Thalamic neuropathology in the chronic pilocarpine and picrotoxin model of epilepsy

Thalamus & Related Systems 2 (2002) 49–53

Thalamic neuropathology in the chronic pilocarpine and picrotoxin model of epilepsy Clement Hamani∗ , Luiz E.A.M. Mello Depto. de Fisiologia da EPM-UNIFESP, São Paulo, SP 04023-900, Brazil Accepted 30 June 2002

Abstract Adult male Wistar rats were injected with 150/0.5, 75/1.5 and 50/2.0 mg/kg of pilocarpine (Pilo) and picrotoxin (PTX) (Pilo/PTX mg/kg). The vast majority of the animals developed status epilepticus (SE), after which they were observed for a period of 120–131 days for the occurrence of spontaneous recurrent seizures (SRS). After the experiments, animals were deeply anesthetized, perfused with a 10% formaldehyde fixative solution and their brains were processed with cresyl violet, Perls and Von Kossa techniques. Cell counts were performed under a regular microscopic grid in diverse anteroposterior levels of the thalamus. Several thalamic nuclei in the epileptic groups, particularly the central medial, central lateral, paracentral, mediodorsal, laterodorsal and lateroposterior, showed intense cell loss, pathologic calcification and iron tissue deposits. Our results are relevant to support the importance of the thalamus in the pathogenesis of the epilepsies. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Thalamus; Pilocarpine; Epilepsy; Epilepsy surgery; Temporal lobe epilepsy

1. Introduction

2. Materials and methods

There is considerable evidence that subcortical structures, particularly the thalamus, are critical for the production and expression of seizures in several models of epilepsy (Jasper and Droogleever-Fortuyn, 1946; Avoli and Gloor, 1982; Bertram et al., 2001; Cassidy and Gale, 1998; Patel et al., 1988). To date, however, most of the published papers addressing this issue, has dealt mostly with absence-like seizures (for a review see Snead, 1995). In our laboratory, we have recently developed a new model of epilepsy in which different dose combinations of pilocarpine (Pilo) and picrotoxin (PTX) were used, mimicking several aspects of human temporal lobe epilepsy (TLE) (Hamani and Mello, 1997, 2002). Notwithstanding, in addition to mesial temporal lobe pathologic features, status epilepticus (SE) induced by pilocarpine–picrotoxin generates a pronounced pattern of acute thalamic destruction (Hamani and Mello, 1997). Due to this intriguing finding, we hereby decided to further investigate the chronic aspects of the thalamic neuropathology in this model of epilepsy (Pilo/PTX).

Adult male EPM-l Wistar rats (150–250 g) were intraperitoneally injected with different dose combinations of pilocarpine and picrotoxin, dissolved together in a 20% ethanol solution (used instead of saline in order to dissolve the picrotoxin). From here on, the solutions used will described as follows: pilocarpine dose/picrotoxin dose (i.e. 150/0.5 mg/kg characterizes a solution containing 150 mg/kg of pilocarpine and 0.5 mg/kg of picrotoxin). Three different dose combinations effective in previous studies in inducing status epilepticus were used (150/0.5, 75/1.5, and 50/2.0 mg/kg) (Hamani and Mello, 1997). Animals injected with pilocarpine and picrotoxin that did not show SE were used as controls (n = 6), since in previous studies no differences were noticed between these animals and the ones injected exclusively with saline (Mello et al., 1993). Eighteen animals (six animals injected with each dose combination) that developed SE after Pilo/PTX and six controls were housed in groups of four and observed for 120– 131 days, 5 h per day, 5 days per week for the occurrence of behavioral spontaneous recurrent seizures (SRS). Assessment of SRS (partial or generalized) and general behavior was performed by a trained observer which recorded all the events displayed by the animals. Ictal events were classified according to Racine (Racine, 1972). A detailed analysis of the electroencelographic features concerning Pilo/PTX

∗ Corresponding author. Present address: 25 Wood Street PH05, Toronto, ON M4Y2P9, Canada. Tel.: +55-11-55792033; fax: +55-11-55792033. E-mail addresses: [email protected] (C. Hamani), [email protected] (L.E.A.M. Mello).

1472-9288/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 4 7 2 - 9 2 8 8 ( 0 2 ) 0 0 0 2 9 - 8

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SRS and the neuropathological aspects related to limbic structures can be found in detail elsewhere and will not be depicted in the present study (Hamani and Mello, 2002). 2.1. Neuropathology and stereological analysis After the above mentioned analysis, animals were deeply anesthetized with thionembutal (50 mg/kg i.p.) and transcardiacally perfused with 0.9% saline, followed by a 10% formaldehyde fixative solution. Coronal sections of 24 ␮m thickness were cut on a cryostat, collected in phosphate buffer, mounted in glass slides and stained with cresyl violet, Von Kossa and Perls techniques for further histological analysis. Cell counts were performed with 10× or 40× objectives, depending on the neuronal density of the analyzed thalamic structure. Counting was achieved with a regular microscopic grid and the cells that touched the lower and right sides of the grid were not considered. Rostrocaudal sections corresponding to each thalamic nucleus were selected according to Paxinos and Watson (1986). For every section, cell counts were performed three times in unilaterally represented nuclei and in both sides of the brain when those displayed a bilateral representation. The average was then considered. Since epileptic animals presented shrinkage in many brain regions (see Section 3 for detail), we have guided the choice of the sections to be analyzed based on structures that had little or no damage, such as the medial habenula and some hypothalamic nuclei. The area for every nucleus in each section was achieved with the stereologic point-counting method (Weibel, 1963) and the density of cells per volume was assessed with the formula Nv = Q/At, where Nv is the neuronal density, Q the number of neurons counted, A the area considered and t is the thickness of the section (West and Gunderesen, 1990). Quantitative results will be described as the percentage of cell loss obtained in epileptic animals when compared to age matched controls, according to the formula (1 − mean epileptic cell counts/mean control cell counts) × 100. Percentage of cell loss was chosen instead of the absolute cell count values for each nucleus and region in as much as this relation consists of a more attractive analysis, since the thalamic neuropathology displayed by the epileptic animals can easily be appreciated. 2.2. Thalamic subdivisions For the purpose of our study, the thalamus was subdivided in an anterior nuclear group, a medial nuclear group, a ventral nuclear complex, a lateral nuclear group, a posterior nuclear group, an intralaminar nuclear group and a midline nuclear group (Faull and Mehler, 1985). The anterior nuclear group was considered as a whole (AD, AV and AM). The medial nuclear group was represented basically by the mediodorsal nucleus (MD). The ventral nuclear complex (ventral tier nuclei) was represented by the ventromedial nucleus (VM), the ventrolateral nucleus (VL),

the ventroposterior posterior complex, comprising its medial (VPM) and lateral (VPL) subdivisions and the gelatinous nucleus (G). The lateral nuclear group was subdivided in a lateroposterior nucleus (LP) and a laterodorsal nucleus (LD). The posterior nuclear group was represented by the posterior nucleus (Po). The intralaminar nuclear group was subdivided in a rostral group (paracentral (PC) and central lateral (CL)), the central medial (CM) and the parafascicular nuclei (PF). The midline nuclear group was represented by two subdivisions; a dorsal group, composed by the paraventricular (PV), paratenial nuclei (PT), interanteromedial (IAM) and intermediodorsal (IMD) nuclei; a ventral group, composed by the rhomboid (Rh) and reuniens (Re) nuclei. The reticular nucleus (Rt), medial (MG) and lateral geniculate bodies (LG) were considered separately. 2.3. Statistical analysis The non-parametric Kruskall–Wallis test was used for the comparison of data among the three different dose combinations and the control group. Statistical significance was achieved when P ≤ 0.05. 3. Results Acute behavioral alterations that supervened immediately after Pilo/PTX injections were similar to previous descriptions (Hamani and Mello, 1997). Following the injections, animals developed mild signs of cholinergic overdose, such as salivation, piloerection and defecation, combined with tremor, akinesia and, in some cases, facial automatisms. Automatisms and tremors increased in intensity and frequency until the development of secondarily generalized tonic–clonic seizures. After 1–5 seizures, approximately 90% of the animals injected with Pilo/PTX developed SE. Following SE, animals went through a latent period (interval between SE induction and the first observed SRS), in which no behavioral epileptiform activity was noticed. A mean latent period of 62 days was observed when the epileptic groups were considered as a whole. Thereafter, a state of “chronic” epilepsy, characterized by the recurrence of the spontaneous seizures (SRS), ensued. During the observational SRS period a total mean of 0.70 ± 0.53 partial SRS per week and 0.76 ± 0.43 generalized seizures were recorded for the group of animals injected with 150/0.5 mg/kg. The group of animals injected with 75/1.5 mg/kg presented a mean of 0.23 ± 0.21 partial and 0.41±0.46 generalized SRS per week. Finally, the group of animals injected with 50/2.0 mg/kg presented a mean of 0.04 ± 0.05 partial and 0.13 ± 0.19 generalized SRS per week (Table 1). Almost all thalamic nuclei assessed in the three epileptic groups suffered some degree of cell loss. Nevertheless, the most affected ones were certain intralaminar nuclei, such as the central medial and the rostral group (central lateral and

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Table 1 Mean and standard deviation of the number of partial, generalized and total (partial + generalized) SRS per week displayed by the three different groups of epileptic animals utilized in our study 150/0.5 (mg/kg)

75/1.5 (mg/kg)

50/2.0 (mg/kg)

Generalized seizures Partial seizures

0.76 ± 0.43 0.70 ± 0.53

0.41 ± 0.46 0.23 ± 0.21

0.13 ± 0.19 0.04 ± 0.05

Total

1.46 ± 0.87

0.64 ± 0.61

0.17 ± 0.23

paracentral), the mediodorsal nucleus and both lateral group nuclei (laterodorsal and lateroposterior) (Fig. 1). In some of the epileptic animals this latter group presented not only a near-total cell loss but also dystrophic calcification, histologically confirmed with the Von Kossa technique (Fig. 2). Perls stained sections revealed small positive dots, denoting iron tissue deposits in the mediodorsal, laterodorsal, lateroposterior and certain intralaminar nuclei (Fig. 3). Both calcium and iron deposits were not found in brain structures other than the thalamus and were not related to any direct exposure of the animals to intracerebral foreign bodies. It is important to mention here that the photomicrographs displayed above represent cases in which extreme levels of tissue destruction were seen. Most of the epileptic animals presented a more constant cytoarchitectural cell loss, in which the thalamic nuclei studied could be easily counted. Percentages of cell loss were acquired by the simple relation between the stereologically corrected cell counts performed in the experimental groups and controls and are depicted in Table 2. Although statistically significant differences in cell counts could be observed in several nuclei when the epileptic groups were compared to controls, those seemed to be related to the intense cell loss depicted by the former animals. Thalamic nuclei that presented high percentages of cell loss always presented statistically significant results when compared to controls. When the three epileptic groups were compared however, we did not achieve significant statistics. Notwithstanding, it was interesting to note that three out of the several nuclei analyzed presented a SRS and cell loss profile that varied in a similar manner. Groups injected with 150/0.5, 75/1.5 and 50/2.0 mg/kg, which respectively presented decreasing seizure frequencies, have also presented statistically significant decreasing percentages of cell loss in the central medial (78, 51 and 25%, respectively), laterodorsal (98, 89 and 64%, respectively) and lateroposterior nuclei (91, 79 and 55%, respectively). Yet as mentioned above, despite of the large variations between some of the percentages of cell loss (i.e. 78% versus 25%), there was no statistically significant differences among the three experimental groups for any given nucleus. 4. Discussion Our results support a relevant role for the thalamus in the Pilo/PTX model of epilepsy. In the present study, almost

Fig. 1. Photomicrographs displaying the thalamus of a control (A) and a chronic epileptic rat injected with 75/1.5 mg/kg of Pilo/PTX (B). In the epileptic rat, severe tissue destruction, necrosis, and dystrophic calcification were largely observed in the lateroposterior and laterodorsal thalamic nuclei (arrow heads), as well as in the intralaminar thalamic nuclear group (arrows). A higher magnification of another Pilo/PTX 75/1.5 mg/kg epileptic animal illustrates the above mentioned findings (C). Sections stained with cresyl violet. Scale bar: 500 ␮m (A, B); 200 ␮m (C).

all thalamic nuclei assessed in the epileptic groups suffered some degree of cell loss. In fact, several regions were severely affected, presenting in addition dystrophic calcification. It is difficult to hypothesize, based in our findings, the actual role of the thalamus in the epileptogenesis of the Pilo/PTX model of epilepsy and any attempt to fully

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C. Hamani, L.E.A.M. Mello / Thalamus & Related Systems 2 (2002) 49–53 Table 2 Percentage of neuronal loss in Pilo/PTX chronic epileptic animals when compared to age matched controls Thalamus 150/0.5 (mg/kg) 13

16

17 (0.09)

Medial nuclear group MD (%)

78

90

60 (0.0001)

Ventral nuclear complex VM (%) VL (%) VPM (%) VPL (%) G (%)

8 13 0 26 49

15 17 18 22 51

12 16 31 18 51

Lateral nuclear group LP (%) LD (%)

91 98

79 89

55 (0.0001) 64 (0.0001)

−10

8

21 (0.068)

73 78 39

50 51 26

52 (0.0001) 25 (0.005) 44 (0.0001)

26

29

28 (0.025)

6 40 34 17

22 13 22 10

14 10 26 21

Intralaminar nuclear group Rostral group (PC, CL) (%) CM (%) PF (%)

Fig. 3. Photomicrograph displaying small iron deposits in the mediodorsal thalamic nucleus of a chronic 150/0.5 mg/kg Pilo/PTX epileptic rat (arrows). Section stained with the Perls technique. Scale bar: 100 ␮m.

50/2.0 (mg/kg)

Anterior nuclear group AD, AV, AM (%)

Posterior nuclear group Po (%)

Fig. 2. Photomicrographs revealing pathologic calcification in the lateroposterior, laterodorsal and the intralaminar thalamic nuclear group of a chronic 150/0.5 mg/kg Pilo/PTX epileptic rat (A). Higher magnification of the same animal displaying calcium deposits in the lateroposterior nucleus (B). Sections stained with the Von Kossa technique. Scale bar: 500 ␮m (A); 200 ␮m (B).

75/1.5 (mg/kg)

Midline nuclear group Dorsal group (PV, PT, IAM, IMD) (%) Ventral group (Rh, Re) (%) Rt (%) MG (%) LG (%)

(0.25) (0.02) (0.005) (0.011) (0.005)

(0.36) (0.024) (0.001) (0.017)

Results were obtained according to the formula (1 − epileptic cell counts/control cell counts) × 100. P-values are represented within parenthesis and were achieved with the non-parametric Kruskall–Wallis test. Negative values represent situations in which the epileptic group presented a higher number of cells than the control one.

elucidate this issue would be merely speculative at this point. In this sense, the central purpose of our study was mainly to provide a detailed description of the thalamic neuropathology in this new model of epilepsy. Despite of the generalized neuropathological alterations mentioned above, our findings have shown that the experimental groups injected respectively with 150/0.5, 75/1.5 and 50/2.0 mg/kg presented both, a decreasing seizure frequency and a statistically significant decreasing percentage of cell loss in the central medial, laterodorsal and lateroposterior nuclei. The central medial thalamic nucleus has been reported as a potent inhibitor of the epileptiform activity in other models of epilepsy (Miller et al., 1989; Miller and Ferrendelli, 1990). In the clinical setting, the electrical stimulation of the centromedian thalamic nucleus has been used for the treatment of difficult-to-control epilepsy in patients presenting multifocal seizures, and was initially described as capable of providing substantial reductions in the number of generalized tonic–clonic motor events (Velasco et al.,

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1995). Controlled studies, however, have failed to show statistically significant sustained seizure reduction, although improvements in seizure frequency of as much as 30% have been reported (Fisher et al., 1992). In the current experiment, both, the laterodorsal and lateroposterior thalamic nuclei, presented extremely high indexes of cell loss and tissue destruction, as well as pathologic calcification. Autoradiographic studies using the pilocarpine model of epilepsy have suggested that the lateroposterior thalamic nucleus might be involved in inhibitory circuits during interictal intervals (Scorza et al., 1998). Our results support this assertion since animals in which elevated cell loss was observed in the lateroposterior nucleus also displayed a high frequency of seizures, which is consistent with the destruction of a structure possibly involved in seizure inhibition. Another pathologic finding observed in our study that deserves particular attention is the presence of iron tissue deposits in the mediodorsal, laterodorsal, lateroposterior and certain intralaminar thalamic nuclei. Various reports have suggested that iron tissue deposits, resultant from microhemorrhages, might occur either after SE or after each particular SRS, contributing to the development and perpetuation of the epilepsy induced in some animal models (Kabuto et al., 1992; Sharma and Singh, 1999). In summary, our study revealed an important thalamic compromise in the Pilo/TX model of epilepsy. Despite of the pivotal role attributed to the hippocampus in human temporal lobe pathology, the seminal work of Margerison and Corsellis in the late 1960s has already focused on the occurrence of thalamic lesions in many of these patients (Margerison and Corsellis, 1966). Therefore, we believe that the rather neglected role of the thalamus in epilepsies other than absence seizures should be reconsidered, since the study of the thalamus as a key cerebral structure concerning its global interactions with ascending and descending projections (McCormick, 1989; Sherman and Koch, 1990; Parent and Hazrati, 1995), might be of great relevance for the understanding of epileptogenesis in TLE and in several other forms of epilepsy. Acknowledgements Supported by PRONEX, FAPESP, CNPq; CH was a fellow from CAPES. Pilocarpine was a gift from Merck-Quimitra (Brazil). The authors wish to thank Ivone de Paulo for the technical assistance. References Avoli, M., Gloor, P., 1982. Interaction of cortex and thalamus in spike and wave discharges of feline generalized penicillin epilepsy. Exp. Neurol. 76, 196–217. Bertram, E.H., Mangan, P.S., Zhang, D.X., Scott, C.A., Williamson, J.M., 2001. The midline thalamus: alterations and a potential role in limbic epilepsy. Epilepsia 42, 967–978.

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