ESTRUCTURA DEL MODELO CAF

Female B6C3Fi mice (Small Animal Breeding Unit, Glaxo Wellcome Research and Development, Ware, Herts, or Charles River, Margate, Kent, or Harlan Olac, Bicester, Oxon), 1 2 -2 0 weeks old, were housed in groups of 5 or 6, bedded on wood shavings, and given diet (Rat and Mouse No. 1, SDS Ltd., Witham, Essex) and drinking water ad libitum. A temperature of 22°C ± 1°C was maintained, with a relative humidity of 45 - 70

% and a 12:12 h light:dark cycle (lights on at 06.00 h). Animals were acclimatised for at least 5 d before the start of each experiment and were observed daily for signs of ill- health, but more frequently after dosing. Animals were randomly assigned to dose groups using computer-generated tables.

2.2 Dosing

2.2.1 Busulphan

BU (Sigma Chemical Co. Ltd., Poole, Dorset) was dissolved in acetone at a concentration of 16.7 mg/mL. The BU solution was administered by ip injection after dilution with distilled water (Water for irrigation, BP) to give a constant dose volume of 10 mlVkg body weight. Control mice were given acetone with distilled water at the same dose volume.

2.2.2 Chloramphenicol Succinate

For administration in drinking water, 100 mg/mL stock solutions of CAPS (Sigma) prepared in distilled water (Water for irrigation, BP) were further diluted in tap water and

prepared dosing solutions. Water bottles were weighed before and after each dosing period to determine the volume consumed. For iv dosing, mice were given CAPS dissolved in distilled water (Water for irrigation, BP) at a rate of 10 mLAg body weight. For gavage dosing, mice were given CAPS in Water for irrigation BP at a constant rate of 10 mlVkg.

2.3 Haematological Measurements

2.3.1 Blood Samples

Animals were killed by CO2 overdose and 0.5 mL blood samples for cell counting were taken from the posterior vena cava and anticoagulated with 1.5 mg dipotassium EDTA/mL blood (Teklab, Sacriston, Durham). For the determination of haemolytic potential, murine blood, taken as above, and blood from healthy human volunteers, taken by antecubital venepuncture, were anticoagulated with heparin at 15 - 20 U/mL (Teklab, Sacriston, Durham).

2.3.2 Peripheral Blood: Full Blood Count, Differential Leucocyte Count and Reticulocyte Count

Full blood counts and differential leucocyte counts were obtained with a Bayer H*1 haematology analyser (Bayer Ltd., Newbury, Berks) (Ross and Bentley, 1986). Optimal separation of platelets and erythrocytes in animal blood is achieved in this system by differentiation of signals generated by scatter of laser light (Zelmanovic et al, 1992; Byrne et al., 1994). The percentages of microcytic and macrocytic erythrocytes were obtained, after isovolumetric sphering and partial fixation, from the forward light scatter signals exceeding cell volume thresholds for the mouse (microcytes <25 fL; macrocytes >75 fL) and of hypochromic erythrocytes from the side scatter signals for cells <18 g

Hb/dL (Tycko et al, 1985). Leucocytes, excluding basophils, were differentiated by cluster analysis of peroxidase content and volume, measured by absorption of right-angle scatter and forward scatter of tungsten light respectively, against an archetype for

leucocytes of the appropriate species (Davies and Fisher, 1991). An estimate of the myeloperoxidase content of neutrophils (MPXI) was generated simultaneously. This index of enzyme activity is expressed in arbitrary units spanning zero, the approximate mean value for man. Basophils were counted in a separate channel as phthalic acid- resistant intact cells; other cells in this channel were detected as stripped nuclei. The basophil channel also generates a measure of nuclear lobularity (lobularity index) in neutrophils and eosinophils compared to mononuclear cells, but owing to the relatively high proportion of lymphocytes in rodent blood, lobularity index is not valid and is not reported here. The H *l, with veterinary software (Versions 1.0 - 3.0; Bayer Ltd, Swords, Dublin), was validated in-house for use with dog, rat and mouse blood (Andrews and Mifsud, 1990; McKinnon, 1994)

Reticulocytes were counted using a Sysmex R-1000 reticulocyte analyser (Sysmex UK Ltd, Milton Keynes, Bucks; Tichelli etal., 1990) with photomultiplier voltage gain adjusted optimally for mouse blood (Fuchs and Eder, 1991 ; Takami and Sakata, 1991). Reticulocytes were differentiated from mature erythrocytes by fluorescence after staining with auramine O. Reticulocyte maturity was assessed according to fluorescence intensity which is subdivided into three fractions: high, mid and low fluorescence ratio (HFR, MFR, LFR). In-house validation of the R-1000 has been performed for dog, rat, mouse and marmoset blood (Hickson, 1992).

2.3.3 Bone Marrow

Bone marrow cellularity was assessed by flushing the contents of one femur into lOmL of phosphate buffered saline, and, after thorough mixing, obtaining the nucleated cell count from the basophil (leucocyte) channel of the H *l. Where the presence of fat particles was evident in the analyser cytogram, correction of the count was performed (Bentley et al.,

1995). Smears made from the tibia or opposite femur were stained with May-Griinwald- Giemsa stain and examined microscopically for morphological assessment and

enumeration of myeloid, erythroid, lymphoid and other cells in 2 0 0 cell differential counts.

2.3.4 Spleen

Nucleated cell counts of spleen suspensions were obtained using the basophil channel of the H *l. Suspensions were prepared by scissor-mincing after removal of capsular material and dispersion by cavitation with a 50 mL syringe. Spleen imprints were made by touching the cut surface of the organ on a microscope slide which was subsequently stained with May-Grünwald-Giemsa stain. Where histological processing was also carried out, spleens were first weighed and cut into two roughly equal portions. Cell counts were performed on one weighed portion and cellularity of the whole spleen extrapolated from the two weights.