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Response characteristics of cutaneous mechanoreceptors in neuropathic rats
Neuroscience Letters 317 (2002) 89–92 www.elsevier.com/locate/neulet
Response characteristics of cutaneous mechanoreceptors in neuropathic rats Aleksandra Bulka, Jing-Xia Hao, Zsuzsanna Wiesenfeld-Hallin* Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden Received 1 September 2001; received in revised form 2 October 2001; accepted 25 October 2001
Abstract The activity of single myelinated afferents was recorded from dorsal roots L4–5 in normal Sprague–Dawley rats and animals that developed mechanical hypersensitivity following ischemic injury to the sciatic nerve. The mechanical response properties and conduction velocity of afferents conducting through the injury site (about 50% of units) were similar to controls. However, the majority of afferents not conducting through the injury site exhibited ongoing activity. The results suggest that mechanical allodynia may be at least partly due to the central integration of activity arising from these two populations of afferents in neuropathic rats. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allodynia; Afferents; Dorsal roots; Ongoing activity; Ab fibers; Conduction velocity; Pain
Neuropathic pain is a major clinical problem because of its chronicity and resistance to treatment [11]. A number of models of partial nerve injury have been developed, such as sciatic nerve constriction [1], partial sciatic nerve ligation [17], spinal nerve ligation [10] and photochemicallyinduced ischemia of the sciatic nerve [6]. In these models some primary afferents are intact, allowing behavioral analysis of the responses of the animals to sensory stimuli applied to the partially deafferented limb. The model of photochemically-induced ischemic injury of the rat sciatic nerve reflects some aspects of neuropathies involving nerve ischemia, such as ischemic neuritis [3], diabetes [5] and nerve compression [18]. Following ischemic nerve injury rats develop abnormal pain-related behaviors, including mechanical, cold and heat allodynia and signs of ongoing pain. The morphological changes at the irradiation site include extensive damage to myelinated and unmyelinated fibers in the irradiated nerve segment surrounding occluded blood vessels [12,20]. The myelinated fibers are more susceptible to injury than the unmyelinated fibers. However, some morphologically normal regions of the nerve, away from the occluded vessels, are also present [20]. Seven days after ischemia the nerve distal * Corresponding author. Tel.: 146-8-585-87085; fax: 146-8-58587050. E-mail address: [email protected]. sll.se (Z. Wiesenfeld-Hallin).
to the irradiation exhibits Wallerian degeneration, whereas at 10 mm proximal to the irradiation the nerve is apparently normal [20]. The aim of the present study was to examine the functional status of myelinated primary afferents in the injured nerve and to compare them to afferents recorded from normal nerves. Adult male Sprague–Dawley rats (Møllegaard, Denmark) weighing 200 g at the start of the experiment were used. Electrophysiological studies were performed on 13 animals with no nerve lesions and 11 animals which developed mechanical allodynia after ischemic injury to the sciatic nerve. The experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and were approved by the local research ethics committee. The procedure for induction of ischemic nerve injury was described in detail previously [12]. Briefly, the rats were anesthetized with chloral hydrate (Sigma, 200 mg/kg, i.p.) and the left sciatic nerve was exposed. The nerve trunk was dissected free over a distance of about 10 mm proximal to trifurcation and irradiated for 90 s with argon laser light at 514 nm with an average power of 170 mW (Innova model 70). Just before the irradiation erythrosyn B (Aldrich, 32.5 mg/kg) was injected i.v. After the surgery the wound was closed in layers. The rats were adapted to the testing situation for 3 days before irradiation and the threshold for mechanically evoked paw withdrawal response was measured. During
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 43 9- 9
90
A. Bulka et al. / Neuroscience Letters 317 (2002) 89–92
testing the rat was placed on a metal grid and the plantar surface of the hind paws was stimulated with a series of calibrated von Frey monofilaments (Stoelting) in ascending order. The lowest force at which the animal withdrew the paw in at least three of five trials was taken as the mechanical withdrawal threshold. The development of mechanical allodynia was tested starting 1 day after irradiation. The electrophysiological experiments were conducted 4– 22 days after the ischemic injury (mean 10 days). The rats were anesthetized with urethane (1.5 g/kg), intubated with a tracheal cannula and ventilated. The electrocardiogram was monitored and the core temperature was maintained as close to 378C as possible. The rats were fixed in a stereotactic spinal unit (Kopf). Pancuronium bromide (Pavulon, Organon, 0.5 mg/kg) was administered i.v., followed by a constant infusion of 0.3 mg/h throughout the experiment to avoid muscle contractions. The left sciatic nerve was exposed at the mid-thigh level; a pool was formed from the skin flaps and filled with paraffin oil. The nerve was stimulated with a bipolar silver electrode at about 15 mm proximal to the nerve trifurcation, proximal to the irradiated segment, with 0.1 ms pulses via a stimulus isolation unit (Grass). The spinal cord and dorsal roots were exposed by a laminectomy and immersed in paraffin oil. Fine filaments were dissected with a watchmaker’s forceps from roots L4 and L5 and were placed on a monopolar silver hook electrode. An electrical search stimulus (0.1 ms) at 1 Hz was used to identify activity in the dorsal root filaments, which was amplified (Neurolog) and displayed on a storage oscilloscope. Activity recorded from single fibers was identified on the basis of an all-or-none response. For each single unit the threshold of electrical stimulation was recorded and the latency of the action potential to the peak was taken. The receptor type, size and location of the receptive field were identified with a hand-held probe. The response threshold to stimulation with von Frey hairs was also assessed and responses to both electrical and mechanical stimulation were monitored on the oscilloscope and recorded on a computer using a Spike 2 program (Cambridge Electronic Devices). The distance between the stimulus and recording sites was measured for each animal at the end of the experiment. Photochemically-induced nerve injury produced hypersensitivity to mechanical stimulation of the hind paw ipsilateral to the irradiation. The response to von Frey hair stimulation was significantly decreased, starting from the first day after the irradiation up to day 22, which is the period when the electrophysiological studies were conducted. The median mechanical threshold before irradiation was 11.1 g and just before the electrophysiological experiments 1.24 g. In the control animals 155 fibers were found by electrical stimulation and classified according to their response to mechanical stimuli (Table 1). The largest group responded to rapid hair movement (n ¼ 53, 34.2%). A similar number of units was activated by joint movement (n ¼ 49, 31.6%).
Rapidly adapting receptors from the glabrous skin represented 6.5% (n ¼ 10). Equal numbers of slowly adapting type I, Pacinian corpuscle and tylotrich hair were recorded (4 units (2.6%) for each group). The remaining units with identifiable receptor properties were D hair (n ¼ 3, 1.9%), muscle and deep tissue (n ¼ 5, 3.2%). Of the entire population of 155 fibers recorded 23 (14.8%) did not respond to mechanical stimulation. The median mechanical threshold of receptors in glabrous skin was 2.42 g. In hairy skin the median mechanical threshold for activating G hair units was 0.19 g and tylotrich hair units 0.047 g. The conduction velocity for primary afferent fibers recorded from the dorsal roots after sciatic nerve stimulation ranged from 16 to 45 m/ s with a mean value of 29.4 m/s (Fig. 1). The joint units exhibited very regular activity which was modified by joint movement. No ongoing activity from any other type of receptor was recorded from the control group (Table 1). An equal number of units (155) was recorded from animals which developed mechanical allodynia 4–22 days after nerve injury. All fibers were activated by sciatic nerve stimulation. The units recorded from allodynic animals had a very different distribution among receptor types in comparison to the control group (Table 1). Almost half of the units (n ¼ 76, 49%) did not respond to any kind of stimulation. Ongoing discharge was recorded in 46 (29.6%) of all recorded fibers and none of these responded to mechanical stimulation. These units represented 60% of the group with no identified receptor field. The most common type of spontaneous discharge was irregular activity, the next most common was bursting and the least common was regular discharge. The most common units with receptor properties recorded in allodynic animals were joint afferents (n ¼ 45, 29%) with response characteristics similar to those recorded in control animals. Of the units with cutaneous receptive fields, receptor sensitivity to mechanical stimulation and the size of receptive fields was not different from normal Table 1 Characteristics of receptor types recorded from control and allodynic rats Receptor type
Control Number
Slowly adapting, glabrous 4 skin Rapidly adapting, glabrous 10 skin Pacinian corpuscle 4 G hair follicle 53 D hair follicle 3 Tylotrich hair 4 Joint 49 Muscle, deep 5 Unexcitable 23 Total 155 Spontaneously active units 0
Allodynic %
Number
%
2.6
1
0.6
6.5
7
4.5
2.6 34.2 1.9 2.6 31.6 3.2 14.8 100.0 0.0
1 18 0 3 45 4 76 155 46
0.6 11.6 0.0 1.9 29.0 2.5 49.3 100.0 29.6
A. Bulka et al. / Neuroscience Letters 317 (2002) 89–92
Fig. 1. Histogram of conduction velocities of afferents recorded from dorsal roots in a sample of units in normal control (A) and allodynic rats (B). N ¼ 155 in each group.
animals. The median threshold of receptors from glabrous skin to stimulation with von Frey hairs was 1.46 g. For G hair units and tylotrich hairs the median threshold value was 0.19 g. The conduction velocity of all fibers recorded from allodynic animals ranged from 14 to 40 m/s (mean 28.3 m/s) and was not significantly different from the controls. Furthermore, there was no difference in the conduction
91
velocity of units with (28.8 m/s) and without receptive fields (27.9 m/s) in the allodynic animals. However, a significant difference (P , 0:05) in conduction velocity was found when units with no receptor fields in the allodynics (27.5 m/s) were compared to the control population (29.4 m/s). The characteristics of the cutaneous afferents were similar to those previously reported in hairy [14] and glabrous [16] skin of the rat hindpaw. Many joint afferents were found in the present study, about 30% in the control and allodynic groups, which was not previously reported. In previous studies the recordings were from the saphenous [14] or tibial [16] nerve whereas in the present study the recordings were from dorsal roots, which may account for this difference. The conduction velocities of the units were similar to those in the previous studies [14,16], but slower than in a study of afferents in the sciatic constriction model [8] where the recordings were also from dorsal roots. Core body temperature was maintained as close to 378C as possible. It is possible that limb temperature was lower than normal, resulting in the slower conduction velocity. Photochemically-induced sciatic nerve ischemia produced mechanical hypersensitivity of the ipsilateral hind paw. Surprisingly, the response characteristics and conduction velocity of mechanoreceptors with receptive fields in the allodynic animals were normal. The response characteristics of afferents in other models of partial nerve injury have not been previously reported. Regenerating afferents were shown to have reduced conduction velocities proximal to the injury site, as well as reduced sensitivity to cutaneous stimuli, following nerve crush [16]. In the sciatic constriction model the conduction velocity of fibers that conducted through the site of injury was normal [8], in agreement with the present results. However, in the present study a slight, but significant, reduction in conduction velocity was observed in afferents that could not be activated from the periphery. Morphological data from the ischemic injury model indicate that the nerve is apparently normal proximal to the injury [20], at the level where it was stimulated. Thus, the slight slowing in conduction velocity was probably due to minor abnormalities undetectable with the morphological techniques used. In the constriction injury model recordings were from Ab, Ad and C fibers [8]. In that study the Ab and Ad fibers were found to be less likely to conduct through the injury site than C fibers, indicating that A fibers are more prone to injury than C fibers. Furthermore, ongoing activity at 1 and 3 days after injury was greatest in Ab, less in Ad and least in C fibers in the constriction model [8]. Similar results were found after total nerve transection where ongoing activity in A fibers had a more rapid onset (a few days) than in C fibers (weeks) [4,7]. In the present study 15% of afferents were unexcitable to mechanical stimuli in control animals and this proportion increased to 50% in the allodynics. Ongoing activity, other than in joint afferents, was not found in any recordings from controls, but was recorded in 30% of fibers in allodynics. Ongoing activity was only recorded in affer-
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ents with no receptive field, constituting 60% of this group in allodynic rats. The origin of the ongoing activity may be the distal portion of the damaged afferents [4] or the dorsal root ganglia [9]. The mechanical threshold was similar in the mechanoreceptors conducting through the injury site recorded from the allodynic animals and in controls. Furthermore, the conduction velocity of fibers with receptive fields recorded from the two groups was not different. Thus, the results suggest that the mechanical allodynia following nerve ischemia at least in part results from the central processing of input mediated by low threshold mechanoreceptors with normal receptive field and response properties. Input from these afferents converges on spinal cord interneurons that receive ongoing activity arising from damaged A fibers, resulting in abnormal ongoing discharges, response and receptive field properties. Such changes have been documented in the sciatic nerve constriction [13,15], partial sciatic nerve ligation [19] and spinal nerve ligation [2] models. We cannot exclude a role for C fibers in the development of allodynia since we did not record from them. Mechanical allodynia in neuropathic rats may at least in part be mediated by activity in low threshold mechanoreceptors that remain in continuity following an ischemic injury to the sciatic nerve. These afferents have normal response characteristics. However, about half of the afferents in neuropathic rats do not conduct through the site of injury and a majority develop ongoing activity, which may contribute to both ongoing and evoked pain. Drugs that block the generation of ongoing activity in damaged afferents, such as sodium channel blockers, may be useful for alleviating neuropathic pain. This work was supported by the Swedish Medical Research Council (07913) and AstraZeneca. The outstanding facilities of the Clinical Research Center at Huddinge University Hospital are gratefully acknowledged. [1] Bennett, G.J. and Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87–107. [2] Chapman, V., Suzuki, R. and Dickenson, A.H., Electrophysiological characterisation of spinal neuronal response properties in anaesthetized rats after ligation of spinal nerves L5-L6, J. Physiol., 507 (1998) 881–894. [3] Daube, J.R. and Dyck, P.J., Neuropathy due to peripheral vascular disease, In P.J. Dyck, P.K. Thomas, E.H. Lambert and R. Bunge (Eds.), Peripheral Neuropathy, W.B. Saunders, Philadelphia, PA, 1984, pp. 1458–1478. [4] Devor, M., The pathophysiology of damaged peripheral nerves, In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, 2nd Edition, Churchill Livingstone, Edinburgh, 1989, pp. 63–81.
[5] Dyck, P.J. and Giannini, C., Pathologic alterations in the diabetic neuropathies of humans: a review, Neuropathol. Exp. Neurol., 55 (1996) 1181–1193. [6] Gazelius, B., Cui, J.G., Svensson, M., Meyerson, B. and Linderoth, B., Photochemically induced ischaemic lesion of the rat sciatic nerve. A novel method providing high incidence of mononeuropathy, NeuroReport, 7 (1996) 2619– 2623. [7] Govrin-Lippmann, R. and Devor, M., Ongoing activity in severed nerves: source and variation with time, Brain Res., 159 (1978) 406–410. [8] Kajander, K.C. and Bennett, G.J., Onset of a painful peripheral neuropathy in rat: a partial and differential deafferentation and spontaneous discharge in Ab and Ad primary afferent neurons, J. Neurophysiol., 68 (1992) 734–744. [9] Kajander, K.C., Wakisaka, S. and Bennett, G.J., Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat, Neurosci. Lett., 138 (1992) 225–228. [10] Kim, S.H. and Chung, J.M., An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat, Pain, 50 (1992) 355–363. [11] Kingerey, W.S., A critical review of controlled clinical trials for peripheral neuropathic pain and complex regional pain syndromes, Pain, 73 (1997) 123–139. [12] Kupers, R., Yu, W., Persson, J.K.E., Xu, X.-J. and Wiesenfeld-Hallin, Z., Photochemically-induced ischemia of the rat sciatic nerve produces a dose-dependent and highly reproducible mechanical, heat and cold allodynia, and signs of spontaneous pain, Pain, 76 (1998) 45–59. [13] Laird, J.M. and Bennett, G.J., An electrophysiological study of dorsal horn neurons in the spinal cord of rats with experimental peripheral neuropathy, J. Neurophysiol., 69 (1993) 2072–2085. [14] Lynn, B. and Carpenter, S.E., Primary afferent units from the hairy skin of the rat hind limb, Brain Res., 238 (1982) 29–43. [15] Palecek, J., Dougherty, P.M., Kim, S.H., Paleckowa, V., Lekan, H., Chung, J.M., Carlton, S.M. and Willis, W.D., Responses of spinothalamic tract neurons to mechanical and thermal stimuli in an experimental model of peripheral neuropathy in primates, J. Neurophysiol., 68 (1992) 1951– 1966. [16] Sanders, K.H. and Zimmermann, M., Mechanoreceptors in rat glabrous skin: redevelopment of function after nerve crush, J. Neurophysiol., 55 (1986) 644–659. [17] Seltzer, Z., Dubner, R. and Shir, Y., A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury, Pain, 43 (1990) 205–218. [18] Sunderland, S., The nerve lesion in the carpal tunnel syndrome, J. Neurol. Neurosurg. Psychiatry, 39 (1976) 615–626. [19] Takaishi, K., Eisele, J.H. and Carstens, E., Behavioral and electrophysiological assessment of hyperalgesia and changes in dorsal horn responses following partial sciatic nerve ligation in rats, Pain, 66 (1996) 297–306. [20] Yu, W., Kauppila, T., Hultenby, K., Persson, J.K.L., Xu, X.-J. and Wiesenfeld-Hallin, Z., Photochemically-induced ischemic injury of the rat sciatic nerve: a light- and electron microscopic study, J. Periph. Nerv. Syst., 5 (2000) 209–217.
Response characteristics of cutaneous mechanoreceptors in neuropathic rats Aleksandra Bulka, Jing-Xia Hao, Zsuzsanna Wiesenfeld-Hallin* Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden Received 1 September 2001; received in revised form 2 October 2001; accepted 25 October 2001
Abstract The activity of single myelinated afferents was recorded from dorsal roots L4–5 in normal Sprague–Dawley rats and animals that developed mechanical hypersensitivity following ischemic injury to the sciatic nerve. The mechanical response properties and conduction velocity of afferents conducting through the injury site (about 50% of units) were similar to controls. However, the majority of afferents not conducting through the injury site exhibited ongoing activity. The results suggest that mechanical allodynia may be at least partly due to the central integration of activity arising from these two populations of afferents in neuropathic rats. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allodynia; Afferents; Dorsal roots; Ongoing activity; Ab fibers; Conduction velocity; Pain
Neuropathic pain is a major clinical problem because of its chronicity and resistance to treatment [11]. A number of models of partial nerve injury have been developed, such as sciatic nerve constriction [1], partial sciatic nerve ligation [17], spinal nerve ligation [10] and photochemicallyinduced ischemia of the sciatic nerve [6]. In these models some primary afferents are intact, allowing behavioral analysis of the responses of the animals to sensory stimuli applied to the partially deafferented limb. The model of photochemically-induced ischemic injury of the rat sciatic nerve reflects some aspects of neuropathies involving nerve ischemia, such as ischemic neuritis [3], diabetes [5] and nerve compression [18]. Following ischemic nerve injury rats develop abnormal pain-related behaviors, including mechanical, cold and heat allodynia and signs of ongoing pain. The morphological changes at the irradiation site include extensive damage to myelinated and unmyelinated fibers in the irradiated nerve segment surrounding occluded blood vessels [12,20]. The myelinated fibers are more susceptible to injury than the unmyelinated fibers. However, some morphologically normal regions of the nerve, away from the occluded vessels, are also present [20]. Seven days after ischemia the nerve distal * Corresponding author. Tel.: 146-8-585-87085; fax: 146-8-58587050. E-mail address: [email protected]. sll.se (Z. Wiesenfeld-Hallin).
to the irradiation exhibits Wallerian degeneration, whereas at 10 mm proximal to the irradiation the nerve is apparently normal [20]. The aim of the present study was to examine the functional status of myelinated primary afferents in the injured nerve and to compare them to afferents recorded from normal nerves. Adult male Sprague–Dawley rats (Møllegaard, Denmark) weighing 200 g at the start of the experiment were used. Electrophysiological studies were performed on 13 animals with no nerve lesions and 11 animals which developed mechanical allodynia after ischemic injury to the sciatic nerve. The experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and were approved by the local research ethics committee. The procedure for induction of ischemic nerve injury was described in detail previously [12]. Briefly, the rats were anesthetized with chloral hydrate (Sigma, 200 mg/kg, i.p.) and the left sciatic nerve was exposed. The nerve trunk was dissected free over a distance of about 10 mm proximal to trifurcation and irradiated for 90 s with argon laser light at 514 nm with an average power of 170 mW (Innova model 70). Just before the irradiation erythrosyn B (Aldrich, 32.5 mg/kg) was injected i.v. After the surgery the wound was closed in layers. The rats were adapted to the testing situation for 3 days before irradiation and the threshold for mechanically evoked paw withdrawal response was measured. During
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 43 9- 9
90
A. Bulka et al. / Neuroscience Letters 317 (2002) 89–92
testing the rat was placed on a metal grid and the plantar surface of the hind paws was stimulated with a series of calibrated von Frey monofilaments (Stoelting) in ascending order. The lowest force at which the animal withdrew the paw in at least three of five trials was taken as the mechanical withdrawal threshold. The development of mechanical allodynia was tested starting 1 day after irradiation. The electrophysiological experiments were conducted 4– 22 days after the ischemic injury (mean 10 days). The rats were anesthetized with urethane (1.5 g/kg), intubated with a tracheal cannula and ventilated. The electrocardiogram was monitored and the core temperature was maintained as close to 378C as possible. The rats were fixed in a stereotactic spinal unit (Kopf). Pancuronium bromide (Pavulon, Organon, 0.5 mg/kg) was administered i.v., followed by a constant infusion of 0.3 mg/h throughout the experiment to avoid muscle contractions. The left sciatic nerve was exposed at the mid-thigh level; a pool was formed from the skin flaps and filled with paraffin oil. The nerve was stimulated with a bipolar silver electrode at about 15 mm proximal to the nerve trifurcation, proximal to the irradiated segment, with 0.1 ms pulses via a stimulus isolation unit (Grass). The spinal cord and dorsal roots were exposed by a laminectomy and immersed in paraffin oil. Fine filaments were dissected with a watchmaker’s forceps from roots L4 and L5 and were placed on a monopolar silver hook electrode. An electrical search stimulus (0.1 ms) at 1 Hz was used to identify activity in the dorsal root filaments, which was amplified (Neurolog) and displayed on a storage oscilloscope. Activity recorded from single fibers was identified on the basis of an all-or-none response. For each single unit the threshold of electrical stimulation was recorded and the latency of the action potential to the peak was taken. The receptor type, size and location of the receptive field were identified with a hand-held probe. The response threshold to stimulation with von Frey hairs was also assessed and responses to both electrical and mechanical stimulation were monitored on the oscilloscope and recorded on a computer using a Spike 2 program (Cambridge Electronic Devices). The distance between the stimulus and recording sites was measured for each animal at the end of the experiment. Photochemically-induced nerve injury produced hypersensitivity to mechanical stimulation of the hind paw ipsilateral to the irradiation. The response to von Frey hair stimulation was significantly decreased, starting from the first day after the irradiation up to day 22, which is the period when the electrophysiological studies were conducted. The median mechanical threshold before irradiation was 11.1 g and just before the electrophysiological experiments 1.24 g. In the control animals 155 fibers were found by electrical stimulation and classified according to their response to mechanical stimuli (Table 1). The largest group responded to rapid hair movement (n ¼ 53, 34.2%). A similar number of units was activated by joint movement (n ¼ 49, 31.6%).
Rapidly adapting receptors from the glabrous skin represented 6.5% (n ¼ 10). Equal numbers of slowly adapting type I, Pacinian corpuscle and tylotrich hair were recorded (4 units (2.6%) for each group). The remaining units with identifiable receptor properties were D hair (n ¼ 3, 1.9%), muscle and deep tissue (n ¼ 5, 3.2%). Of the entire population of 155 fibers recorded 23 (14.8%) did not respond to mechanical stimulation. The median mechanical threshold of receptors in glabrous skin was 2.42 g. In hairy skin the median mechanical threshold for activating G hair units was 0.19 g and tylotrich hair units 0.047 g. The conduction velocity for primary afferent fibers recorded from the dorsal roots after sciatic nerve stimulation ranged from 16 to 45 m/ s with a mean value of 29.4 m/s (Fig. 1). The joint units exhibited very regular activity which was modified by joint movement. No ongoing activity from any other type of receptor was recorded from the control group (Table 1). An equal number of units (155) was recorded from animals which developed mechanical allodynia 4–22 days after nerve injury. All fibers were activated by sciatic nerve stimulation. The units recorded from allodynic animals had a very different distribution among receptor types in comparison to the control group (Table 1). Almost half of the units (n ¼ 76, 49%) did not respond to any kind of stimulation. Ongoing discharge was recorded in 46 (29.6%) of all recorded fibers and none of these responded to mechanical stimulation. These units represented 60% of the group with no identified receptor field. The most common type of spontaneous discharge was irregular activity, the next most common was bursting and the least common was regular discharge. The most common units with receptor properties recorded in allodynic animals were joint afferents (n ¼ 45, 29%) with response characteristics similar to those recorded in control animals. Of the units with cutaneous receptive fields, receptor sensitivity to mechanical stimulation and the size of receptive fields was not different from normal Table 1 Characteristics of receptor types recorded from control and allodynic rats Receptor type
Control Number
Slowly adapting, glabrous 4 skin Rapidly adapting, glabrous 10 skin Pacinian corpuscle 4 G hair follicle 53 D hair follicle 3 Tylotrich hair 4 Joint 49 Muscle, deep 5 Unexcitable 23 Total 155 Spontaneously active units 0
Allodynic %
Number
%
2.6
1
0.6
6.5
7
4.5
2.6 34.2 1.9 2.6 31.6 3.2 14.8 100.0 0.0
1 18 0 3 45 4 76 155 46
0.6 11.6 0.0 1.9 29.0 2.5 49.3 100.0 29.6
A. Bulka et al. / Neuroscience Letters 317 (2002) 89–92
Fig. 1. Histogram of conduction velocities of afferents recorded from dorsal roots in a sample of units in normal control (A) and allodynic rats (B). N ¼ 155 in each group.
animals. The median threshold of receptors from glabrous skin to stimulation with von Frey hairs was 1.46 g. For G hair units and tylotrich hairs the median threshold value was 0.19 g. The conduction velocity of all fibers recorded from allodynic animals ranged from 14 to 40 m/s (mean 28.3 m/s) and was not significantly different from the controls. Furthermore, there was no difference in the conduction
91
velocity of units with (28.8 m/s) and without receptive fields (27.9 m/s) in the allodynic animals. However, a significant difference (P , 0:05) in conduction velocity was found when units with no receptor fields in the allodynics (27.5 m/s) were compared to the control population (29.4 m/s). The characteristics of the cutaneous afferents were similar to those previously reported in hairy [14] and glabrous [16] skin of the rat hindpaw. Many joint afferents were found in the present study, about 30% in the control and allodynic groups, which was not previously reported. In previous studies the recordings were from the saphenous [14] or tibial [16] nerve whereas in the present study the recordings were from dorsal roots, which may account for this difference. The conduction velocities of the units were similar to those in the previous studies [14,16], but slower than in a study of afferents in the sciatic constriction model [8] where the recordings were also from dorsal roots. Core body temperature was maintained as close to 378C as possible. It is possible that limb temperature was lower than normal, resulting in the slower conduction velocity. Photochemically-induced sciatic nerve ischemia produced mechanical hypersensitivity of the ipsilateral hind paw. Surprisingly, the response characteristics and conduction velocity of mechanoreceptors with receptive fields in the allodynic animals were normal. The response characteristics of afferents in other models of partial nerve injury have not been previously reported. Regenerating afferents were shown to have reduced conduction velocities proximal to the injury site, as well as reduced sensitivity to cutaneous stimuli, following nerve crush [16]. In the sciatic constriction model the conduction velocity of fibers that conducted through the site of injury was normal [8], in agreement with the present results. However, in the present study a slight, but significant, reduction in conduction velocity was observed in afferents that could not be activated from the periphery. Morphological data from the ischemic injury model indicate that the nerve is apparently normal proximal to the injury [20], at the level where it was stimulated. Thus, the slight slowing in conduction velocity was probably due to minor abnormalities undetectable with the morphological techniques used. In the constriction injury model recordings were from Ab, Ad and C fibers [8]. In that study the Ab and Ad fibers were found to be less likely to conduct through the injury site than C fibers, indicating that A fibers are more prone to injury than C fibers. Furthermore, ongoing activity at 1 and 3 days after injury was greatest in Ab, less in Ad and least in C fibers in the constriction model [8]. Similar results were found after total nerve transection where ongoing activity in A fibers had a more rapid onset (a few days) than in C fibers (weeks) [4,7]. In the present study 15% of afferents were unexcitable to mechanical stimuli in control animals and this proportion increased to 50% in the allodynics. Ongoing activity, other than in joint afferents, was not found in any recordings from controls, but was recorded in 30% of fibers in allodynics. Ongoing activity was only recorded in affer-
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ents with no receptive field, constituting 60% of this group in allodynic rats. The origin of the ongoing activity may be the distal portion of the damaged afferents [4] or the dorsal root ganglia [9]. The mechanical threshold was similar in the mechanoreceptors conducting through the injury site recorded from the allodynic animals and in controls. Furthermore, the conduction velocity of fibers with receptive fields recorded from the two groups was not different. Thus, the results suggest that the mechanical allodynia following nerve ischemia at least in part results from the central processing of input mediated by low threshold mechanoreceptors with normal receptive field and response properties. Input from these afferents converges on spinal cord interneurons that receive ongoing activity arising from damaged A fibers, resulting in abnormal ongoing discharges, response and receptive field properties. Such changes have been documented in the sciatic nerve constriction [13,15], partial sciatic nerve ligation [19] and spinal nerve ligation [2] models. We cannot exclude a role for C fibers in the development of allodynia since we did not record from them. Mechanical allodynia in neuropathic rats may at least in part be mediated by activity in low threshold mechanoreceptors that remain in continuity following an ischemic injury to the sciatic nerve. These afferents have normal response characteristics. However, about half of the afferents in neuropathic rats do not conduct through the site of injury and a majority develop ongoing activity, which may contribute to both ongoing and evoked pain. Drugs that block the generation of ongoing activity in damaged afferents, such as sodium channel blockers, may be useful for alleviating neuropathic pain. This work was supported by the Swedish Medical Research Council (07913) and AstraZeneca. The outstanding facilities of the Clinical Research Center at Huddinge University Hospital are gratefully acknowledged. [1] Bennett, G.J. and Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87–107. [2] Chapman, V., Suzuki, R. and Dickenson, A.H., Electrophysiological characterisation of spinal neuronal response properties in anaesthetized rats after ligation of spinal nerves L5-L6, J. Physiol., 507 (1998) 881–894. [3] Daube, J.R. and Dyck, P.J., Neuropathy due to peripheral vascular disease, In P.J. Dyck, P.K. Thomas, E.H. Lambert and R. Bunge (Eds.), Peripheral Neuropathy, W.B. Saunders, Philadelphia, PA, 1984, pp. 1458–1478. [4] Devor, M., The pathophysiology of damaged peripheral nerves, In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, 2nd Edition, Churchill Livingstone, Edinburgh, 1989, pp. 63–81.
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