Bilaterally enhanced dorsal horn postsynaptic currents in a rat model of peripheral mononeuropathy

ELSEVIER

Neuroscience Letters 207 (1996) 29-32

N[UIMLH LIIIB

Bilaterally enhanced dorsal horn postsynaptic currents in a rat model of peripheral mononeuropathy L . A . C o l v i n a, M . A . M a r k b, A . W . D u g g a n b,* aDepartment of Anaesthetics. University of Edinburgh, The Royal Infirmary, Lauriston Place, Edinburgh EH3 9YW, UK bDepartment of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh. Summerhall, Edinburgh EH9 IQH, UK

Received 10 November 1995; revised version received 16 February 1996; accepted 16 February 1996

Abstract

Surface compound potentials were recorded from the surgically exposed lumbar spinal cord in anaesthetized rats which had had one sciatic nerve loosely ligatured 12-15 days previously, resulting in unilateral allodynia and hyperalgesia, as assessed behaviourally. These cord dorsum potentials were recorded in response to electrical stimulation of the ligatured and non-ligatured sciatic nerve, respectively, on both the ipsi- and contralateral side with respect to the stimulated nerve. Compared to potentials produced by stimulation of the non-ligatured sciatic nerve, electrical stimulation of large diameter fibres proximal to the ligatures resulted in a smaller afferent fibre input arriving at the spinal cord. However, larger net postsynaptic currents in the contralateral dorsal horn and a larger net postsynaptic current per unit of afferent fibre input were found in the ipsilateral and contralateral spinal cord. Such changes may result from structural changes or increased synaptic efficacy in the dorsal horn following peripheral nerve injury. Keywords: Sciatic nerve; Nerve ligature; Neuropathy; Neuropathic pain; Cord dorsum potentials

The neural basis for the neuropathic pain syndromes are poorly understood as it is only recently that animal models for these conditions have been developed [3, t2, 17]. In the procedure of Bennett and Xie [3], four loose ligatures are placed around one sciatic nerve of the rat resulting in many of the characteristic phenomena of neuropathic pain such as allodynia, mechanical and thermal hyperalgesia and guarding behaviour suggestive of spontaneous pain. There are many histological reports of changes induced both peripherally and in the spinal cord by such ligatures. Thus, at around 14 days post-ligature there is near-complete loss of large myelinated fibres and a significant degeneration of small myelinated fibres distal to the ligature [2,7,9,14]. As with peripheral nerve section, there are alterations in the neuropeptides contained within dorsal root ganglion neurons [ 15,19,21,25] suggesting changes in what is released at the central terminals of primary afferent fibres in response to peripheral stimulation. There is evidence for the loss of the central connections of some primary afferent fibres and their * Corresponding author. Fax: +44 131 6506576.

replacement by others [22,23]. Considerable evidence points to changes in the activity and synaptic connections of the fibres proximal to the ligature as being responsible for many of the sensory abnormalities produced by nerve ligature [10,11 ]. The present experiments have found that the cord dorsum potentials recorded in rats with peripheral nerve injury show important differences from those of normal animals, indicating bilateral enhanced responses to impulses in large diameter afferents. For the production of nerve injuries, adult male Wistar rats were anaesthetized with sodium pentobarbitone (50 mg/kg i.p.). Using full aseptic technique, four 4/0 chromic gut loose ligatures were placed around the right sciatic nerve, proximal to its trifurcation. In the sham group, the right sciatic nerve was exposed and manipulated but not ligated. Post-operatively, the International Association for the Study of Pain guidelines for the care of experimental animals were followed [26]. The animals used displayed the changes associated with this model such as hopping, licking, everted paw and ventroflexed toe. An analgesymeter (Ugo Basile) was used to test for the development of mechanical allodynia. Rats were

0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304-3940(96)12480-9

30

L.A. Colvin et al. / Neuroscience Letters 207 (1996) 2 9 - 3 2

tested prior to surgery and then at 1-3 day intervals over the next 2 weeks. A difference score was calculated for paw withdrawal thresholds obtained from right and left hind paws, as this gives a more consistent value [3]. The mean difference (_SEM) preoperatively was -0.23 _+ 0.16 g and post-operatively was 10.36_ 1.7 g. This difference is significant (P < 0.001). For the electrophysiological experiments rats ( 1 2 15 days post ligature) were initially anaesthetized with urethane (1.25g/kg i.p.) incremented when necessary. Cannulae were inserted into the trachea, a carotid artery and an external jugular vein. Body temperature was maintained between 36-38°C, and humidified supplemental oxygen was given. Both sciatic nerves were mounted on platinum stimulating electrodes, placed proximal to the site of the ligatures on the right side and a comparable position on the left. The lumbar spinal segments were exposed and covered with agar-saline. The dura was removed with sterile forceps and the spinal cord covered with paraffin oil. The threshold stimulus voltage (T) for the fastest conducting fibres to produce a cord dorsum potential was determined for each nerve, which was then stimulated at 2 - 3 T (1 Hz, pulse width 0.5 ms). A silver ball surface electrode, applied just medial to the line of entry of the dorsal roots, was used to obtain cord dorsum potentials. These potentials were measured at three rostrocaudal positions, on both sides of the cord over segments L3 to L6. Eight 25 ms duration responses to nerve stimulation were averaged using a Neurolog NL750 Averager. The band pass of the recording amplifier was 5 Hz to 50 kHz and the gain ranged from x 200 to x 2000. The final averages were read out on a pen recorder and also preserved on magnetic digital tape. When electrical stimulation is restricted to large myelinated fibres, the cord dorsum potentials have two constant, predominantly negative, waves and a later, more variable, positive wave [5,6]. The first wave is a triphasic deflection produced by the action potentials in the dorsal columns and hence it is an index of the number of excited large diameter primary afferent fibres (see V0 in Fig. I). The second, short latency negative wave (N in Fig. 1) results predominantly from postsynaptic currents in monosynaptically activated neurones of the dorsal horn adjacent to the recording ball electrode [4] with a probable late contribution from polysynaptic potentials. These deflections may be followed by a positive wave produced by primary afferent depolarization which is not the subject of the present communication. In each animal both the ligatured and non-ligatured nerves were stimulated separately and potentials recorded bilaterally. Potentials generated on one side will be recorded bilaterally with appropriate attenuation according to the conductivity of the intervening structures. If, however, differences in ipsilaterai/contralateral ratios are observed for a particular parameter when stimulating the ligatured or non-ligatured nerves, then deductions can be made on the relative im-

portance of contralateral sources to contralaterally recorded potentials. A digitizing tablet was used to obtain the areas (~V x ms integrals) of the two waveforms studied. When V0 was clearly triphasic only the area of the negative component was measured. In some instances the later part of Vo merged with the start of N (see Fig. 1). In these cases the point of inflection was regarded as the end of V0 and the start of N. Because large myelinated fibres were stimulated (with small spread of conduction velocities) we do not believe this is a significant error. The following indices were derived for the nonligatured and ligatured nerves: (a) The area of the ipsilateral V0 wave. (b) The area of the ipsilateral N wave. (c) The N / V o ratio. This is an index of net post-synaptic current per unit of afferent fibre input. (d) All of the indices (a), (b) and (c) were also calculated for potentials recorded on the contralateral side. (e) The ratio V0 (contralateral)/V0 (ipsilateral). If this ratio is larger for one nerve than the other it suggests a larger contralateral projection by that nerve. (f) The ratio N (contralateral)/N (ipsilateral). If this ration is larger for one nerve that the other it suggests a larger contralaterai synaptic influence by that nerve. The threshold for activation of the fastest conducting fibres in the non-ligatured nerve was 90 _+ 13 mV (SEM). The corresponding threshold for the ligatured nerve was 384_+ 86 mV. This difference, which may result from oedema of the loosely ligated nerve close to the site of constriction [4], made it important to use identical multiples of threshold when stimulating the normal and ligatured nerves. Table 1 contains the values calculated for the parameters measured. The significant findings are: (a) The ipsilateral afferent fibre input from stimulation of the ligatured nerve was smaller than that from the non-ligatured nerve. (b) The ligatured nerve generated a larger net postsynap-

Non ligalured Nerve

>,lim

Ipsdateral Cord

~

o ......

i'

Ligatured Nerve

~liul

~'\

i!"

' "~ ~:

j:,

V,. j

'

\~,i' :~J

ContralateraE Cord Dorsum

"x

I mV:

I ':

;~ f",

'\;\" :

i ~'-,,_.~

/1",

~ ' ",

'-,~;"bi

2 ms ",~

,,.,~

Fig. 1. (A) Records from stimulation of a previously ligatured sciatic nerve (×3 threshold, 1 Hz). (B) Records from stimulation of the opposite, non-ligatured nerve (x3 threshold, I Hz). Each trace is a pen recording of the average of eight responses; average sweep time. 25 ms.

L.A. Colvin et al. / Neuroscience Letters 207 (1996) 29-32

Table 1 Integrals derived from cord dorsum potentials

Ipsi c.d.p.

Vo(n= 16) N ( n = 17) N/V 0 (n = 15) Conlra c.dp. Vo(n = 15) N ( n = 16) NIV0 (n = 15) V0 contr~dV 0 ipsi (n = 15) N conlra/N ipsi (n = 15)

Non-ligatured nerve

Ligatured nerve

217.18+29 1279.99+ 126 6.06 _+0.7 93.92_+ I I 450.82_+46 5.68-+ 0.9 0.44 _+0.04 0.40 _+0.03

148.11 -+26* 1415.98_+ 131 I 1.93 +- 1.3'* 80.40_+ 12 795.52_+ 100"* 12.28 -+ 2.1" 0.55 - 0.04* 0.56 _+0.06*

Means (~,nd standard errors of the means) of the/~V × ms integrals, and intcgral ratios, from the wave forms V0 and N within the measured cord dorsum polentials (see Fig. 1). lpsi, ipsilateral; contra, contralateral; c.d.p., cord dorsum potential. The significance of the differences between the means was determined by Student's paired t-tests: *P < 0.05; **P < 0.005.

tic current contralaterally in the spinal cord when compared to the non-ligatured nerve. (c) The V0 contralateral/Vo ipsilateral ratio was larger for the ligatured than for the non-ligatured nerve. (d) Both ipsilaterally and contralaterally the ligatured nerve generated a greater net postsynaptic current per unit of afferent fibre input than did the non-ligatured nerve. There is only one study in the rat [13] comparable to the present results. Laird and Bennett [13] found no significant difference between the non-ligatured and ligatured nerves in terms of the N wave generated ipsilaterally. This is in agreement with the present study but these investigators did not measure contralaterally evoked potentials and did not determine the N/V o ratio. There are several explanations for our finding of increased postsynaptic currents per unit of afferent fibre input on the side ipsilateral to the ligatured nerve. It may be a functional correlate to recent anatomical findings of reorganization in the dorsal horn following peripheral axotomy. The use of the marker, growth-associated protein (GAP-43) suggests that the spinal terminations of some large myelinated fibres migrate dorsally from their normal termination sites in laminae Ill-IV to establish new connections in laminae I-II [22-24], a process termed reactive collateral sprouting [8]. Such new large fibre connections may result in larger monosynaptic excitation of dorsal horn neurones compared to the normal situation. Not all functional studies support this proposal. Zimmermann and Westerman [27] found no difference in the spinal N waves recorded early (11-45 days) and late (3-4 months) following sectioning of one dorsal root in the cat. A reduction in the short latency inhibitory potentials produced by impulses in large diameter primary afferents I161 could explain an increased N/Vo. Others have suggested a loss of inhibitory neurons in the dorsal horn is relevant to allodynia following nerve injury [18]. An

31

additional factor may be altered synaptic efficacy, since many changes have been reported in the neuroactive compounds synthesized by dorsal root ganglion neurones following peripheral nerve injury [ 15,19,21,25]. Our finding of a greater V0 contralateral/V0 ipsilateral ratio for the ligatured relative to the non-ligatured nerve implies some contralateral projection of fibres in the ligatured nerve. This may explain the larger N wave recorded contralaterally from stimulation of the ligatured nerve relative to that from the non-ligatured nerve. Alternatively, disinhibition of intra-spinal pathways may have contributed to this result. There are several findings supportive of our proposal of altered bilateral effects from the ligatured nerve. These include the occasional occurrence of bilateral behaviourai changes following unilateral nerve constriction [1 ], the description of bilateral cell death (small dark cells) [18] and bilateral increases in spinal cord dynorphin levels again with unilateral nerve constriction [20]. It has been suggested that the ingrowth of large diameter primary afferent fibres into lamina I - I I results in these fibres accessing pathways resulting in pain perception and provides a basis for the allodynia observed in rats with peripheral nerve constriction [23]. The present results suggest that the excitatory post-synaptic effects of impulses in large fibre primary afferents are enhanced bilaterally in such animals. This work was supported by Glaxo Research Ltd and a British Journal of Anaesthesia Fellowship. [I] Attal, N., Jazat, F., Kayser, V. and Guilbaud, G., Further evidence for pain-related behaviours in a model of unilateral peripheral mononeuropathy, Pain, 41 (1990) 235-251. [2] Basbaum, A.I., Gautron, M., Jazat, F., Mayes, M. and Guilbaud, G., The spectrum of fiber loss in a model of neuropathic pain in the rat: an electron microscopic study, Pain, 47 (1991) 359367. [3] 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. [4] Bernhard, C.G., Analysis of spinal cord potentials in leads from the cord dorsum. In J.L. Malcolm and J.A.B. Gray (Eds.), The Spinal Cord, Churchill, London, 1953, pp. 43-62. [5] Bernhard, C.G., The spinal cord potentials in leads from the cord dorsum in relation to the peripheral source of afferent stimulation, Acta Physiol. Scand. 29 (Suppl. 106) (1953) 1-29. [6] Bernhard, C.G. and Widen, L., On the origin of the negative and positive spinal cord potentials evoked by stimulation of low threshold cutaneous fibres, Acta Physiol. Scand., 29 (Suppl. 106) (1953) 42-54. 17l Carlton, S.M., Dougherty, P.M., Pover, C.M. and Coggeshall, R.E., Neuroma formation and numbers of axons in a rat model of experimental peripheral neuropathy, Neurosci. Len., 131 (1991) 88-92. [8] Coggeshall, R.E., A possible relation between neuropathic pain and central sensory sprouting following peripheral nerve lesions. In J.M. Besson, G. Guilband and H. Ollat (Eds.), Peripheral Neurons in Nociception, John Libbey, Montrouge, 1994, pp. 201208. [9] Coggeshall, R.E., Dougherty, P.M., Pover, C.M. and Carlton, S.M., Is large myelinated fiber loss associated with hyperalgesia

32

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

L.A. Colvin et a l . / Neuroscience Letters 207 (1996) 2 9 - 3 2

in a model of experimental peripheral neuropathy in the rat, Pain, 52 (1993) 233-242. 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. 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. Kim, S.H. and Chung, J.M., An experimental model for peripheral neuropathy produced by segmental nerve ligation in the rat, Pain, 50 (1992) 355-363. Laird, J.M.A. and Bennett, G.J., Dorsal root potentials and afferent input to the spinal cord in rats with an experimental neuropathy, Brain Res., 584 (1992) 181-190. Munger, B.L., Bennett, G.J. and Kajander, K.C., An experimental painful peripheral neuropathy due to nerve constriction, I: axonal pathology in the sciatic nerve, Exp. Neurol., 118 (1992) 204-214. Nahin, R.L., Ren, K., De Le6n, M. and Ruda, M., Primary sensory neurons exhibit altered gene expression in a rat model of neuropathic pain, Pain, 58 (1994) 95-108. Price, DD., lntracellular responses of dorsal horn cells to cutaneous and sural nerve A and C fibre stimuli, Exp. Neurol., 33 (1971) 291-309. Seltzer, Z., Dubner, R. and Shir, Y., A novel behavioural model of neuropathic pain disorders produced in rats by partial sciatic nerve injury, Pain, 43 (1990) 205-218. Sugimoto, T., Bennett, G.J. and Kajander, K.C., Strychnineenhanced transsynaptic degeneration of dorsal horn neurons in rats with an experimental painful neuropathy, Neurosci. Lett., 98 (1989) 139-143. Villar, M.J., Wiesenfeld-Hallin, Z., Xu, X.-J., Theodorsson, E., Emson, P.C. and Hrkfelt, T., Further studies on galanin-, substance P-, and CGRP-Iike immunoreactivities in primary sensory

[20]

I21]

[22]

[23]

[24]

125]

[26] [27]

neurons and spinal cord: effects of dorsal rhizotomies and sciatic nerve lesions, Exp. Neurol., 112 (1991) 29-39. Wagner, R., DeLeo, J.A., Coombs, D.W., Willenbring, S. and Fromm, C., Spinal dynorphin immunoreactivity increases bilaterally in a neuropathic pain model, Brain Res., 629 (1993) 323326. Wakisaka, S., Kajander, K.C., and Bennett, G.J., Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy, Neurosci. Lett., 124 (1901) 200203. Woolf, C.J., Reynolds, M.L., Molander, C., O'Brien, C., Lindsay, R.M and Benowitz, L.I., The growth associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury, Neuroscience, 34 (1990) 465-478. Woolf, C.J.. Shortland, P. and Coggeshall, R.E., Peripheral nerve injury triggers central sprouting of myelinated afferents, Nature, 335 (1992) 75-78. Woolf, C.J., Shortland, P., Reynolds, M., Ridings, J., Doubell, T. and Coggeshall, R.E., Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy, J. Comp. Neurol., 360 (1995) 121-134. Zhang, X., Bean, A.J., Wiesenfeld-Hallin, Z. and Hrkfelt, T., Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord, IV: effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptide/ peptide histidine isoleucine, Neuroscience, 64 (1995) 917-94 I. Zimmermann, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 (1983) 109-110. Zimmermann, M. and Westerman, R.A., Partial deafferentation of the spinal dorsal horn does not induce changes of synaptic connectivity as revealed by recording cord dorsum potentials in adult and newborn cats. In L.M. Pubols and B.J. Sessle (Eds.), Effects of Injury on Trigeminal and Spinal Somatosensory Systems, Liss, New York, 1987, pp. 495-498.