CONVERSIÓN DEL ARCHIVO STEP 7 A TIA PORTAL V13

2.10.1 Binding (Competition) Experiments.

The amount of radiolabelled ligand bound (i.e. that retained on the filter) in disintegrations per minute (dpm) in duplicate or triplicate were meaned for total and non-specific binding, and also for each concentration of displacing compound. Specific

binding was defined as the difference between total and non-specific binding. The amount of radioligand specifically bound was finally expressed in femtomoles per milligram protein (fmol/mg), calculated from the specific activity of the ligand and the amount of protein in the assay.

The inhibition produced by the various standard compounds was expressed as a percentage of total specific binding (i.e. in the absence of unlabelled DM). These values were then fitted to a sigmoidal dose-response curve using the program 'GraphPad' InPlot v.4 (ISI Software, Philadelphia, PA). This iterative curve-fitting program uses least squares non-linear regression analysis, and allows the user to define constant values for zero or 0% (i.e. non-specific binding in the presence of an excess of unlabelled ligand) and maximum specific binding or 100% (i.e. in the absence of any

competing compound), respectively. The results obtained were expressed as the IC50

value, which was defined as the concentration required to produce 50% inhibition of the maximum specific binding of radioligand. This value corresponds to the mean of

the IC50S obtained from a number (n) of experiments. The slope of the displacement curve, corresponding to the Hill coefficient, was also determined.

For experiments with DM and carbetapentane analogues, an overall IC50 value was obtained by calculating the mean percent of total specific binding at each concentration from a number of experiments. This mean curve was then fitted to a sigmoidal curve as described above. Although this method does not allow the calculation of standard

errors as such, the program calculates 95% confidence limits, which is a good indication of the variability of the data, and the square of the correlation coefficient (r^), which represents the goodness of fit of the curve.

Where the Hill coefficients were significantly less than one, a mean displacement curve was constructed as described above and fitted to both one-site and two-site models to obtain the IC50 values and percentage bound at each site, using 'GraphPad' InPlot v.4 (non-linear regression, 1- and 2-site competition curve option).

2.10.2 Binding (Saturation) Experiments.

The total amount of free and bound radioligand in dpm was converted into moles of ligand per milligram protein, using the equation given above (2.10.1). The affinity dissociation constant (Kj) and the total number of binding sites (Bn^ax)» were calculated by linear transformation of the data, the Scatchard Plot (Bennett and Yamamura, 1985), which states that the relationship between the ratio of bound to free ligand (B/F) and the concentration of bound ligand (B) is according to the following equation:

B/F = (Bjjj^x " B)/Kj

Thus a plot of B/F (ordinate) against B (abscissa) will give a straight line where the slope is equal to the affinity dissociation constant and the intercept with the abscissa corresponds to the total concentration of binding sites in the reaction. This data was analysed by linear regression using the program GraphPad v.4. Where more than one binding site was suggested by non-linearity of the Scatchard Plot, the data were fitted to a two-site model of binding using the program "EnzFitter", ligand binding 2 sites option.

2.10.3 Autoradiography Experiments.

The developed films were analysed for radiographic density using a Quantimet 970 (Cambridge Instruments). The density was then converted to radioactivity in terms of nanocuries per milligram (nCi/mg) tissue equivalent by reference to a set of commercially available radioactive standards (Amersham, Batch 8) developed with the tissue samples. The values for total and non-specific binding from a total of 3-4 sections per brain area at each anatomical plane studied were meaned and a value for specific binding in nCi/mg was calculated from these. This was then converted from nCi/mg to units of receptor density in fmoles/mg tissue equivalent using the specific activity of the [^H]DM (Ci/mmole). The data from 4-5 rats were then meaned to give the overall amount of [^H]DM specifically bound.

2.10.4 Rat MCA Occlusion Experiments.

Photographic negatives were taken of both sides of each stained brain section, and analysed using the Quantimet 970 image analyser, program 'RHAT'. This program measures the area within a chosen region, in this case, the infarct and hemisphere. All measurements were performed "blind", i.e. without prior knowledge of the treatment.

The size of the infarct was quantified as follows:

a) the surface area at the stereotactic coronal plane at which the infarct was measured, and the total surface area of the infarct throughout the brain (i.e. the sum

of the surface area values at each brain level).

b) an estimate of the total volume of the infarct, calculated from the area of the infarct at each level and the distance between each section.

Results were expressed both in absolute terms of total area and volume (mm^ and mm^), and also as a percentage of the ipsilateral hemisphere (relative to the occluded MCA). It should be noted, however, that these values are probably an overestimation

of the true values for infarcted tissue, as the measurements include the effects of swelling from brain oedema.

An estimation of the relative potency of the compounds was obtained by extrapolation

of the linear regression analysis of the data points. An "ED5 0" value was obtained which was defined as the dose required to reduce the total infarct area to 50% of that obtained in control (vehicle-treated) rats. This was done because the small number of data points (due to limited supply of compound) meant that the maximum response could not be defined. While the values obtained from this analysis were obviously not precise ED50 values, they were used to make approximate comparisons between the

potency of the compounds.

2.10.5 Statistical Analysis.

All results represent the mean and standard error of the mean (s.e.m.) of the data from a number (n > 3) of experiments. For binding experiments, statistical comparisons were made between groups using either the Students 't' test (two-tailed), or the Mann Whitney 'U' test (for non-parametric data), whichever was appropriate. For comparisons between one- and two-site curves, the F-test (which compares the root mean square error of each fit) was used to determine which model gave the best fit to the data. In vivo neuroprotection experiments were analysed using Analysis of Variance (ANOVA), followed by Dunnett's test for comparisons between control

(vehicle) and test (drug-treated) groups. A probability level of p < 0.05 was considered to be significant.

CHAPTER 3

THE CHARACTERISTICS AND DISTRIBUTION OF DM