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In the experimental status epilepticus, 14-17 days after stimulation, animals were deeply anaesthetised with pentobarbitone. The heart was exposed and a cannula was placed in the aorta via the left ventricle; an incision was also made in the right auricle. Saline was administered via the cannula at a pressure o f 1-2 bar until the animal had been exsanguinated (usually 1-2 minutes). Then freshly made, cold 4% paraformaldehyde was administered via the cannula at a pressure o f 1-3 bar for 10-15 minutes. The head was then removed and the brain was exposed and placed in 4% paraformaldehyde for 12-24 hours before undergoing further dissection freeing the brain from the skull. The olfactory bulbs and cerebellum were removed, and the brain was cut coronally through the optic chiasm, so

giving a consistent reference point. The brains were then placed in 4% paraformaldehyde for 18-24 hours before being embedded in paraffin wax. 16pm coronal sections were then taken using a Leitz sledge microtome, and two sections every 320pm were dewaxed in xylene, placed in alcohol, washed and then stained with cresyl fast violet for 10 minutes at 60°C. Cresyl fast violet stains Nissl granules, and cell nuclei through its bonding with anionic nucleic acids. Neurons were distinguished from other cell types on the basis o f their size, the presence o f Nissl granules in the cytoplasm, and a large pale staining nucleus with one or more nucleoli.

When neuronal damage was assessed, it was done so in a blinded fashion so that the observer was unaware o f what treatments the rat had received or whether it was a treated or control rat. Neuronal damage was assessed in a semi-quantitive fashion as described by Kelly and McIntyre (1994). Neurons and glia were counted in a 50pm x 60pm grid. For each rat, two sections 320pm apart were selected such that the sections were either side o f the recording electrode. The counts were averaged from two randomly chosen locations for each structure (C A l, CA3 or hilus) on each section. Neuronal loss and gliosis were scored as 0- no damage, 1-slight damage, 2-moderate damage and 3-severe damage. These were defined as: slight damage - 50% increase o f glial cells and/or a few necrotic neurons in the cell field beyond the grid; moderate damage - 50-200% increase in glial cells and/or a 50% reduction o f neurons, and severe damage - more than 200% increase o f glial cells and/or a 90-100% loss o f neurons.

In situations where no damage was obvious, this was further confirmed using detailed cell counts. An optical dissector microscope (Zeiss) with a digital length gage (Heidenhaim, Ilinois) was used. Neurons in three dimensional boxes 70pm x 70pm X 5pm for the hilus, 20pm x 20pm x 5pm for C A l and CA3 were counted

according to the rules described by Williams & Rakic (1988): 1) Cell nuclei completely inside the box were counted; 2) Cell nuclei completely outside the box were not counted;

3) Cell nuclei that touch the bottom, front and left planes o f the counting box were not counted;

4) Cell nuclei that touch top, back or right plains were counted.

Rows of boxes that spanned from one blade o f the dentate fascia to the other were used to count the neurons within the hilus (figure 2.13).

Dentate

Figure 2.13: Section from rat hippocampus showing approximate positions o f CAl, CA2, CA3, hilus and dentate granule cells. Section was stained with Nissl stain.

Every other row was missed out, resulting in approximately 100 boxes being used per hilus. The hilus contains cells from CA3 and CA4. The rest o f CA3 was counted using 4 boxes (dimensions above) across the cell layer every 70pm so that 32 boxes were counted in all. The change from CA2 to CAl was distinguishable by a change in the density and width o f the cell layer. Neurons were counted in CAl using 3 boxes (dimensions above) to span the cell layer, every 70pm and a total o f 24 boxes were counted. The number and size of boxes were chosen to decrease inter-observer variability. Using the method above the repeat measure variability for one observer on two occasions was <5% The same observer was used for all subsequent counting. For each rat, three sections 320pm apart were selected such that the sections were either side o f the recording electrode.

The area between the blades o f the dentate fascia, and the lengths o f C A l and CA3 were also calculated using a Kontron mini-MOP in order to determine if there was significant shrinkage accounting for difference in cell density between cell groups.

Neuronal cell counts were expressed as three dimensional cell densities. Results for cell density and area and length measurement for each region were compared using a multifactorial analysis o f variance (ANOVA). M ain effects were section number, side, and stimulation. The data were modelled using main effects, and the interactions between stimulation and side, and between stimulation and slice position.

2.6 Audit methods