Address correspondence and reprint requests to Dr. H.G. Niessen at Otto-von-Guericke University Magdeburg, Department of Neurology II, Leipziger Str. 44, 39120 Magdeburg, Germany. E-mail: firstname.lastname@example.org
Summary: Purpose: After pilocarpine-induced epilepsy in rats, volumetric magnetic resonance imaging (MRI) reveals significant morphologic changes in functionally relevant structures of the brain. To relate structural changes to functional alteration, we studied the correlation of regional brain atrophy (e.g., of the hippocampus) with lesion-induced learning deficits in the Morris water maze.
Methods: MRI experiments were performed on an MR scanner at 4.7 Tesla. For volumetric analysis, various cerebral structures were segmented in horizontal and coronal T2-weighted MR images. Before the MRI investigations, animals were trained for 10 days in a Morris water maze.
Results: Volumetric MRI revealed a significant loss in hippocampal size in both the dorsal and ventral parts, correlated with an increase in ventricular size. Furthermore, significant losses were found in the relative size of thalamus, putamen, cortex, and the combined areas of perirhinal, entorhinal, and piriform cortices adjacent to the hippocampus. A significant correlation of learning performance in the Morris water maze with the relative hippocampal area and not with other areas tested was observed in pilocarpine-treated animals.
Conclusions: The data provide a quantitative analysis of functionally relevant structural alterations in rats with chronic epilepsy. Water maze performance of pilocarpine-treated animals correlates with the degree of hippocampal but not with the degree of cortical damage, demonstrating the potential of this method for the investigation of cognitive impairments in relation to cerebral changes. In addition, the data point to an important role of even the residual hippocampus in memory formation.
Pilocarpine-induced epileptic seizures in rats, a frequently used animal model to study the underlying mechanisms of human temporal lobe epilepsy (TLE) (1–4), have yielded much valuable information with respect to epilepsy-related behavioral changes (5), neurochemical changes (6,7), and pathologic alterations (8,9). Besides other behavioral disturbances, the learning performance of pilocarpine-treated animals is strongly impaired (10). The pilocarpine treatment provokes the onset of a status epilepticus (SE). After a “latent” phase of ∼2 weeks, the animals develop spontaneous recurrent seizures (SRS) lasting for their whole life. The seizure activity observed in human TLE is best represented by the rat's later chronic phase.
Clinical studies have used magnetic resonance imaging (MRI) to elucidate and quantify various structural changes caused by epileptic seizures in human TLE (11–13). Important parameters in the quantification of such alterations are T2-relaxation times (relaxometry), apparent diffusion coefficients (ADC), and the volumetric segmentation of certain brain areas. So far, volumetric MRI studies in humans have been used successfully in the assessment of damage in the hippocampus and the amygdala (14–16), but mostly focusing on the hippocampus.
In contrast to the expanding literature on MRI in human epilepsy research, only a sparse number of contributions exist for related epilepsy models in rats, such as pilocarpine-induced (in combination with lithium or without) or kainic acid–induced epileptic seizures. Although affected structures are known to some extent, a detailed quantitative MRI-based analysis of these structural alterations and especially their relation to behavioral impairments in the corresponding pilocarpine rat model of TLE is still lacking. So far, the development of the prominent hippocampal atrophy has been described in detail for the lithium pilocarpine model of human TLE (17,18).
Here, we describe in detail structural alteration and damage in various regions of the rat brain caused by pilocarpine-induced epilepsy. The volumetric analysis was performed for a number of structures, such as hippocampus, putamen (striatum), thalamus, cortex, tectum, and cerebellum, thereby expanding the current basis of volumetric MRI data in the literature. Furthermore, we show that the degree of the hippocampal atrophy correlates with lesion-induced learning deficits in the Morris water maze (19).
For the generation of the animal model of epilepsy, male Wistar rats (n = 5, 32–36 days of age; 120–170 g; Charles River, Germany) were injected with pilocarpine (340 mg/kg i.p.; Sigma-Aldrich, Deisenhofen, Germany) 30 min after the injection of methylscopolamine (1 mg/kg i.p.; Sigma-Aldrich). All pilocarpine-treated rats showed an onset of seizure activity 20–40 min after pilocarpine administration. After 40 min, the SE was terminated by injection of diazepam (DZP; 4 mg/kg i.p.; Sigma-Aldrich). Control rats (n = 6) were injected with methylscopolamine, DZP, and physiologic salt solution instead of pilocarpine. Video monitoring was performed during the 12-h light phases for an average of 125 h between day 20 and day 30 after pilocarpine injection to detect the occurrence of spontaneous seizures. Only treated animals that developed spontaneous seizures after 20 days were used for behavioral and MRI experiments.
Water maze learning trials were performed between days 30 and 40 after pilocarpine injection. After an initial habituation trial, animals had to find a hidden platform for 10 days, with six trials per day. Maximum trial duration was 60 s; the platform sojourn time was 30 s. The platform stayed at the same location for each animal. Rats that did not find the platform were gently placed on it after 60 s. The intertrial interval was 60 s.
For the subsequent in vivo MRI experiments, rats were anesthetized with 1.5–2% isoflurane (in 70:30 N2O:O2; volume ratio) and secured by using a head-holder with bite bar to reduce motion artifacts. MRI experiments were performed on a Bruker Biospec 47/20 scanner at 4.7 T (free-bore diameter of 20 cm) equipped with a BGA 12 (200 mT/m) gradient system. For sample excitation, a 72-mm birdcage resonator was used; for detection, an anatomically shaped 3-cm surface coil was used. Eight coronal and six horizontal T2-weighted spin-echo images were measured consecutively by using a rapid-acquisition relaxation-enhanced sequence (RARE) (20) with the following parameters: TR, 2,000 ms; TE, 15 ms; slice thickness, 1 mm; distance between slices, 0.2 mm; FOV, 30 × 30 mm; matrix, 256 × 256; RARE factor, 8; number of averages (NEX), 16. The total scanning time was 17 min for each horizontal and coronal set of MR images. All of these animal experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Volumes and areas of the various brain structures were measured on the MR images by using the public domain Java-based imaging-processing and -analysis program ImageJ (http://rsb.info.nih.gov/ij/). For the analysis of structural changes within a certain horizontal section of the rat brain, the area of the selected structures was divided by the whole cranial area of the brain, yielding a ratio that is independent of the animal's brain size (21). For volumetric analyses of the hippocampus, total volumes were obtained by combining the results from a series of MRI slices. All structures were segmented in accordance with the rat anatomy atlas (22). The resulting data are expressed as mean ± SD. Correlation between variables was first obtained by calculating nonparametric Spearman tests. When nonparametric correlations were found to be significant, parametric Pearson correlation coefficients (rp) between variables were chosen for graphic and statistical representation. The statistical significance of differences between controls and the epileptic rats was evaluated by using a multivariate analysis of variance (MANOVA). To determine whether significant associations were present between the size of a certain brain structure and water maze performance of pilocarpine-treated animals, stepwise multiple correlation analyses were performed with the sum of latencies during first daily trials, and during daily trials 2–6 as dependent variables, and brain area sizes as independent variables. All correlation analyses were calculated by using SPSS for Windows; p values of <0.05 were considered significant.
T2-weighted MRI was used to elucidate structural changes during the chronic phase of epilepsy 60 days after pilocarpine-induced SE. A horizontal slice (bregma position, −3.7 mm) of the rat brain was segmented into the structures of hippocampus (Hc), ventricles (Ve), putamen (Pu), cortex (Co), cerebellum (Cb), and tectum (Te), excluding the olfactory bulb (Fig. 1). For the specified brain structures, the ratio between the area of the respective structure and total cranial area was calculated (Table 1). With this straightforward method, an animal-independent quantification was obtained (21).
Table 1. Relative areas of the brain structures segmented inFig. 1as ratio between the structure's area and total cranial area
Relative area (%), control (n = 6)
Relative area (%), pilocarpine-treated (n = 5)
Relative change (%)
Values (mean ± SD) for ventricles, hippocampus, putamen, tectum, and cerebellum are taken from the section shown in Fig. 1; values for the thalamus (Th) from an adjacent section (bregma position, −4.9 mm, not shown). Values for total cortex and combined area for perirhinal (Pr), entorhinal (En), and piriform (Pi) cortices are taken from the coronal slices (bregma position, −3.6 mm) shown in Fig. 2C and D.
ap < 0.001; bp < 0.05; cp < 0.01.
2.18 ± 0.32
9.24 ± 1.42
16.65 ± 0.84
11.23 ± 0.61
9.90 ± 0.61
8.56 ± 0.76
7.26 ± 0.46
6.40 ± 0.47
10.61 ± 0.59
11.35 ± 0.96
25.20 ± 0.63
24.44 ± 1.53
Cortex (Co), total
40.79 ± 1.17
35.92 ± 1.84
Pi, Pr, En cortices
13.32 ± 0.35
8.32 ± 0.74
In addition, comparison of coronal (bregma position, −3.6 mm) and horizontal (bregma position, −7.3 mm) brain sections of a control (Fig. 2A and C, respectively) and a pilocarpine-treated rat (Fig. 2B and D) revealed major damage in the perirhinal (Pr), ectorhinal (Ec), entorhinal (En) (Fig. 2B), and piriform (Pi) (Fig. 2D) cortices after recurrent epileptic seizures. By using the coronal slice shown in Fig. 2C and D, cortical areas for the total cortex (Co) and the combined area containing perirhinal (Pr), entorhinal (En), and piriform (Pi) cortices were calculated. By using the rhinal fissure (rf; Fig. 2E and F) as an anatomic landmark, the specified cortices can be reliably located.
Table 1 shows the data obtained from the brain regions shown in Figs. 1 and 2E and F. Significant changes in relative sizes of hippocampus, putamen, cortex, the combined area of perirhinal, entorhinal, and piriform cortices, and thalamus were observed after pilocarpine treatment; the values for the change in thalamic area (Th) were obtained from an slice adjacent to the one shown in Fig. 1 (bregma position, −4.9 mm; segmentation not shown). Although a moderate decrease in relative size of the putamen (F= 7.58; p = 0.025) and the thalamus (F= 7.15; p = 0.028) was observed with almost similar significance, the changes in relative size of the cortex (F= 24.90; p = 0.0011) and the combined area of perirhinal, entorhinal, and piriform cortices (F= 187.65; p < 10−6) are more pronounced. In addition, for the ventricles and the hippocampus, a highly significant increase (F= 214.72; p < 10−6) and decrease (F= 166.94; p < 10−5) in relative size was observed. For cerebellum (F= 1.49; p = 0.26) and tectum (F= 3.24; p = 0.11), no significant difference between controls and pilocarpine-treated rats was found, leaving these structures as unaffected control areas. Further analysis revealed a significant correlation (rp=−0.95; p = 0.013) between the loss in relative hippocampal area and the increase in relative ventricular area (by using the data from Table 1); however, no correlation with the increase in ventricular size was found for any other structure. The hippocampus–ventricle correlation suggests that the expansion of the ventricles can be attributed to hippocampal atrophy (17). Furthermore, the significant decrease in cortical area can be explained by a reduction in its piriform, entorhinal, and perirhinal subregions (rp= 0.95; p = 0.013), which are located adjacent to the atrophic hippocampus (Hc) and the expanded ventricles (Ve) in Figs. 1B and 2D.
Further to characterize the pilocarpine-induced atrophy of the hippocampal formation, the volume change for the total hippocampus and its dorsal (−2.12 to −3.8 mm from bregma) and its ventral part [−4.5 to ∼−6.8 mm from bregma (22)] were calculated from consecutive coronal MRI slices. Whereas the total brain volume did not differ between untreated (2,049 ± 29 mm3) and pilocarpine-treated rats (2,034 ± 32 mm3; p > 0.5), the total hippocampus volume changed from 104.9 ± 5.3 mm3 for controls to 79.1 ± 3.0 mm3 for pilocarpine-treated rats (−24.6%; p < 10−5). Furthermore, for controls and pilocarpine-treated rats, the loss in hippocampal volume is a volume change from 35.4 ± 1.7 mm3 to 22.0 ± 2.0 mm3 (−37.9%; p < 10−6) in the dorsal part and from 69.5 ± 4.6 mm3 to 57.1 ± 1.6 mm3 (−17.9%; p < 10−3) in the ventral part, respectively.
Pilocarpine-treated animals were severely impaired in the Morris water maze and showed considerable variance in performance. Whereas control animals learned reliably to find and to climb onto the hidden platform after 3–4 days, many pilocarpine-treated animals fail to do so, even after prolonged training for 10 days (see Fig. 3, for daily trials 2–6). Stepwise multiple regression revealed highly negative correlations between relative hippocampal size and performance (trial 1: F= 14.46, rp=−0.91, p = 0.032; trials 2–6: F= 23.83, rp=−0.94, p = 0.016; Fig. 3), whereas no correlation was found between the size of the combined area of perirhinal, entorhinal, and piriform cortices (rp=−0.68; p = 0.21) or any other brain area and the performance in the Morris water maze.
In this study, we applied volumetric MRI to determined structural alterations in rat brains after pilocarpine-induced epilepsy. In comparison with control animals, significant volumetric changes for hippocampus, thalamus, cortex (total and its perirhinal, entorhinal, and piriform subregions), putamen, and ventricles were found in pilocarpine-treated animals, whereas cerebellum and tectum remained unaffected. Although the entire hippocampal atrophy is strongly correlated with the expansion of the ventricles, a loss in cortical volume can be attributed mainly to damages in the perirhinal, entorhinal, ectorhinal, and piriform cortices (Fig. 2B and D), which have been reported previously (9,18,17,23,24). In humans, a participation of perirhinal, entorhinal, and piriform cortices has been described based on an observed signal increase in T2-weighted MR images (11,16).
In good agreement with histologic and neurochemical investigations in this animal model (25) and with indications from clinical MRI investigations in humans (26,27), a significant change in the size of the putamen is reported here for the first time in the pilocarpine model of epilepsy by MRI. Furthermore, a change in thalamic volume, as recently reported in human TLE patients (14,28), also was found in our study for the pilocarpine animal model. In agreement with previous histologic studies on pilocarpine-treated rats (29) and MRI studies on humans (13), our study shows a significant loss in total hippocampal volume and differences between the ventral and dorsal parts (30).
Impairment in water maze performance of pilocarpine-lesioned animals was found to be correlated only with the degree of hippocampal damage but not with the degree of damage in the combined areas of perirhinal, entorhinal, and piriform cortices (31); furthermore, no correlation was seen between the relative size of any other tested brain areas and the maze performance. This observation indicates that even a residual hippocampus in epileptic animals is still involved in memory formation (10,32). Similarly, in human TLE, predominant deficits in hippocampus-mediated cognitive capacities, such as verbal, visuofigural, and spatial memory (33) appear to be progressive in the course of the disease, similar to the hippocampal damage (34).
In summary, our study demonstrates that, in contrast to tedious histologic evaluations, noninvasive volumetric MRI can quantify the degree of atrophy of various brain structures easily within 1-h measuring time without dealing with any forms of artifacts during tissue processing, such as distortions and shrinkage. Based on the noninvasive nature of MRI, the progress of atrophy in different structures can be monitored over time. Therefore this way to quantify functionally relevant structural alterations is fast and repeatable during recurrent epileptic seizures after pilocarpine treatment and might become a valuable tool to validate therapeutic studies in TLE.
Acknowledgment: This work was supported by the German Research Council (SFB/TR3, TP A7) and the Center of Advanced Imaging Magdeburg (CAI, BMBF-grant 01GO0202).