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Antiepileptic effect of electroacupuncture vs. vagus nerve stimulation in the rat thalamus
Neuroscience Letters 441 (2008) 183–187
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Antiepileptic effect of electroacupuncture vs. vagus nerve stimulation in the rat thalamus Jian-Liang Zhang, Shi-Ping Zhang, Hong-Qi Zhang ∗ School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
a r t i c l e
i n f o
Article history: Received 10 January 2008 Received in revised form 22 May 2008 Accepted 12 June 2008 Keywords: Epilepsy Vagus nerve stimulation Electroacupuncture Thalamus Pentylenetetrazole
a b s t r a c t Our previous study has shown that both electroacupuncture (EA) and vagus nerve stimulation (VNS) can inhibit cortical epileptiform activities induced by pentylenetetrazole (PTZ). The current study compared the effects of EA and VNS on thalamic neuronal responses to PTZ-induced epileptiform activities. Under general anesthesia, extracellular single unit recordings were made from 49 single neurons in the rat ventrobasal (VB) thalamus. The left vagus nerve was stimulated at 30 Hz, 1 or 3 mA for 5 min. For EA, “Dazhui” acupoint (GV14) was stimulated with the same parameters. It was found that (1) the VB thalamic neurons showed epileptiform activities after PTZ injection; (2) VNS and EA could predominantly inhibit the PTZ-induced epileptiform activities in the thalamic neurons. The higher intensity stimulation (3 mA) in either VNS or EA was, however, not associated with a greater inhibition. Our study suggests that both EA and VNS reduce epileptiform activities at the thalamic level, and EA may be an alternative to VNS. © 2008 Elsevier Ireland Ltd. All rights reserved.
Vagus nerve stimulation (VNS) has been increasingly used in recent years for the treatment of epilepsy. Though VNS can reduce seizures in ∼60% of patients refractory to conventional antiepileptic drugs, it is nevertheless an invasive and expensive procedure that may cause complications. Furthermore, the exact antiepileptic mechanisms of VNS are still unclear, and the roles played by subcortical stations remain to be delineated. Acupuncture has been used to treat epilepsy in China since ancient times. In our recent study [17], we found that both VNS and electroacupuncture (EA) could comparably inhibit experimental cortical epileptiform activities in rats, suggesting that EA could be a good alternative to VNS in the management of epilepsy. However, the question of where in the central nervous system this inhibition takes place remains to be answered. It has been shown that VNS leads to bilateral general changes of neuronal activities in the brain in which the thalamus is considered to play an important role [16]. Using positron emission topography (PET), Henry et al. [3] demonstrated that although VNS might alter the cerebral blood flow (CBF) in a number of nuclei in the medulla, the diencephalons and the limbic system, only the bilateral thalami showed significant CBF change in association with seizure–frequency alternation. Immediately after the initial VNS, single photon emission computed tomography (SPECT) showed a significant inhibition of activities in the
∗ Corresponding author. Tel.: +852 34112431; fax: +852 34112461. E-mail address: [email protected] (H.-Q. Zhang). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.06.032
left thalamus, which was in line with the prominent reduction of seizures [15]. Thus, it has been proposed that VNS might inhibit the seizure onset or propagation by means of inhibiting the thalamic relay center [13]. The VNS influence on thalamic activity and blood perfusion appears to depend on the prestimulation conditions. That is, in the acute, initial setting, the left thalamus could be deactivated along with the right hippocampus and parahippocampal gyrus whereas acute stimulation after long-term VNS resulted in significant left thalamic activation [14]. As the last somatosensory relay to the cortex, the discharge patterns of the ventrobasal (VB) thalamic nuclei appear to correlate well with cortical seizure activities. In the cat experiencing generalized epilepsy induced by penicillin, spiky epileptiform activity could be readily shown by extracellular recordings from the thalamic VB nuclei [7]. In pyridoxine-deficient rats, microinjection of either picrotoxin or PTZ into the VB thalamus induced irregular unit activities with frequent bursts among electrosilent periods, and severe seizure discharge activities [12]. Indirect support for the role of VB thalamus in seizure came from the observation that bilateral electrolytic lesions of the VB thalamus might significantly attenuate the duration of PTZ- and gamma-hydroxybutyric acidinduced spikes and wave discharges in both the thalamus and the cortex [1]. From the available data, the VB thalamus appears to be an important subcortical site involved in epilepsy. Thus, in the current study we set out to examine VB thalamic responses in order to determine the role of the thalamus in the antiepileptic effect of EA as well as
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Fig. 1. Experimental arrangement. (A) After the first i.v. administration of PTZ, the thalamic neuronal activities were enhanced and the discharge pattern was altered in comparison with the background control. Below the original spike waveforms are the corresponding PSTHs. (B and C) The recordings before and after the treatment by EA (B) and VNS (C), and corresponding impulse counts are the average for the whole 5 min following the stimulation though only 2 min recordings are shown.
VNS in the rat. The hypothesis to be tested was that EA, like VNS, could inhibit epileptiform activities in the thalamus. Experiments were performed on 18 male SD rats weighing 200–280 g after approval by the institute research ethics committee. The animals were anesthetized by intraperitoneal injection of ketamine (40 mg/kg) and xylazine (4 mg/kg) and maintained with intravenous infusion of the same agents (ketamine ∼6 mg/(kg h) and xylazine ∼0.6 mg/(kg h)). The trachea was intubated by tracheostomy to ensure smooth respiration. The jugular vein was cannulated for administration of drugs. The body temperature was maintained at ∼38 ◦ C by a thermostatically controlled heating blanket. The left cervical vagus nerve was isolated from the cortical sheath and placed on a bipolar platinum hook electrode which was insulated from the surrounding tissue by a piece of soft plastic sheet for stimulation. Then the animal was fixed on a stereotaxic frame and the left parietal cortex was exposed by craniotomy. The dura mater was removed and the exposed cortex was protected with warm paraffin oil in a skin pool made from the scalp. The VB thalamus was accessed stereotaxically guided by stereotaxic coordinates [9]. Epileptiform activities were induced by slow injection (within 2 min) of pentylenetetrazole (PTZ, Sigma, USA) via the jugular vein at 60 mg/100 g body weight [11]. Repeated injections with the same dose of PTZ were given when the epileptiform activities diminished, usually 1–2 h after previous injection. Epileptiform thalamic neuronal activities usually appeared 2–5 min after i.v. administration of PTZ and lasted for one-half to several hours. The epileptiform activities were characterized by increase in neuronal discharge frequency with irregular and clustered firing pattern, particularly after repeated injections of PTZ (Fig. 1A). For VNS, the electrical stimuli delivered from an A-M2100 System (USA) were applied to the left cervical vagal trunk via the bipolar hook platinum electrode at low (1 mA) and/or high (3 mA) intensities at a fixed frequency of 30 Hz with 0.5 ms bipolar pulse width for 5 min. For EA stimulation, a pair of stainless steel acupuncture needles 2 mm apart were inserted about 5 mm deep into skin at the location equivalent to acupoint “Dazhui” in humans (Governor Vessel, GV14) which, located at the midline on
the back between the last cervical and the first thoracic vertebral spinous processes, is one of the most commonly used acupoints for the treatment of epilepsy. The EA stimuli were generated by an EA stimulator (CEFAR ACUS II, Sweden) and delivered via the inserted needles with the same parameters as VNS. The stimuli were delivered randomly, such that in some experiments the EA was given first, usually starting with low intensity at 1 mA, and followed by VNS at the same intensity, or vice versa. Extracellular recordings were carried out with the use of tungsten microelectrodes (125 m shank, 5–10 M). When the microelectrode was driven 5 mm below the surface of cerebral cortex, the body parts on the right side were gently tapped to search for VB thalamic neurons as guided by the somatotopic organization. Only the neurons that were responsive to gentle tapping or brushing were selected for further investigation, as they were most likely to be tactile sensitive VB neurons. The well-isolated activities of individual neurons were captured and monitored online using a data acquisition system (CED1401 Spike2, Cambridge, England) that could effectively filter out large stimulation artifacts with proper setting of the window discriminator. The neuronal responses were analyzed on- and off-line by constructing peri-stimulus time histograms (PSTH) with Spike2 software. The recording paradigm was similar to those previously described for cortical recording [17]. Briefly, a 5 min continuous recording of the baseline activities was taken first as a control. Then, PTZ was administered to induce the epileptiform activities (Fig. 1A). Only the neurons with their activities increased by >10% after PTZ were selected for further study by EA and/or VNS. EA or VNS was applied for 5 min followed by another 5 min post-treatment recording (Fig. 1B and C). The impulse counts were averaged over the whole 5 min period and expressed as mean ± S.E. The spike counts in the 5 min post-stimulation period were compared with those immediately prior to the stimulation for the same duration. Represented in a formula, the treatment effect = (B − A)/A where B was post-stimulation responses and A was pre-stimulation control activities. If the impulse count after stimulation was 10% more or less than the control before the stimulation, the effects were considered to be excitatory or inhibitory, respectively [17]. For statistical analysis, the software SPSS for Windows (Version 11.5) was used. To compare data among multiple groups or trials, one-way ANOVA was adopted in which P < 0.05 was considered statistically significant. If equal variances were assumed, the LSD method was used; otherwise Dunnett’s T3 method was chosen. For histological verification of recording sites in the thalamus, electrolytic lesions (3500 Lesion Making Device, Stoelting, Italy) were made at the tip of the recording track by passing DC current at 30 A for 30 s at the end of experiments, and/or a marker electrode was sometimes cut and left in place at the known coordinates. The rats were overdosed with anesthetics and perfused with normal saline followed by 3% paraformaldehyde. The areas of the brain containing lesions were cut coronally into 40 m sections. The locations of neurons were verified by reconstruction of the microelectrode tracks and checking recording coordinates with reference to the rat brain atlas [9]. Forty-nine single thalamic neurons were isolated and studied successfully from 18 rats. The neuronal receptive fields (RFs) of the majority (42/49) were distributed contralateral to the thalamic recording site at the caudal parts of body on the right side, that is, the low back and abdomen, the perineum, hind limb, tail and foot. RFs of 4 neurons (4/49) were located around the upper parts of the body including the right chest, forelimb, ear and face. The remaining 3 (3/49) had large RFs covering almost the entire right side of the body.
J.-L. Zhang et al. / Neuroscience Letters 441 (2008) 183–187
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Fig. 3. An example of the effect of 1 mA stimulation on PTZ-induced epileptiform activities in a same thalamic neuron. (A) PTZ led to a gradual increase of the thalamic neuronal activities; (B) 1 mA VNS inhibited the epileptiform activities; (C) 1 mA EA stimulation of “Dazhui” acupoint strongly inhibited the neuronal activities; (D) the neuronal epileptiform activities gradually returned after the cessation of EA at time zero.
Fig. 2. Neuronal locations in the thalamus. (A) Microphotograph showing an electrode track in the rat brain. A lesion was made at the end of recording. The locations of the thalamic neurons were reconstructed based on the recording coordinates, taking into account the fixation shrinkage factor of 15%. (B) Summary of the locations of recorded neurons in the rat thalamus. The recordings were carried out at coronal plane 3 ± 0.5 mm posterior to the bregma. VPL: ventroposterior lateral nucleus; VPM: ventroposterior medial nucleus; VM: ventrmedial nucleus; VL: ventrolateral nucleus; PO: posterior thalamic nuclei group; ZI: zona incerta; RT: reticular thalamic nucleus; IC: internal capsule. Triangles represent neuronal locations in the VPL, and dots in the VPM.
In terms of sensitivity, about half (23/49) of the neurons could be activated by 8 mg von Frey hair, 19 by 20 mg and the rest 7 neurons by >20 mg von Frey hairs. That is, the thalamic neurons recorded were predominantly tactile sensitive neurons. After the recording, 20 neurons were successfully verified for their locations in the thalamus by checking histological sections. Fifteen were located in the VPL and the remaining 5 were in the VPM, the two subnuclei of the VB thalamus (Fig. 2). Among the 49 neurons tested for their responses to PTZ administration, 34 (69.4%) had their neuronal discharge increased by 244.6 ± 56.6% (range 13–1568%), 9 neurons (18.4%) were inhibited by 47.9 ± 6% (range 28–82%) and the remaining 6 neurons (12.2%) had little change (<10%). That is, the thalamic VB neurons showed mixed responses to PTZ, but the majority had excitatory response. On average, the activities of all 49 neurons increased by 160.8 ± 43.2% after PTZ application. Among the 34 neurons that had excitatory response to PTZ, 32 neurons were recorded successfully for the effect of 1 mA VNS on the PTZ-induced epileptiform activities. Twenty-three (23/32, or 71.9%) were inhibited by 1 mA VNS, with an inhibition rate of
42.2 ± 4.8% on average (range 10–83.5%). Excitation after VNS was seen in 4 neurons (12.5%) with a mean increase of 32 ± 9.2% on average (range 13.6–55.8%), and the remaining 5 neurons (15.6%) had little change (<10%). Counting all 32 neurons together, the PTZ-induced epileptiform neuronal activities were reduced by 26.6 ± 5.9% (P < 0.05) on average after 1 mA VNS (Fig. 3B). Thus, the effect of 1 mA VNS was predominantly inhibitory. At 3 mA VNS, an inhibition of neuronal epileptiform activity ranging from 10.3% to 67.1% (mean ± S.E. 33.9 ± 6.5%) was seen in 9 out of 20 neurons tested. Four neurons showed excitation by 3 mA VNS, ranging from 12.2% to 55.8% (38 ± 9.2%), whereas the remaining 7 neurons had little change. Overall for the 20 neurons, there was a 7.7 ± 7.1% reduction on average of epileptiform activities after 3 mA VNS (P > 0.05) (Fig. 4C). In comparison with 1 mA VNS, 3 mA VNS produced less inhibition on the thalamic epileptiform activities (P < 0.05).
Fig. 4. An example of the effect of 3 mA stimulation on PTZ-induced epileptiform activities in a same thalamic neuron. (A) PTZ led to the vigorous epileptiform neuronal activities; (B) 3 mA EA stimulation of “Dazhui” acupoint inhibited the epileptiform neuronal activities; (C) 3 mA VNS also inhibited the neuronal activities.
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After 1 mA EA stimulation of “Dazhui” acupoint, the PTZinduced epileptiform activities were inhibited in 13 out of 29 neurons (44.8%) tested, with a decrease of 48.2 ± 9% on average (range 10.2–96.7%). Six neurons (20.7%) showed excitation with an increase of 39.7 ± 6.5% (range 19.5–61.7%) while the remaining 10 neurons (34.4%) had little change. Overall, the inhibition rate for all 29 neurons together was 13.5 ± 7.7% on average at this intensity, and it was statistically significant (P < 0.05) in comparison with the pre-treatment control (Fig. 3C). In comparison with 1 mA VNS, the effect was comparable, with no statistically significant difference between the two groups (P > 0.05). At 3 mA EA stimulation, an inhibition ranging from 23.8% to 59.2% was seen in 10 out of 20 neurons (50%), with an inhibition rate of 48.2 ± 5.2% on average. Eight neurons (40%) showed excitation ranging from 10.2% to 143.2% with an increase of 54.9 ± 17.5% on average. The remaining 2 neurons showed little change. Overall, there was a 1.8 ± 13.3% reduction (P > 0.05) on average of the epileptiform activities in all 20 neurons together after 3 mA EA (Fig. 4B). This overall effect was not significantly different from that of 1 mA EA, nor from that of VNS at the same intensity, i.e. 3 mA (P > 0.05). In summary, the current work demonstrated that the VB thalamic neurons displayed epileptiform activities after PTZ injection. VNS and EA had a mixed effect but predominantly inhibited the PTZ-induced epileptiform activities in the VB neurons, suggesting that both VNS and EA could bring about antiepileptic effect by, at least partial inhibition at the level of the thalamus. However, the higher intensity stimulation of either VNS or EA was not associated with a greater level of inhibition. The thalamic cells responded to either VNS or EA in a similar way, suggesting that EA could be a good alternative to VNS for acute abortion of epilepsy, at least in this animal model. The results were consistent with what we observed in the cerebral cortex [17], and support our hypothesis that the VB thalamus plays an important role in the antiepileptic effect of either VNS or EA. In this study, the neurons tested were predominantly located in the thalamic VB nucleus as judged by the response characteristics of the neurons and by the histological verifications. Thus, the VB thalamus appears to be involved in certain forms of epilepsy. This is in line with some of the previous reports [1,7,12] and clinical observations that some seizures are preceded by a variety of auras including sensory illusion and hallucinations. Of course, other thalamic nuclei might also take part, as shown by a recent long-term follow-up study [6] in which electrical stimulation of the anterior nucleus of thalamus produced a significant reduction of seizures. An early study showed that stimulation of the thalamic midline structures could induce cortical responses very similar to the spiky wave discharges of generalized absence epilepsy in association with an underlying thalamocortical oscillation [8]. The fact that VB neuronal response to EA and VNS was predominantly inhibitory posed the possibility that they could interrupt the genesis and spread of epileptiform activities at this subcortical gate to the cerebral cortex. At present, it is not entirely clear where this inhibition is initiated. Though the thalamus might be related to epileptic activities, we do not at all imply that the thalamus is the only important subcortical site that mediates epilepsy. Furthermore, there are many types of epilepsy that may have different mechanisms. Other potential sites include the hippocampus, amygdale and the nucleus of solitary tract (NTS). Since the latter is a primary site for convergence of vagal afferents and afferents from facial, scalp and auricular regions via trigeminal, cervical spinal and glossopharyngeal nerves [2], it could be an important structure involved in the antiepileptic effects of EA and VNS, and thus could be a potential candidate for further investigations. The antiepileptic effect observed in this study was instant and short-lived. The acute inhibitory effect is in line with pre-
vious observations that reduction of the interictal epileptiform discharges in human subjects was more prominent during the actual VNS period and the first interstimulation period [4]. This acute effect is most likely due to mobilization of the inhibitory circuit in the CNS. After cessation of either VNS or EA stimulation, the epileptiform thalamic activities may recover gradually. For example, Fig. 3D shows that the epileptiform activity returned about ∼13 min after cessation of EA. Mechanisms other than direct mobilization of the inhibitory circuit must also play a role too as in most cases VNS only gradually achieves a progressive prophylactic effect clinically, suggesting the involvement of various neurotransmitters, neuromodulators, cytokines or even morphological changes [10]. In the current experimental setting, repeated testing was difficult to carry out since the recovery time was variable and the later tests became somewhat unreliable. Chronic recording may circumvent this problem to a certain extent. The somatic RFs of many thalamic neurons recorded did not cover the location of acupoint GV14 in the current study. This observation suggests that the meridian theory, which is an important foundation for acupuncture practice, cannot simply be explained by somatotopical organization in modern neuroanatomical terms. Nevertheless, in the practice of Chinese medicine, acupuncture is often applied to a set of acupoints that can be remote from the diseased site and bear little relationship with nerve trajectory or convergence. For epilepsy treatments, acupoints from all over the body can be selected, though GV14 is one of the most commonly used. Although the anatomical trajectory for the interaction is not exactly clear, it is conceivable that EA may exert effects on a wide range of brain structures that may directly or indirectly affect thalamic neuronal responses. In this study, fixed frequency was used because clinic evidence has indicated that VNS at 20–30 Hz was most effective for the management of epilepsy. This is somewhat different from acupuncture analgesia where different EA frequencies would invoke different mechanisms; for example, low frequency EA chiefly leads to the release of enkephalin to induce analgesia via and ␦ receptors at the supraspinal levels, whereas high frequency EA (100 Hz) works by stimulating dynorphin release acting on receptors in the spinal cord. So far there is no evidence suggesting that different frequency of VNS would invoke different mechanisms. In further studies, however, the effects of different frequencies of VNS and EA could be investigated, and neurochemical analysis should be carried out to examine molecular mechanisms of these treatments. With regards to the stimulation intensity, our results indicated that EA at either low or high intensity produced similar effects in terms of the extent of inhibition and the proportion of neurons inhibited. For VNS, however, low intensity stimulation gave rise to significantly stronger inhibition in comparison with high intensity in both the extent of inhibition and the proportion of neurons inhibited. It was also noted that for both VNS and EA, high intensity (3 mA) also caused more excitation of thalamic neurons than 1 mA in terms of the discharge rate (EA: 55% vs. 40%; VNS: 38% vs. 32%), as well as the proportion of neurons excited (EA: 40% vs. 21%; VNS: 20% vs. 13%). Thus, it appears that high intensity stimulation could activate not only inhibitory circuits but also excitatory circuits to some extent. This observation is in line with the clinical observation that increased VNS current has weak and insignificant correlation with seizures reduction but more side effects, which could be due to activation of high threshold unmyelinated C fibers [5]. As the antiepileptic effects of VNS are assumed largely due to the activation of myelinated fibers rather than unmyelinated C fibers in the cervical vagus nerve, 3 mA might cause activation of more unmyelinated fibers than 1 mA and might in turn offset to some extent the beneficial effect of VNS. Thus, care should be taken clini-
J.-L. Zhang et al. / Neuroscience Letters 441 (2008) 183–187
cally in deciding the parameters of VNS in order to achieve the best curative efficacy with minimal side effects. It is interesting that EA, in comparison with VNS, is less likely to activate excitatory neurons at the higher intensity. This is perhaps due to the fact that the EA is not directly applied on a nerve truck as VNS is. Clinical EA application is usually not painful, and is unlikely to activate nociceptive C fibers. In contrast, the vagus nerve contains a large proportion of unmyelinated C fibers that could be readily activated by direct electrical stimulation at high intensities. As the vagus nerve trunk of the rat is far finer than that of humans, 3 mA intensity could well activate all nerve fibers in it. Based on the current data, we conclude that both VNS and EA might acutely inhibit PTZ-induced epileptiform activities in the VB thalamus that appears to be involved in epilepsy. Taken together with our recent observation that EA was similarly effective as VNS in inhibiting epileptiform activities in the cerebral cortex [17], and with the concern of potential side effects, comfort and the financial costs of VNS, it appears that acupuncture may be a good alternative therapy to VNS in the management of epilepsy. Nevertheless, much more work is still needed to document its clinical efficiency, particularly in chronic settings, and to understand the mechanisms of the acupuncture treatment. Acknowledgements This work was supported by HKBU FRG/04-05/I-16 and FRG/0506/II-55. References [1] P.K. Banerjee, O.C. Snead, Thalamic mediodorsal and intralaminar nuclear lesions disrupt the generation of experimentally induced generalized absencelike seizures in rats, Epilepsy Res. 17 (1994) 193–205. [2] Y.O. Cakmak, Epilepsy, electroacupuncture and the nucleus of the solitary tract, Acupunct. Med. 24 (2006) 164–168.
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[3] T.R. Henry, J.R. Votaw, P.B. Pennell, C.M. Epstein, R.A. Bakay, T.L. Faber, S.T. Grafton, J.M. Hoffman, Acute blood flow changes and efficacy of vagus nerve stimulation in partial epilepsy, Neurology 52 (1999) 1166–1173. [4] R. Kuba, M. Guzaninova, M. Brazdil, Z. Novak, J. Chrastina, I. Rektor, Effect of vagal nerve stimulation on interictal epileptiform discharges: a scalp EEG study, Epilepsia 43 (2002) 1181–1188. [5] D.M. Labiner, G.L. Ahern, Vagus nerve stimulation therapy in depression and epilepsy: therapeutic parameter settings, Acta Neurol. Scand. 115 (2007) 23– 33. [6] S.N. Lim, S.T. Lee, Y.T. Tsai, I.A. Chen, P.H. Tu, J.L. Chen, H.W. Chang, Y.C. Su, T. Wu, Electrical stimulation of the anterior nucleus of the thalamus for intractable epilepsy: a long-term follow-up study, Epilepsia 48 (2007) 342–347. [7] R.S. McLachlan, P. Gloor, M. Avoli, Differential participation of some ‘specific’ and ‘non-specific’ thalamic nuclei in generalized spike-and-wave discharges of feline generalized penicillin epilepsy, Brain Res. 307 (1984) 277–287. [8] E. Niedermeyer, E.R. Laws, A.E. Walker, Depth EEG findings in epileptics with generalized spike–wave complexes, Arch. Neurol. 21 (1969) 51–58. [9] G. Paxinos, G.C. Watson (Eds.), The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 2005. [10] S.C. Schachter, D. Schmidt (Eds.), Vagus Nerve Stimulation, Martin Dunitz, London/New York, 2003. [11] H. Schmoll, I. Badan, G. Crecksch, L. Walker, C. Kessler, A. Popa-Wagner, Kindling status in Sprague–Dawley rats induced by pentylenetetrazole: involvement of a critical development period, Am. J. Pathol. 162 (2003) 1027–1034. [12] S.K. Sharma, B. Bolster, K. Dakshinamurti, Picrotoxin and pentylene tetrazole induced seizure activity in pyridoxine-deficient rats, J. Neurol. Sci. 121 (1994) 1–9. [13] K. Van Laere, K. Vonck, P. Boon, B. Brans, T. Vandekerckhove, R. Dierckx, Vagus nerve stimulation in refractory epilepsy: SPECT activation study, J. Nucl. Med. 41 (2000) 1145–1154. [14] K. Van Laere, K. Vonck, P. Boon, J. Versijpt, R. Dierckx, Perfusion SPECT changes after acute and chronic vagus nerve stimulation in relation to prestimulus condition and long-term clinical efficacy, J. Nucl. Med. 43 (2002) 733– 744. [15] K. Vonck, P. Boon, K. Van Laere, M. D’Have, T. Vandekerckhove, S. O’Connor, B. Brans, R. Dierckx, J. De Reuck, Acute single photon emission computed tomographic study of vagus nerve stimulation in refractory epilepsy, Epilepsia 41 (2000) 601–609. [16] K. Vonck, K. Van Laere, S. Dedeurwaerdere, J. Caemaert, J. De Reuck, P. Boon, The mechanism of action of vagus nerve stimulation for refractory epilepsy: the current status, J. Clin. Neurophysiol. 18 (2001) 394–401. [17] J.L. Zhang, S.P. Zhang, H.Q. Zhang, Antiepileptic effects of electroacupuncture vs. vagus nerve stimulation on cortical epileptiform activities, J. Neurol. Sci. 270 (2008) 114–121.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Antiepileptic effect of electroacupuncture vs. vagus nerve stimulation in the rat thalamus Jian-Liang Zhang, Shi-Ping Zhang, Hong-Qi Zhang ∗ School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
a r t i c l e
i n f o
Article history: Received 10 January 2008 Received in revised form 22 May 2008 Accepted 12 June 2008 Keywords: Epilepsy Vagus nerve stimulation Electroacupuncture Thalamus Pentylenetetrazole
a b s t r a c t Our previous study has shown that both electroacupuncture (EA) and vagus nerve stimulation (VNS) can inhibit cortical epileptiform activities induced by pentylenetetrazole (PTZ). The current study compared the effects of EA and VNS on thalamic neuronal responses to PTZ-induced epileptiform activities. Under general anesthesia, extracellular single unit recordings were made from 49 single neurons in the rat ventrobasal (VB) thalamus. The left vagus nerve was stimulated at 30 Hz, 1 or 3 mA for 5 min. For EA, “Dazhui” acupoint (GV14) was stimulated with the same parameters. It was found that (1) the VB thalamic neurons showed epileptiform activities after PTZ injection; (2) VNS and EA could predominantly inhibit the PTZ-induced epileptiform activities in the thalamic neurons. The higher intensity stimulation (3 mA) in either VNS or EA was, however, not associated with a greater inhibition. Our study suggests that both EA and VNS reduce epileptiform activities at the thalamic level, and EA may be an alternative to VNS. © 2008 Elsevier Ireland Ltd. All rights reserved.
Vagus nerve stimulation (VNS) has been increasingly used in recent years for the treatment of epilepsy. Though VNS can reduce seizures in ∼60% of patients refractory to conventional antiepileptic drugs, it is nevertheless an invasive and expensive procedure that may cause complications. Furthermore, the exact antiepileptic mechanisms of VNS are still unclear, and the roles played by subcortical stations remain to be delineated. Acupuncture has been used to treat epilepsy in China since ancient times. In our recent study [17], we found that both VNS and electroacupuncture (EA) could comparably inhibit experimental cortical epileptiform activities in rats, suggesting that EA could be a good alternative to VNS in the management of epilepsy. However, the question of where in the central nervous system this inhibition takes place remains to be answered. It has been shown that VNS leads to bilateral general changes of neuronal activities in the brain in which the thalamus is considered to play an important role [16]. Using positron emission topography (PET), Henry et al. [3] demonstrated that although VNS might alter the cerebral blood flow (CBF) in a number of nuclei in the medulla, the diencephalons and the limbic system, only the bilateral thalami showed significant CBF change in association with seizure–frequency alternation. Immediately after the initial VNS, single photon emission computed tomography (SPECT) showed a significant inhibition of activities in the
∗ Corresponding author. Tel.: +852 34112431; fax: +852 34112461. E-mail address: [email protected] (H.-Q. Zhang). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.06.032
left thalamus, which was in line with the prominent reduction of seizures [15]. Thus, it has been proposed that VNS might inhibit the seizure onset or propagation by means of inhibiting the thalamic relay center [13]. The VNS influence on thalamic activity and blood perfusion appears to depend on the prestimulation conditions. That is, in the acute, initial setting, the left thalamus could be deactivated along with the right hippocampus and parahippocampal gyrus whereas acute stimulation after long-term VNS resulted in significant left thalamic activation [14]. As the last somatosensory relay to the cortex, the discharge patterns of the ventrobasal (VB) thalamic nuclei appear to correlate well with cortical seizure activities. In the cat experiencing generalized epilepsy induced by penicillin, spiky epileptiform activity could be readily shown by extracellular recordings from the thalamic VB nuclei [7]. In pyridoxine-deficient rats, microinjection of either picrotoxin or PTZ into the VB thalamus induced irregular unit activities with frequent bursts among electrosilent periods, and severe seizure discharge activities [12]. Indirect support for the role of VB thalamus in seizure came from the observation that bilateral electrolytic lesions of the VB thalamus might significantly attenuate the duration of PTZ- and gamma-hydroxybutyric acidinduced spikes and wave discharges in both the thalamus and the cortex [1]. From the available data, the VB thalamus appears to be an important subcortical site involved in epilepsy. Thus, in the current study we set out to examine VB thalamic responses in order to determine the role of the thalamus in the antiepileptic effect of EA as well as
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Fig. 1. Experimental arrangement. (A) After the first i.v. administration of PTZ, the thalamic neuronal activities were enhanced and the discharge pattern was altered in comparison with the background control. Below the original spike waveforms are the corresponding PSTHs. (B and C) The recordings before and after the treatment by EA (B) and VNS (C), and corresponding impulse counts are the average for the whole 5 min following the stimulation though only 2 min recordings are shown.
VNS in the rat. The hypothesis to be tested was that EA, like VNS, could inhibit epileptiform activities in the thalamus. Experiments were performed on 18 male SD rats weighing 200–280 g after approval by the institute research ethics committee. The animals were anesthetized by intraperitoneal injection of ketamine (40 mg/kg) and xylazine (4 mg/kg) and maintained with intravenous infusion of the same agents (ketamine ∼6 mg/(kg h) and xylazine ∼0.6 mg/(kg h)). The trachea was intubated by tracheostomy to ensure smooth respiration. The jugular vein was cannulated for administration of drugs. The body temperature was maintained at ∼38 ◦ C by a thermostatically controlled heating blanket. The left cervical vagus nerve was isolated from the cortical sheath and placed on a bipolar platinum hook electrode which was insulated from the surrounding tissue by a piece of soft plastic sheet for stimulation. Then the animal was fixed on a stereotaxic frame and the left parietal cortex was exposed by craniotomy. The dura mater was removed and the exposed cortex was protected with warm paraffin oil in a skin pool made from the scalp. The VB thalamus was accessed stereotaxically guided by stereotaxic coordinates [9]. Epileptiform activities were induced by slow injection (within 2 min) of pentylenetetrazole (PTZ, Sigma, USA) via the jugular vein at 60 mg/100 g body weight [11]. Repeated injections with the same dose of PTZ were given when the epileptiform activities diminished, usually 1–2 h after previous injection. Epileptiform thalamic neuronal activities usually appeared 2–5 min after i.v. administration of PTZ and lasted for one-half to several hours. The epileptiform activities were characterized by increase in neuronal discharge frequency with irregular and clustered firing pattern, particularly after repeated injections of PTZ (Fig. 1A). For VNS, the electrical stimuli delivered from an A-M2100 System (USA) were applied to the left cervical vagal trunk via the bipolar hook platinum electrode at low (1 mA) and/or high (3 mA) intensities at a fixed frequency of 30 Hz with 0.5 ms bipolar pulse width for 5 min. For EA stimulation, a pair of stainless steel acupuncture needles 2 mm apart were inserted about 5 mm deep into skin at the location equivalent to acupoint “Dazhui” in humans (Governor Vessel, GV14) which, located at the midline on
the back between the last cervical and the first thoracic vertebral spinous processes, is one of the most commonly used acupoints for the treatment of epilepsy. The EA stimuli were generated by an EA stimulator (CEFAR ACUS II, Sweden) and delivered via the inserted needles with the same parameters as VNS. The stimuli were delivered randomly, such that in some experiments the EA was given first, usually starting with low intensity at 1 mA, and followed by VNS at the same intensity, or vice versa. Extracellular recordings were carried out with the use of tungsten microelectrodes (125 m shank, 5–10 M). When the microelectrode was driven 5 mm below the surface of cerebral cortex, the body parts on the right side were gently tapped to search for VB thalamic neurons as guided by the somatotopic organization. Only the neurons that were responsive to gentle tapping or brushing were selected for further investigation, as they were most likely to be tactile sensitive VB neurons. The well-isolated activities of individual neurons were captured and monitored online using a data acquisition system (CED1401 Spike2, Cambridge, England) that could effectively filter out large stimulation artifacts with proper setting of the window discriminator. The neuronal responses were analyzed on- and off-line by constructing peri-stimulus time histograms (PSTH) with Spike2 software. The recording paradigm was similar to those previously described for cortical recording [17]. Briefly, a 5 min continuous recording of the baseline activities was taken first as a control. Then, PTZ was administered to induce the epileptiform activities (Fig. 1A). Only the neurons with their activities increased by >10% after PTZ were selected for further study by EA and/or VNS. EA or VNS was applied for 5 min followed by another 5 min post-treatment recording (Fig. 1B and C). The impulse counts were averaged over the whole 5 min period and expressed as mean ± S.E. The spike counts in the 5 min post-stimulation period were compared with those immediately prior to the stimulation for the same duration. Represented in a formula, the treatment effect = (B − A)/A where B was post-stimulation responses and A was pre-stimulation control activities. If the impulse count after stimulation was 10% more or less than the control before the stimulation, the effects were considered to be excitatory or inhibitory, respectively [17]. For statistical analysis, the software SPSS for Windows (Version 11.5) was used. To compare data among multiple groups or trials, one-way ANOVA was adopted in which P < 0.05 was considered statistically significant. If equal variances were assumed, the LSD method was used; otherwise Dunnett’s T3 method was chosen. For histological verification of recording sites in the thalamus, electrolytic lesions (3500 Lesion Making Device, Stoelting, Italy) were made at the tip of the recording track by passing DC current at 30 A for 30 s at the end of experiments, and/or a marker electrode was sometimes cut and left in place at the known coordinates. The rats were overdosed with anesthetics and perfused with normal saline followed by 3% paraformaldehyde. The areas of the brain containing lesions were cut coronally into 40 m sections. The locations of neurons were verified by reconstruction of the microelectrode tracks and checking recording coordinates with reference to the rat brain atlas [9]. Forty-nine single thalamic neurons were isolated and studied successfully from 18 rats. The neuronal receptive fields (RFs) of the majority (42/49) were distributed contralateral to the thalamic recording site at the caudal parts of body on the right side, that is, the low back and abdomen, the perineum, hind limb, tail and foot. RFs of 4 neurons (4/49) were located around the upper parts of the body including the right chest, forelimb, ear and face. The remaining 3 (3/49) had large RFs covering almost the entire right side of the body.
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Fig. 3. An example of the effect of 1 mA stimulation on PTZ-induced epileptiform activities in a same thalamic neuron. (A) PTZ led to a gradual increase of the thalamic neuronal activities; (B) 1 mA VNS inhibited the epileptiform activities; (C) 1 mA EA stimulation of “Dazhui” acupoint strongly inhibited the neuronal activities; (D) the neuronal epileptiform activities gradually returned after the cessation of EA at time zero.
Fig. 2. Neuronal locations in the thalamus. (A) Microphotograph showing an electrode track in the rat brain. A lesion was made at the end of recording. The locations of the thalamic neurons were reconstructed based on the recording coordinates, taking into account the fixation shrinkage factor of 15%. (B) Summary of the locations of recorded neurons in the rat thalamus. The recordings were carried out at coronal plane 3 ± 0.5 mm posterior to the bregma. VPL: ventroposterior lateral nucleus; VPM: ventroposterior medial nucleus; VM: ventrmedial nucleus; VL: ventrolateral nucleus; PO: posterior thalamic nuclei group; ZI: zona incerta; RT: reticular thalamic nucleus; IC: internal capsule. Triangles represent neuronal locations in the VPL, and dots in the VPM.
In terms of sensitivity, about half (23/49) of the neurons could be activated by 8 mg von Frey hair, 19 by 20 mg and the rest 7 neurons by >20 mg von Frey hairs. That is, the thalamic neurons recorded were predominantly tactile sensitive neurons. After the recording, 20 neurons were successfully verified for their locations in the thalamus by checking histological sections. Fifteen were located in the VPL and the remaining 5 were in the VPM, the two subnuclei of the VB thalamus (Fig. 2). Among the 49 neurons tested for their responses to PTZ administration, 34 (69.4%) had their neuronal discharge increased by 244.6 ± 56.6% (range 13–1568%), 9 neurons (18.4%) were inhibited by 47.9 ± 6% (range 28–82%) and the remaining 6 neurons (12.2%) had little change (<10%). That is, the thalamic VB neurons showed mixed responses to PTZ, but the majority had excitatory response. On average, the activities of all 49 neurons increased by 160.8 ± 43.2% after PTZ application. Among the 34 neurons that had excitatory response to PTZ, 32 neurons were recorded successfully for the effect of 1 mA VNS on the PTZ-induced epileptiform activities. Twenty-three (23/32, or 71.9%) were inhibited by 1 mA VNS, with an inhibition rate of
42.2 ± 4.8% on average (range 10–83.5%). Excitation after VNS was seen in 4 neurons (12.5%) with a mean increase of 32 ± 9.2% on average (range 13.6–55.8%), and the remaining 5 neurons (15.6%) had little change (<10%). Counting all 32 neurons together, the PTZ-induced epileptiform neuronal activities were reduced by 26.6 ± 5.9% (P < 0.05) on average after 1 mA VNS (Fig. 3B). Thus, the effect of 1 mA VNS was predominantly inhibitory. At 3 mA VNS, an inhibition of neuronal epileptiform activity ranging from 10.3% to 67.1% (mean ± S.E. 33.9 ± 6.5%) was seen in 9 out of 20 neurons tested. Four neurons showed excitation by 3 mA VNS, ranging from 12.2% to 55.8% (38 ± 9.2%), whereas the remaining 7 neurons had little change. Overall for the 20 neurons, there was a 7.7 ± 7.1% reduction on average of epileptiform activities after 3 mA VNS (P > 0.05) (Fig. 4C). In comparison with 1 mA VNS, 3 mA VNS produced less inhibition on the thalamic epileptiform activities (P < 0.05).
Fig. 4. An example of the effect of 3 mA stimulation on PTZ-induced epileptiform activities in a same thalamic neuron. (A) PTZ led to the vigorous epileptiform neuronal activities; (B) 3 mA EA stimulation of “Dazhui” acupoint inhibited the epileptiform neuronal activities; (C) 3 mA VNS also inhibited the neuronal activities.
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After 1 mA EA stimulation of “Dazhui” acupoint, the PTZinduced epileptiform activities were inhibited in 13 out of 29 neurons (44.8%) tested, with a decrease of 48.2 ± 9% on average (range 10.2–96.7%). Six neurons (20.7%) showed excitation with an increase of 39.7 ± 6.5% (range 19.5–61.7%) while the remaining 10 neurons (34.4%) had little change. Overall, the inhibition rate for all 29 neurons together was 13.5 ± 7.7% on average at this intensity, and it was statistically significant (P < 0.05) in comparison with the pre-treatment control (Fig. 3C). In comparison with 1 mA VNS, the effect was comparable, with no statistically significant difference between the two groups (P > 0.05). At 3 mA EA stimulation, an inhibition ranging from 23.8% to 59.2% was seen in 10 out of 20 neurons (50%), with an inhibition rate of 48.2 ± 5.2% on average. Eight neurons (40%) showed excitation ranging from 10.2% to 143.2% with an increase of 54.9 ± 17.5% on average. The remaining 2 neurons showed little change. Overall, there was a 1.8 ± 13.3% reduction (P > 0.05) on average of the epileptiform activities in all 20 neurons together after 3 mA EA (Fig. 4B). This overall effect was not significantly different from that of 1 mA EA, nor from that of VNS at the same intensity, i.e. 3 mA (P > 0.05). In summary, the current work demonstrated that the VB thalamic neurons displayed epileptiform activities after PTZ injection. VNS and EA had a mixed effect but predominantly inhibited the PTZ-induced epileptiform activities in the VB neurons, suggesting that both VNS and EA could bring about antiepileptic effect by, at least partial inhibition at the level of the thalamus. However, the higher intensity stimulation of either VNS or EA was not associated with a greater level of inhibition. The thalamic cells responded to either VNS or EA in a similar way, suggesting that EA could be a good alternative to VNS for acute abortion of epilepsy, at least in this animal model. The results were consistent with what we observed in the cerebral cortex [17], and support our hypothesis that the VB thalamus plays an important role in the antiepileptic effect of either VNS or EA. In this study, the neurons tested were predominantly located in the thalamic VB nucleus as judged by the response characteristics of the neurons and by the histological verifications. Thus, the VB thalamus appears to be involved in certain forms of epilepsy. This is in line with some of the previous reports [1,7,12] and clinical observations that some seizures are preceded by a variety of auras including sensory illusion and hallucinations. Of course, other thalamic nuclei might also take part, as shown by a recent long-term follow-up study [6] in which electrical stimulation of the anterior nucleus of thalamus produced a significant reduction of seizures. An early study showed that stimulation of the thalamic midline structures could induce cortical responses very similar to the spiky wave discharges of generalized absence epilepsy in association with an underlying thalamocortical oscillation [8]. The fact that VB neuronal response to EA and VNS was predominantly inhibitory posed the possibility that they could interrupt the genesis and spread of epileptiform activities at this subcortical gate to the cerebral cortex. At present, it is not entirely clear where this inhibition is initiated. Though the thalamus might be related to epileptic activities, we do not at all imply that the thalamus is the only important subcortical site that mediates epilepsy. Furthermore, there are many types of epilepsy that may have different mechanisms. Other potential sites include the hippocampus, amygdale and the nucleus of solitary tract (NTS). Since the latter is a primary site for convergence of vagal afferents and afferents from facial, scalp and auricular regions via trigeminal, cervical spinal and glossopharyngeal nerves [2], it could be an important structure involved in the antiepileptic effects of EA and VNS, and thus could be a potential candidate for further investigations. The antiepileptic effect observed in this study was instant and short-lived. The acute inhibitory effect is in line with pre-
vious observations that reduction of the interictal epileptiform discharges in human subjects was more prominent during the actual VNS period and the first interstimulation period [4]. This acute effect is most likely due to mobilization of the inhibitory circuit in the CNS. After cessation of either VNS or EA stimulation, the epileptiform thalamic activities may recover gradually. For example, Fig. 3D shows that the epileptiform activity returned about ∼13 min after cessation of EA. Mechanisms other than direct mobilization of the inhibitory circuit must also play a role too as in most cases VNS only gradually achieves a progressive prophylactic effect clinically, suggesting the involvement of various neurotransmitters, neuromodulators, cytokines or even morphological changes [10]. In the current experimental setting, repeated testing was difficult to carry out since the recovery time was variable and the later tests became somewhat unreliable. Chronic recording may circumvent this problem to a certain extent. The somatic RFs of many thalamic neurons recorded did not cover the location of acupoint GV14 in the current study. This observation suggests that the meridian theory, which is an important foundation for acupuncture practice, cannot simply be explained by somatotopical organization in modern neuroanatomical terms. Nevertheless, in the practice of Chinese medicine, acupuncture is often applied to a set of acupoints that can be remote from the diseased site and bear little relationship with nerve trajectory or convergence. For epilepsy treatments, acupoints from all over the body can be selected, though GV14 is one of the most commonly used. Although the anatomical trajectory for the interaction is not exactly clear, it is conceivable that EA may exert effects on a wide range of brain structures that may directly or indirectly affect thalamic neuronal responses. In this study, fixed frequency was used because clinic evidence has indicated that VNS at 20–30 Hz was most effective for the management of epilepsy. This is somewhat different from acupuncture analgesia where different EA frequencies would invoke different mechanisms; for example, low frequency EA chiefly leads to the release of enkephalin to induce analgesia via and ␦ receptors at the supraspinal levels, whereas high frequency EA (100 Hz) works by stimulating dynorphin release acting on receptors in the spinal cord. So far there is no evidence suggesting that different frequency of VNS would invoke different mechanisms. In further studies, however, the effects of different frequencies of VNS and EA could be investigated, and neurochemical analysis should be carried out to examine molecular mechanisms of these treatments. With regards to the stimulation intensity, our results indicated that EA at either low or high intensity produced similar effects in terms of the extent of inhibition and the proportion of neurons inhibited. For VNS, however, low intensity stimulation gave rise to significantly stronger inhibition in comparison with high intensity in both the extent of inhibition and the proportion of neurons inhibited. It was also noted that for both VNS and EA, high intensity (3 mA) also caused more excitation of thalamic neurons than 1 mA in terms of the discharge rate (EA: 55% vs. 40%; VNS: 38% vs. 32%), as well as the proportion of neurons excited (EA: 40% vs. 21%; VNS: 20% vs. 13%). Thus, it appears that high intensity stimulation could activate not only inhibitory circuits but also excitatory circuits to some extent. This observation is in line with the clinical observation that increased VNS current has weak and insignificant correlation with seizures reduction but more side effects, which could be due to activation of high threshold unmyelinated C fibers [5]. As the antiepileptic effects of VNS are assumed largely due to the activation of myelinated fibers rather than unmyelinated C fibers in the cervical vagus nerve, 3 mA might cause activation of more unmyelinated fibers than 1 mA and might in turn offset to some extent the beneficial effect of VNS. Thus, care should be taken clini-
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cally in deciding the parameters of VNS in order to achieve the best curative efficacy with minimal side effects. It is interesting that EA, in comparison with VNS, is less likely to activate excitatory neurons at the higher intensity. This is perhaps due to the fact that the EA is not directly applied on a nerve truck as VNS is. Clinical EA application is usually not painful, and is unlikely to activate nociceptive C fibers. In contrast, the vagus nerve contains a large proportion of unmyelinated C fibers that could be readily activated by direct electrical stimulation at high intensities. As the vagus nerve trunk of the rat is far finer than that of humans, 3 mA intensity could well activate all nerve fibers in it. Based on the current data, we conclude that both VNS and EA might acutely inhibit PTZ-induced epileptiform activities in the VB thalamus that appears to be involved in epilepsy. Taken together with our recent observation that EA was similarly effective as VNS in inhibiting epileptiform activities in the cerebral cortex [17], and with the concern of potential side effects, comfort and the financial costs of VNS, it appears that acupuncture may be a good alternative therapy to VNS in the management of epilepsy. Nevertheless, much more work is still needed to document its clinical efficiency, particularly in chronic settings, and to understand the mechanisms of the acupuncture treatment. Acknowledgements This work was supported by HKBU FRG/04-05/I-16 and FRG/0506/II-55. References [1] P.K. Banerjee, O.C. Snead, Thalamic mediodorsal and intralaminar nuclear lesions disrupt the generation of experimentally induced generalized absencelike seizures in rats, Epilepsy Res. 17 (1994) 193–205. [2] Y.O. Cakmak, Epilepsy, electroacupuncture and the nucleus of the solitary tract, Acupunct. Med. 24 (2006) 164–168.
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