Cell loss and degeneration at day 3 post pilocarpine.

<p>HSP72 is heavily expressed in a large population of pyramidal cells (A, B) and in some mossy cells, indicating that they were exposed to excitotoxic insult. In contrast, hardly any granule cells are stained (arrows) (C) compared to 1 day post SE. Gallyas silver impregnation reveals that in the early latent period principal cell loss begins at a large scale (orange cells are alive). Dark silver deposit is present in neurons irreversibly damaged (D, E, F). Argyrophilic cell degeneration can be seen in the vulnerable regions of the hippocampus, like the CA1 pyramidal cell layer (E) and the hilar region (F) (arrows in the CA1, arrowheads in the hilus). (s.o.: str. oriens, s.p. str. pyramidale, s.r.: str. radiatum, s.m.: str. moleculare, s.g.: str. granulosum) Scale: A, D 200 µm, B, E, F, C and F: 50 µm.</p

Table A describes seizure behavioral scoring for each Racine scale value.

<p>Graphs B, C and D show the correlation between seizure intensity (Racine scale value) and cell loss (color code indicates cell loss from 0 to 3, where 0 refers to no cell loss, 1: cell-loss under 10%, 2: cell-loss between 10% and 50%, 3: cell-loss above 50%) in CA1, CA3 and the hilus respectively. Correlation between Racine scale value and cell loss in the regions proved to be significant using Pearson correlation (p<0.05).</p

Anatomical and physiological changes in pilocarpine induced epilepsy.

<p>Light micrographs of control (A) and epileptic (B, C) animals immunostained for NeuN. Animals survived for 1 month after pilocarpine treatment. Compared to control mice (A), in non-sclerotic “weak” animals (B) only mild, restricted changes (arrow) are seen occasionally in the hippocampus. In the sclerotic hippocampi of “strong” animals (C) a characteristic pattern of neuronal damage appears. The principal cell loss is over 50% (sclerosis) mostly in CA1 and CA3 (arrows) and in the hilus. In the EEG recordings of control animals no ictal or interictal acitivity can be seen (D), however, in weak animals (E) interictal spikes (arrowheads) occur frequently. In strong animals (F) recurrent seizures are observed in most cases, both interictal and ictal (arrows) activity appeares. An increase in frequency and decline in amplitude can be seen immediately preceding and after the seizures. Scale (A, B, C): 200 µm.</p

Changes of CB1-R-staining in the acute phase.

<p>In control samples intense staining of CB1-R-positive fibers can be seen in CA1 stratum pyramidale and radiatum (A) and in the molecular layer of the dentate gyrus (C). In the acute phase of epilepsy (2 hours after pilocarpine injection) the hippocampi of strongly epileptic animals show decrease of CB1-R staining (B, D) compared to controls (A, C). The change is more remarkable in CA1 than in the DG, where the faint diffuse staining of the inner str. moleculare is hardly affected. Scale: 50 µm.</p

Changes in CB1-R-staining in the chronic phase.

<p>CB1-R immunostaining in the hippocampi of control (A, C) and chronically epileptic, sclerotic mice (1 month post pilocarpine)(B, D). In the sclerotic samples the general CB1-R immunostaining is much stronger throughout the hippocampus. In the CA1 (A) a dense immunostained axonal plexus can be seen in the sclerotic CA1 region (layers cannot be distinguished) (B). In the dentate gyrus stained fibers establish more dense meshworks (D). Scales: A, C, D: 50 µm B: 20 µm.</p

Reversible changes in CB1-R-staining in slice preparation (acute phase).

<p>In CA1 region of surviving brain slices 2 hours post pilocarpine a massive decrease is seen in CB1-R staining in strongly epileptic animals (B) compared to scopolamine treated controls (A) as it was seen in perfusion-fixed epileptic animals. In contrast, the slices from the same animals which were incubated in ACSF for an additional 2 hours the CB1-R staining in the epileptic tissue (D) did not differ from control slices (C). Scale: 50 µm.</p

Different behaviour and survival of knock out and wild type animals.

<p>Graph showing the number of animals as a function of seizure strength demonstrates that knock out animals had more severe seizures compared to wild types and never survived tonic-clonic (Racine 5) seizures.</p

Demonstration of excitotoxic insult 1 day after pilocarpine treatment.

<p>In animals of the “weak” group (B) HSP72-immunostaining is control-like (A), only a slight increase in background-staining can be seen, but no stained cells with processes appear. Although there are no morphological signs of neuronal degeneration at this early stage in the hippocampi of “strong” epileptic animals (C) either, the vulnerable cells as well as the resistant granule cells start to express HSP72, and are stained in a Golgi-like manner. Faint homogeneous staining of CA1 pyramidal cells can also be seen. Scale: 200 µm.</p

Quantitative changes of CB1-Rs in the acute and chronic phase of epilepsy.

<p>Graph <b>A</b> shows changes in the ratio of CB1-R immunopositive terminals establishing symmetric versus asymmetric synapses in control and epileptic tissue in str. moleculare of the DG. No difference was found in the hippocampi of strong animals in the acute phase compared to controls. However, in strong animals in the chronic phase a significant increase occurred in the ratio of stained symmetric versus asymmetric synapses. Graph <b>B</b> shows changes in the absolute number of symmetric versus asymmetric synapses in control and epileptic tissue. Significant decrease was found in the number of stained asymmetric and symmetric synapses in the hippocampi of strong animals in the acute phase, compared to controls. In contrast, in strong animals in the chronic phase, the number of immunostained asymmetric and symmetric synapses significantly increased. Graph <b>C</b> shows changes in the normalized number of gold particles in control and epileptic tissue. No difference appears in the mean quantity of gold particles in asymmetric synapses in control or in epileptic tissue (acute or chronic phase). No difference was seen in case of symmetric synapses between the hippocampi of control and acute epileptic mice either. However, in chronically epileptic tissue the number of gold particles located in symmetric terminals increased significantly. Statistics were calculated with Mann-Whitney test (p<0.05). High power electron micrographs of CB1-R immunolabelled axon terminals from the str. moleculare of the DG of controls (D,G) acute and chronic animals 2 hours (E, H) and 1month (F, I) post pilo. Our antibody labels both CB1-R-positive terminals giving symmetric (D, E, F) and asymmetric (G, H, I) synapses. Gold particles are located extrasynaptically. In the chronic, strong tissue the number of gold particles on terminals forming symmetric synapses has increased (F). Scale: 200 nm.</p