TEMAS TRANSVERSALES, VALORES Y ACTITUDES PRIORIZADOS

expression of iNOS protein was detected by Western blotting in extracts of lung, heart, liver and RTAs from post-mortem control and LPS-treated animals (t= 4, 6, 12, 24, 72hrs; see Appendix 3.1). Fig. 3.9 shows lung extract from t= 24hr LPS-treated rats expressing high levels of iNOS as evident by the densely staining band at 130KDa. Lung extract from t= 24hr LPS-treated rats was used as a positive control for iNOS expression until the commercially available positive cell lysate arrived. The validity of the lung extract was confirmed with both bands detected at 130KDa (see fig. 3.10). Fig. 3.10 also reveals a time course of iNOS expression in LPS-treated RTAs, with a maximum at t= 4-6hrs, gradually declining until it was barely detectable by t= 24hrs and undetectable by t= 72hrs. Dot blot analysis further revealed that liver extracts from t= 24hr LPS-treated rats also expressed iNOS (see fig. 3.11).

Several histochemical and immunohistochemical techniques were used to detect eNOS and iNOS expression in tissue sections (see figs. 3.12-3.18 and Appendix 3.2). Immunoflourescent labelling revealed intensely stained macrophage-like cells scattered throughout the lung tissue of t= 24hr LPS-treated rats (fig. 3.12). Intensely fluorescent cells were often seen in lung tissue from control animals but not to the same extent.

Fig 3.9 Western blot analysis using a specific antibody to iNOS enzyme of lung

extract from t= 24hr LPS-treated rat reveals an intensely staining band at 130 KDa, suggesting that iNOS is being expressed.

130 Kl);i

12 3 4 5 6 7

Fig. 3.10 Western blot analysis using a specific antibody to iNOS of LPS- treated RTAs at t= 4hrs (lane 1), 6hrs (lane 2) 12hrs (lane 3) 24hrs (lane 4) and 72hrs (lane 5). Lung extract from t= 24hr LPS-treated rats and activated- macrophage lysate are used as positive controls (lanes 6 & 7 respectively). iNOS expression is visibly reduced at t= 24hrs after LPS treatment.

Fig. 3.11 shows a dot blot of lung and liver extracts from t= 24hr LPS-treated rats demonstrating a more intense signal for iNOS with increasing protein concentration.

Fig. 3.11 Dot blot analysis with anti-serum to iNOS on lung ([proteinjs, 1.7,

6.6, 7.6g/l; Nos 1-3 respectively) and liver ([proteinjs, 1, 7.6 and 30g/l; Nos 6-4 respectively) extracts from t- 24hr LPS-treated rats.

Fig. 3.12 Cryostat section (5-7pm) of lung tissue from a t= 24hr LPS-treated rat immunostained with fluorescently-labelled (TRITC) 2° antibody. Intensely stained macrophage-like cells are scattered throughout the lung tissue (x400).

Fig. 3.13 shows that iNOS positive cells were not seen within bronchioles or their associated epithelial lining but were instead interposed between the alveolar network in the surrounding visceral pleura.

Fig. 3.13 TRITC-labelled cryostat section of lung from t= 24hr LPS-treated rat

showing the intensely fluorecent cells in the visceral pleura (A) but absent from the nearby bronchiole (B; x400).

Fig. 3.14 Similarly, immunostaining by the ABC method on sectioned lung tissue from a t-24hr LPS-treated rat showed iNOS positive cells (M) as large rounded black dots within the visceral pleura (x400).

Cryostat sections of the same area in the lung as shown in fig. 3.14 were also stained for NADPH-diaphorase activity (see Appendix 3.4) and resulted in a large number darkly stained cells within the visceral pleura and attached to the

Fig. 3.15 Cells within lung tissue from t= 24hr LPS-treated rat stain intensely

for NADPH-diaphorase. NADPH-diaphorase-Positive cells are also evident on the epithelial lining of a bronchiole (B; x400).

LPS-treated RTAs at t= 6, 24 and 72hrs were immunostained with anti-serum to eNOS to determine whether eNOS expression persisted throughout the LPS- mediated induction of iNOS (see figs. 3.16-3.18). Fig. 3.16 shows an LPS- treated RTA at t= 6hrs immunostained using the ABC method. iNOS is highly expressed at t= 4-6hrs and it is clear from fig. 3.16 that eNOS is also present at this time.

Fig. 3.16 Paraffin-sectioned (5-7pm) RTA from an LPS-treated rat at t- 6hrs immunostained with anti-serum to eNOS using the ABC method. Endothelial cells (E) which express eNOS are seen here as a thin, darkly stained layer facing towards the lumen of the vessel (L; xlOOO).

Fig. 3.17 Paraffin-sectioned (5-7pm) RTA from an LPS-treated rat at t- 24hrs

immunostained with anti-serum to eNOS and detected using a fluorescently- labelled (FITC) 2° antibody. Endothelial cells (E) which express eNOS are seen here as a thin, brightly stained layer facing towards the lumen of the vessel (L;

Fig. 3.18 Paraffin-sectioned (5-7pm) RTA from an LPS-treated rat at t- 72hrs immunostained with anti-serum to eNOS and detected using a fluorescently- labelled (FITC) 2° antibody. Endothelial cells (E) which express eNOS are seen here as a thin, brightly stained layer facing towards the lumen of the vessel (L; xlOOO).

It is important to realise that little quantitative information regarding the relative expression of eNOS at t= 6, 24 and 72hrs post-LPS is provided by these sections

as staining will depend very much on a) the type of detection method used b)

the condition of the RTA, which will vary from one preparation to the next; and finally c) the quality of the photograph. It can however be concluded that eNOS is expressed in RTAs at t= 6, 24 and 72hrs post LPS.

Part B

3.6 Quantitative analysis of hyporesponsive vessels isolated from

animals in endotoxic shock.

It will be seen later (Chapter 7) that isolated vessels that previously supplied a solid tumour exhibit a similar diminished sensitivity to PE when compared to their respective controls: superficially, at least, the hyporesponsiveness shown by LPS-treated vessels and tumour supply vessels appears very similar. However, a more detailed analysis (below) suggests that there may be qualitative differences between the two, a point which is discussed again later (Chapter 9).