filaments and tubules in myelinated and unmyelinated axons in relation to increasing age.     

Selective fiber vulnerability in acute ischemic neuropathy

To study the relationship between fiber size and vulnerability to ischemia, we produced partial infarction of the proximal posterior tibial nerve of Wistar rats by injecting arachidonic acid into the ipsilateral femoral artery. In the resulting lesion degenerating fibers were concentrated in the center of the nerve. Distal to the infarct, surviving myelinated and unmyelinated fibers were evenly distributed throughout both fascicles. Smaller myelinated fibers (<6μm in diameter) were more severely depleted than larger ones (>6μm). Unmyelinated fibers were reduced in number in proportion to or to a greater extent than myelinated fibers. These findings demonstrate that large myelinated fibers are relatively less vulnerable to peripheral nerve ischemia than smaller fibers, and do not support the contention that unmyelinated fibers are inherently resistant to acute ischemia.     

Conduction velocity distribution of afferent fibers innervating the rat urinary bladder

The conduction velocities of individual afferent fibers innervating the rat urinary bladder were determined by the antidromic stimulation of dorsal roots while recording from bladder postganglionic nerves. Conduction velocities ranged from 0.5 to 21.0 m/s; 70% of the velocities were less than 2.5 m/s. The distribution within the dorsal roots was ipsilateral with 84% in L₆ and 16% in S₁. Neuroanatomical tracing with horseradish peroxidase applied to individual bladder postganglionic nerves resulted in ipsilateral labeling of dorsal root ganglion cells with 77% in L₆, 20% in S₁, and 3% in L₁–L₂. Ultrastructural examination of bladder postganglionic nerves revealed some myelinated fibers (average diameter: 2.6 μm) and many unmyelinated fibers. Therefore, in the rat, most of the bladder afferent fibers appear to be unmyelinated, although a population of myelinated afferent fibers is also present. Bladder afferent fibers enter the spinal cord mainly in segment L₆ with a minor fraction entering in S₁.     

The effect of bupivacaine on compound action potential parameters of sciatic nerve fibers

The aim of this study was to document the effects of the local anesthetic agent bupivacaine on individual fibers of peripheral nerve. To accomplish this objective, compound action potentials (CAPs) were recorded from isolated frog sciatic nerves treated with bupivacaine for seven individual concentration levels. Numerical and fast Fourier transform (FFT) analysis were performed on these recordings. The areas, latency periods, maximum and minimum derivatives, and power spectrums of the CAPs were computed. The results show that the area and the absolute values of maximum and minimum derivatives decrease linearly as bupivacaine concentration increases. The power spectrum of the CAPs, which resides in the 0-1000 Hz interval, initially shifts to higher frequencies then returns to lower frequency region again with increasing bupivacaine concentration. Due to this result, it is thought that bupivacaine inhibits nerve fibers in a dose-dependent manner. It primarily affects the fibers having the least myelin sheets (motor fibers), then it begins to depress the fast conducting (neurosensorial) fibers as the bupivacaine concentration increases, and finally blocks the unmyelinated C-fibers.     

Axonal thermosensitivity and mechanosensitivity of cutaneous afferent neurons

We hypothesized that cutaneous afferent myelinated fibers (A-fibers) and afferent unmyelinated fibers (C-fibers) respond to the same natural stimuli applied to their axons as to their terminals in the skin. In anesthetized rats, activity was recorded from afferent axons in strands isolated proximally from the sural nerve. Mechanical, cold or heat stimuli were applied to the skin or along a 15-mm length of the distal sural nerve. One-hundred and eighteen A-fibers and 109 C-fibers were characterized by their conduction velocity and/or shape of their action potentials, and by their responses to natural stimulation of the skin. Then, these fibers were tested for their responses to the same stimuli applied to the nerve. In some cases, the nerve was crushed distally after the nerve fibers had been characterized by their responses to physiological stimulation of the skin, and the responses to stimuli applied to the nerve proximal to the lesion were tested again. Almost all non-nociceptive cold-sensitive (type 1) C-fibers (97%) could be activated by cold stimuli applied to the nerve. Of nociceptive cold-sensitive (type 2) C-fibers, 39% were activated by cold stimuli applied to the nerve. Furthermore, 34% of heat-sensitive C-fibers could be activated by heating the nerve. In contrast, only 2-4% of mechanosensitive A-fibers and C-fibers responded to mechanical stimuli applied to the nerve. In conclusion, cold and heat sensitivity of cutaneous afferent neurons is not restricted to their terminals in the skin, but often extends along the axons in the nerve. Mechanosensitivity is restricted to the afferent endings in the skin.     

Regeneration of dorsal root fibers into the adult rat spinal cord

Regeneration of dorsal root nerve fibers into the spinal cord of adult rat was studied with the electron microscope after crushing the roots. Regenerated dorsal root myelinated fibers were observed in the substantia gelatinosa and posterior funiculi around the tenth week after lesion. Cytoplasmic processes of oligodendrocytes were often found close to the young myelinated nerve fibers. The astrocytic response subsided as regeneration progressed. Clusters of small, circular profile of cellular processess were found in the neuropil about the sixth week and are considered to be regenerated unmyelinated axons. At this period, groups of segments of cellular processes containing clear vesicles were also encountered in the substantia gelatinosa and resembled growth cones described in peripheral regenerating nerves.     

The threshold for eliciting the jaw opening reflex in rats is not increased by neonatal capsaicin

Neonatal capsaicin administration has previously been shown to increase the threshold for withdrawal and escape response to noxious thermal, chemical and mechanical stimuli. The reason for these effects is destruction of afferent C-fibers and associated reduction in central substance P levels. In contrast, neonatal capsaicin treatment did not eliminate the "jaw opening reflex" (JOR) or even significantly increases its threshold when tested in rats 2-8 months of age. There was no significant difference in the voltage required to elicit the JOR nor was there any difference in its latency, in the animal that had received an intraperitoneal injection of 50 mg/kg capsaicin on the second day of life. A reduction of 85% unmyelinated fibers was found as compared with untreated animals. This data support earlier indications that upon incisor tooth pulp chamber stimulation in rats, the JOR is affectively activated by low-threshold myelinated fibers in the tooth pulp and surrounding periodontal ligament. Destruction of tooth pulp C-afferents, therefore, does not affect jaw opening reflex threshold.