Roles of the Frontal Cortex and Subcortical Structures in the Mechanisms Underlying the Development of Pilocarpine-Induced Seizures in Rats N. V. Kopiyova1 Received May 27, 2014. We studied the pathogenetic role of central structures belonging to epileptogenic and antiepileptogenic systems, namely the ventral hippocampus (VH), superior colliculus (SC), and frontal cortex, in the pathogenesis of pilocarpine-induced status epilepticus (with the presence of spontaneous seizures, SSs). Using a stereotaxic technique, the above-mentioned structures were subjected to preliminary local destruction (by microinjections of ibotenic acid) or electrostimulation in the course of the experiment. Pilocarpine-induced SSs were readily recorded in rats subjected to stimulation of the VH and frontal cortex, as well as to destruction of the SC. At the same time, in rats with destruction of the VH and frontal cortex but with activation of the SC, we observed no SS episodes. Therefore, the above-mentioned central structures play significant pathogenetic roles in the mechanisms underlying the development of the pilocarpine-induced epileptic state; the VH and frontal cortex are in reciprocal relations with the SC.
INTRODUCTION Injection of pilocarpine (PC) leads to the development of a chronic seizure syndrome with ictal convulsive manifestations, interictal behavioral changes, and emotional and cognitive dysfunctions in animals, which can, in the future, be transformed into an acute convulsive stage (development of the status eplepticus) with spontaneous seizures (SSs) . This attracts special attention of researchers to a cholinergic model of chronic epilepsy in order to elucidate pathophysiological mechanisms of chronic epileptogenesis [2-4]. It has been demonstrated that the midline thalamic nuclei (mediodorsal and connective) are involved in the development of spontaneous PC-induced seizures [5, 6]. The question of pathogenesis of SSs under conditions of this experimental model is complicated and remains little studied. There are grounds to believe that certain subcortical structures and separate cortical areas are most likely involved in the process of formation of SSs. Scrupulous experimental examination of pathogenetic mechanisms of SSs Odessa National Medical University, Ministry of Public Health of Ukraine, Odessa, Ukraine. Correspondence should be addressed to N. V. Kopiyova ([email protected]). 1
can promote the development of sophisticated and complex pathogenetically directed therapy of this form of chronic convulsive syndrome. We studied the role of cerebral structures belonging to the epileptogenic and antiepileptogenic systems, namely the ventral hippocampus (VH), superior colliculus (SC), and frontal cortex, in the pathogenesis of PC-induced SSs.
METHODS Chronic experiments were carried out on Wistar male rats under conditions of chronic experiments. Under ketamine anesthesia using coordinates of the stereotaxic atlas, we performed local destruction (provided by microinjections of ibotenic acid) and electrical stimulation (60 sec –1, 0.1 msec, 400-450 µA, duration 1 sec) of the VH, SC, and frontal cortex in the above rats. Pilocarpine was diluted with NaCl solution (pH 7.4) immediately prior to the beginning of experiments and i.p. injected (dose 380 mg/kg). Thirty minutes before this procedure, we injected M-scopolamine (1.0 mg/kg, subcutaneously) to prevent the development of peripheral cholinomimetic effects. The behavioral phenomena
282 (seizure activity in particular) of animals were visually controlled during 42 days from the moment of PC injection. To decrease the lethality, the rats were injected i.p. with 10 mg/kg diazepam 45 min after cessation of electrical stimulation. The development of SSs in rats injected with PC was confirmed only in the case where at least one SS episode during the observation period was noticed. The animals were divided into the following groups (18 rats in each): group 1, control (injection of PC); group 2, destruction of the VH + PC; group 3, stimulation of the VH + PC; group 4, destruction of the SC + PC; group 5, stimulation of the SC + PC; group 6, destruction of the frontal cortex + PC, and group 7, stimulation of the frontal cortex + PC. The data obtained were treated statistically using parametric (one-variant ANOVA accompanied by the Newman–Keuls post-hoc test) and nonparametric (Kruskal–Wallis test) criteria. Differences were considered significant at P < 0.05.
RESULTS AND DISCUSSION During the first 2-5 min after injection of PC, autonomic disturbances (hypersalivation, intensification of grooming, sniffing, tachypnea, and more frequent acts of defecation) and increased anxiety and motor activity prevailed in most rats of all groups. Then, on the 12th to 25th min, all animals demonstrated seizures; the latter were initially manifested as myoclonic flinching and tremor of muscles of the muzzle, head, and forelimbs. During the subsequent 7-15 min, such symptoms were transformed into clonic contractions of muscles of the body, forelimbs, and hindlimbs; 80
12 11 10 9 8 7 6 5 4 3 2 1 0
70 60 50 40
then, generalized clonico-tonic attacks developed in all animals. The maximum duration of an acute stage of the PC-induced status epilepticus (SE) was observed in rats of the control group (72.3 ± ± 6.7 min) and in animals with activation of the VH, destruction of the SC, and activation of the frontal cortex (Fig. 1). Values of the above-mentioned index were significantly smaller in animal groups with destruction of the VH (P < 0.01), activation of the SC (P < 0.001), and destruction of the frontal cortex (P < 0.001; Fig. 1). In 12 of 18 rats of the control group, which survived after the acute SE stage, spontaneous convulsive reactions were observed during the subsequent 20-25 days; such reactions were manifested as mild low-amplitude myoclonic seizures (muscle contractions in the forelimbs). The development of SSs was also observed in rats with activation of the VH, destruction of the SC, and activation of the frontal cortex (Fig. 2). None of the rats belonging to the group with VH destruction, activation of the SC, and destruction of the frontal cortex demonstrated episodes of SSs during 35 days of the observation period (P < 0.01; Fig. 2). The mean numbers of SS episodes in rats with activation of the VH, destruction of the SC, and activation of the frontal cortex were, on average, 7, 6, and 5, respectively, while the mean duration of such episodes was 36 ± 6 sec (Fig. 2). Therefore, the obtained data allow us to interpret some pathophysiological mechanisms underlying the formation of SSs induced by pilocarpine. Recently, we reported that these seizures were formed during 20-25 days, on average; the degree of their development demonstrated a close relationship
F i g. 1. Duration (min) of an acute stage of the pilocarpine-induced epileptic state under experimental conditions. Horizontal scale) Animal groups 1-7 correspond to those mentioned in the text. **P < 0.01 and ***P < 0.001, cases of significant differences of the examined index compared with that in the control.
** ** 2
** ** ** ** 5
F i g. 2. Characteristic of pilocarpine-induced spontaneous seizures (SSs) under experimental conditions. Horizontal axis) Animal groups 1-7 (according to the groups listed in the text). Open columns correspond to the number of rats that demonstrated SSs; filled columns correspond to the number of episodes of SSs. Other designations are the same as in Fig. 1.
Roles of Various Brain Structures in Generation of Pilocarpine-Induced Seizures to the duration of the acute stage of the pilocarpineinduced SE . The above-described data show that the process of spreading of PC-induced convulsive activity is mediated by subcortical structures and a frontal part of the cerebral cortex. This conclusion is confirmed by the characteristics of the development of SSs under conditions of activation of the VH and frontal cortex and destruction of the SC, as well as by a longer duration of PC-induced SE in rats of the above-mentioned groups; these effects were comparable with the respective indices in control rats. Opposite results were obtained in the case of destruction of the VH and frontal cortex and activation of the SC. These data are intriguing from the aspect of the theory of systemic mechanisms underlying the development of neuropathological syndromes, including convulsive ones . We should take into account that the VH belongs to structures of the epileptogenic system, while the SC belongs to the antiepileptogenic one . Dynamic shifts of the intensity of functional activity of these structures are indices of the power of chronic convulsive activity . The pathogenetic role of the midline thalamic nuclei in the generation of SSs under conditions of the PC-induced model of chronic epileptic activity  allows us to postulate that this pathological process is mediated by the frontal cortex. It was demonstrated that there are thalamo-cortical synaptic projections to layers I, III, and VI of the prefrontal cortex; terminal projections of the midline thalamic nuclei form a diffuse neuronal network in the cortex . It is known that reciprocal projections return to the thalamus from layers ІІ and V of the prefrontal cortex and in such a way form a neuronal mechanism responsible for thalamic recruit rhythms . The latter modulate the functional state of the cerebral cortex in toto . Therefore, injections of PC induced the development of the SE in rats; this was characterized by the appearance of acute seizures and delayed spontaneous convulsive reactions. Pilocarpineinduced seizure reactions were recorded in rats after activation of the VH and frontal cortex, as well as after destruction of the SC. No SS episodes were recorded in rats subjected to destruction of the VH
and frontal cortex and to stimulation of the SC. The state of the VH, VHМ, and frontal parts of the cerebral cortex plays a significant pathogenetic role in the mechanisms underlying the development of pilocarpine-induced SSs. Experiments were carried out in accordance with the International Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1985), as well as with the regulations of the Ethics Committee of the Odessa National Medical University (Ministry of Public Health of Ukraine).
REFERENCES 1. L. Turski, C. Ikonomidou, W. A. Turski, et al., “Cholinergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy,” Synapse, 3, No. 2, 154171 (1989). 2. L. R. Zenkov, Clinical Epileptology (with Elements of Neurophysiology) [in Russian], OOO Med. Inform. Agency, Moscow (2002). 3. O. A. Shandra amd O. A. Kashchenko, “Models and main pathophysiological mechanisms underlying chronic epilepsy,” Dosyagnennya Biol. Med., No. 1, 80-88 (2003). 4. S. A. Chepurnov, N. E. Chepurnova, O. M. Redkozubova, and S. A. Saakyan, “Status epilepticus – novel mechanisms and pathways of inhibition (lithiumpilocarpine model),” Usp. Fiziol. Nauk, 36, No. 1, 68-84 (2005). 5. R. S. Vast’yanov and N. V. Kopiyova, “Examination of the role of midline thalamic nuclei in the mechanisms of the development of spontaneous seizures,” Zdobutky Klin. Éksp. Med., No. 1 (14), 118-120 (2011). 6. O. A. Shandra and N. V. Kopiyova, “Pathophysiological mechanisms of the development of spontaneous seizure activity,” Visn. Psikhiatr. Psikhofarmakoter., No. 2 (14), 7-17 (2008). 7. L. Negyessy and P. S. Goldman-Rakie, “Morphometric characterization of synapses in the primate prefrontal cortex formed by afferents from the mediodorsal thalamic nucleus,” Exp. Brain Res., 164, 148-154 (2005). 8. D. M. Sloan and E. D. Bertram 3rd, “Changes in midline thalamic recruiting responses in the prefrontal cortex of the rat during the development of chronic limbic seizures,” Epilepsia, 50, No. 3, 556-565 (2009). 9. R. P. Vertes, “Differential projections of the intralimbic and prelimbic cortex in the rat,” Synapse, 51, No. 1, 52-58 (2004).