Lithium pilocarpine-induced status epilepticus in postnatal day 20 rats results in greater neuronal injury in ventral versus dorsal hippocampus

Abstract

Many quantitative animal studies examining the possible relationship between hippocampal neuronal loss and the development of epilepsy have examined only the dorsal hippocampus. The ventral hippocampus, however, represents the more homologous structure to the anterior hippocampus in humans, which is the area associated with the maximal damage in patients with temporal lobe epilepsy. This study tested the hypothesis that the ventral hippocampus has greater neuronal injury than the dorsal hippocampus in an animal model of chemoconvulsant-status epilepticus at postnatal day 20. Status epilepticus was induced in postnatal day 20 Sprague-Dawley rat pups with the chemoconvulsant lithium-pilocarpine and brain tissue was examined with Fluoro-Jade B. Horizontal sections (n=7) favoring a visualization of the ventral hippocampus showed marked Fluoro-Jade B staining in CA1, CA3, and hilar region. Coronal sections favoring a visualization of the dorsal hippocampus did not consistently show as robust a staining pattern in these regions. In coronal sections where both the dorsal and ventral hippocampus could be viewed, greater staining was always seen in ventral versus dorsal hippocampus. Quantitative analysis of cell counts demonstrated a significant difference between ventral and dorsal hippocampus in CA1 and CA3, but not hilus. These results demonstrate that in ventral hippocampus, lithium pilocarpine-induced status epilepticus consistently results in hippocampal neuronal injury in postnatal day 20 rats. This study shows the importance of including the ventral hippocampus in any analysis of seizure-induced hippocampal neuronal injury, and raises concerns about the accuracy of studies quantifying hippocampal neuronal loss when only the dorsal hippocampus is examined.

Figure 1

Lithium pilocarpine induces status epilepticus at P20. Representative EEG recording showing initiation of status epilepticus. Arrows indicate beginning of expanded time scale for tracings prior to seizure activity (A), at initiation of one early seizure (B), and during status epilepticus (C). Note the slowly increasing amplitude in trace B which is characteristic of electrographic seizure activity. D shows trace at point C of top trace at even more expanded time scale in order to visualize individual spike activity during status epilepticus.

Figure 2

Orientation of the rat hippocampus in relationship to surrounding cortical structures (A). Sections cut in coronal orientation allowed optimal visualization of the dorsal hippocampus, which becomes distorted as one progresses toward the temporal pole (B). Sections cut in horizontal orientation allowed optimal visualization of the ventral hippocampus, which maintains orientation perpendicular to septo-temporal axis in the temporal pole (C). Figure adapted from Amaral and Witter. In: Paxinos. The Rat Nervous System, 1995.

Figure 3

Fluoro-jade B predominantly stains neuronal soma in ventral versus dorsal hippocampus. Horizontal (A, C, E, and G) and coronal (B, D, F, and H) sections allowed optimal visualization of the ventral and dorsal hippocampal formation, respectively. Ventral hippocampus (A) showed much more staining than dorsal hippocampus (B). Figures A and B are taken at approximate level of cross section of Figure 2C and first cross section of Figure 2B, respectively. Flourescent staining could be seen predominantly in the stratum pyramidale layers CA1-CA3. The granule layer of the dentate gyrus was spared, although some mossy cells in the hilus were visualized. Dorsal hippocampus also demonstrated more “patches” of Fluoro-jade B staining (compare A and B). C and D show absence of staining in untreated control animals. (E and F) Higher magnification of stratum pyramidale layer. At even higher magnification (G and H) individual pyramidal neuron soma and apical dendrites are visualized. DG, dentate gyrus; Str Pyr, stratum pyramidale layer.

Figure 4

Representative coronal section that includes portions of both dorsal and ventral hippocampal formation (approximately at 2^nd cross section in fig 2B). Sections with little to no staining in the dorsal hippocampus showed definite staining in the ventral hippocampus despite distortions in anatomical orientation.

Figure 5

Quantitative analyses of Fluoro-jade B staining shows significant difference between ventral and dorsal hippocampus in CA1 and CA3 region. (A and B) Areas of interest for CA1, CA3, and hilus chosen for quantitative analysis. (C-K) Bar graphs of Fluoro-jade B positive cell bodies. Counts from three separate sections for each animal were assessed for ventral (C, F, I) and dorsal (D, G, J) hippocampus for CA1, CA3, and hilus in order to show variability between tissue sections and animals. (E, H, K) Mean counts for ventral and dorsal hippocampus for all tissue sections samples show significant difference between staining in ventral vs dorsal hippocampus in CA1 and CA3, but not hilus. Error bars represent SEM. *= significance (p<0.001)