SEROPREVALENCIA DEL VIRUS DE LA RINOTRAQUEITIS

8.1 Introduction

8.2 Methods

8.2.1 Photosensitizers

8.2.2 Light distribution in ex vivo lungs 8.2.3 Photodynamic Therapy

8.3 Results

8.3.1 Light distribution in the lung 8.3.2 Macroscopic appearances of PDT

8.3.2.1 Acute effects

8.3.2.2 Chronic effects 8.3.3 Adverse effects

8.1 Introduction

Having determined the distribution of drug in the lung parenchyma in the previous chapter I will now discuss the findings when PDT is performed on rat lungs. Drug was distributed throughout the lung parenchym a and therefore PDT lesions were to be expected in a lung. The relationship between the quantity of drug in the lung at different time points after injection and the subsequent tissue effects are also analysed in this chapter.

8.2 Methods

8.2.1 Photosensitizers

The three photosensitizers used were ALA, AIS2PC, and mTHPC. The doses of each were 200mg/kg, 1 mg/kg, and 0.3mg/kg respectively. These are typical doses used in rat experiments previously. ALA was prepared as a solution with phosphate buffered saline. AIS2PC solution was prepared by adding the powder to O.IM NaOH and 0.01 M N aH 2 P 0 4 . mTHPC was prepared immediately prior to injection by dissolving in a solution composed of ethanol, polyethylene glycol, and distilled water. All photosensitizers were injected slowly via the tail vein. Thereafter the rats were protected from direct light to prevent skin photosensitivity.

8.2.2 Light distribution in ex vivo lungs

This was performed by inflating an ex vivo rat lung and a porcine lung, and inserting the PDT laser fibre through the lung surface. The distribution of light in the lung could therefore be directly observed.

8.2.3 Photodynamic Therapy

Rats were placed under a general anaesthetic and the left side of the chest shaved and sterilized with a topical preparation. The rat was placed in the same frame as used for the ILP experiments and again the optimum site of placement into the left lung was chosen by fluoroscopy. Once this was determined a small incision was made and the introducer needle passed into the lung parenchyma. The laser fibre within the introducer needle was then held stationary while the introducer needle itself was retracted a short distance leaving the bare laser fibre tip exposed in the lung parenchyma. The laser light source was a copper vapour pumped dye laser (Oxford

Lasers), which is tunable so that the different wavelengths for each photosensitizer could be obtained. The laser fibre was a 400 um internal diameter bare-tipped glass fibre with silastic cladding. The fibre was cleaned between each treatment and carefully recalibrated to check the power before and after each treatment. After sacrifice by CO2 inhalation lungs were fixed and sectioned as described in Chapter 5. The largest diameter of necrosis (black tissue) was measured. Lung volumes were also measured as described in Chapter 5.

Four animals were treated with laser light alone without prior administration of photosensitizer to determine if any thermal effects were produced on the tissues by the laser power used. The light dose was 100 mW for 1000 seconds ( 100 joules) at a wavelength of 635nm. These animals were killed three days after treatment for histological examination. Further control rats were also kept up to 6 months after insertion of the laser fibre to exclude any effect on macroscopic appearance of this intervention.

The wavelengths for ALA, AIS2PC, and MTHPC were 635, 675, and 652nm respectively. All treatments were performed using a laser output of 100 mW. In the first set of experiments the drug light intervals were varied between 2 and 8 hours for ALA, 1 and 72 hours for AIS2PC, and 1 and 6 days for mTHPC. These experiments were to determine the time at which PDT damage measured 3 days after treatment was greatest with each sensitizer. In the subsequent experiments one treatment time was chosen and the light dose varied between 20 and 300 Joules. The treatment time selected was that which gave the largest lesion size in the above experiments. For mTHPC this was chosen at 3 days due to marked vascular effects of PDT at earlier times which may have confounded lesion size measurements. Also previous reports have shown that the best ratios of mTHPC between tumour and normal tissue are seen at 72 hours after sensitization and therefore information obtained at this drug light interval was more relevant than the effects at 24 hours, since the 72 hour interval would be the likely interval chosen in tumour treatments (Ris et al., 1993a).

The effects of PDT were assessed between 3 days and 6 months after initial PDT treatment. An assessment of healing after PDT was made with all photosensitizers however most of this assessm ent was perform ed with mTHPC. Histology was performed at 10 days, 3 weeks, and 2 months after ALA and ALS2PC, however mTHPC was also assessed at 6 months. Groups of

five animals were used at each time point with mTHPC at times up to 6

months.

Fluoroscopy was performed immediately after treatment to determine if pneumothorax had occurred. Conventional chest Xrays were used post­ mortem to determine if late pneumothorax occurred. These were taken on groups of rats at each time point up to 6 months. Angiograms of pulmonary arteries were performed as described in Chapter 5. These were done on lungs removed 2 months after mTHPC PDT.

8.3 Results

8.3.1 Light distribution in the lung

Inserting the laser fibre into an inflated lung ex vivo resulted in uniform illumination of the lung ( Figure 8.1 ).The light appeared to be scattered in all directions in the lung ; it did not simply continue in a forward direction from the laser. The pleura appeared to internally reflect the light from the laser back into the lung. This was demonstrated by putting the laser fibre through the lung to the opposite pleural surface. The light still gave a diffuse glow over the pleural surface and inside the lung even when the tip of the fibre was touching the pleural surface. When the fibre was advanced through the pleural surface the point source of the light immediately became apparent and the light shone in a forward direction onto adjacent structures.

Figure 8.1 Light distribution in an ex vivo lung illuminated by 652nm wavelength light. In the foreground is the right lung which has not had the fibre inserted.

8.3.2 Macroscopic appearances of PDT

8.3.2.1 Acute effects:

On the cut surface perpendicular to the line of the laser fibre the PDT effect with all photosensitizers was seen as a circular uniformly dark area centred around where the tip of the fibre had been placed. There was a clear distinction between PDT lesions and the surrounding untreated parenchyma (Figure 8.3). As with ILP the PDT effect from the parenchymal lesion was also visible on the adjacent lung surface (Figure 8.2).The pleural surface had a normal contour and there was no shrinkage of the lung at the lesion site. No adhesions between visceral and parietal pleura were seen at 3 days. The PDT effect was confined to the lung and visceral pleura and there was never any PDT effect on the parietal pleura (Figure 8.4). Also, the mediastinal structures such as the great vessels or oesophagus were not damaged by the PDT occurring in the immediately adjacent lung (Fig 8.5). An exception to this was the few cases where the laser fibre had inadvertently been placed through the medial surface of the visceral pleura (see below, adverse effects).

The remainder of the lung appeared normal in virtually all cases apart from some rats treated with mTHPC with a drug-light interval of 24 hours. Here there was evidence of vascular congestion on the lung surface and on sectioning the lung appeared oedematous (Figure 8.6)

Lesion sizes were highly reproducible. The lesion diameter in 10 rats treated with mTHPC with a 3 day drug light interval and 80 J was 9.8mm, standard deviation 1 mm. The effect of changing drug light interval is shown for each of the photosensitizers in figures 8.7 a, 8.8a and 8.9a. The effect of increasing the light dose when a single drug light interval is used is shown in figures 8.7b,8.8b and 8.9b. With mTHPC a 72 hour drug light interval was chosen as although the results with a 24 hour interval were slightly larger there was the potential for lesions to be obscured by thrombosis and oedema seen using this earlier treatment time. With each drug there was a plateau above which increasing the light dose does not increase the diameter of PDT necrosis. The largest lesion seen was with mTHPC and this was 12mm in diameter. The drug light interval for ALS2PC was 1 hour. Only 1 animal was used for the 300 J treatment with ALS2PC. For ALA 4 hours was chosen as there was a smaller standard error than for 3 hours even though the mean results were very similar.

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Figure 8.2 Macroscopic appearance of lungs 3 days after mTHPC PDT.(A) Light dose 40 J. (B) Light dose 100 .1 Both treated with a 3 day drug light interval.

Figure 8.3 Appearance of the pleural surface superficial to the interstitial PDT lesion shown in Figure 8.3 a.

Figure 8.4 Lung and chest wall post mortem 3 days after PDT demonstrating PDT effect on visceral pleura but no effect on parietal pleura.

Figure 8.5 PDT effect in lung adjacent to oesophagus which is unaffected. Treated with 80 .1 with 3 day drug light interval.

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