Disruption of the blood-brain barrier following ALA-mediated photodynamic therapy

Abstract

Background and objective: Photodynamic therapy (PDT) is a local antineoplastic treatment with the potential for tumor cell specificity. PDT using either hematoporphyrin derivatives or 5-aminolevulinic acid (ALA) has been reported to induce brain edema indicating disruption of the blood-brain barrier (BBB). We have evaluated the ability of ALA-mediated PDT to open the BBB in rats. This will permit access of chemotherapeutic agents to brain tumor cells remaining in the resection cavity wall, but limit their penetration into normal brain remote from the site of illumination.

Study design/materials and methods: ALA-PDT was performed on non-tumor bearing inbred Fischer rats at increasing fluence levels. Contrast T(1)-weighted high field (3 T) magnetic resonance imaging (MRI) scans were used to monitor the degree of BBB disruption which could be inferred from the intensity and volume of the contrast agent visualized.

Results: PDT at increasing fluence levels between 9 and 26 J demonstrated an increasing contrast flow rate. A similar increased contrast volume was observed with increasing fluence rates. The BBB was found to be disrupted 2 hours following PDT and 80-100% restored 72 hours later at the lowest fluence level. No effect on the BBB was observed if 26 J of light was given in the absence of ALA.

Conclusion: ALA-PDT was highly effective in opening the BBB in a localized region of the brain. The degradation of the BBB was temporary in nature at fluence levels of 9 J, opening rapidly following treatment and significantly restored during the next 72 hours. No signs of tissue damage were seen on histological sections at this fluence level. However, higher fluences did demonstrate permanent tissue changes localized in the immediate vicinity of the light source.

(c) 2008 Wiley-Liss, Inc.

Figures

Fig. 1

a: T1-weighted MR images of a PDT-treated rat along with four calibration tubes with Gd concentrations of 0, 0.5, 1, and 2 mg/ml. b: Normalized signal intensity as a function of Gd concentration.

Fig. 2

T1-weighted MRI contrast enhanced images (a, b) showing focal contrast enhancement centered around the area of light treatment. PDT treatment to a fluence level of 9 (a) and 17 J (b), at a fluence rate of 10 mW was performed 4 hours following ALA administration (125 mg/kg i.p.). Both scans were performed 3–4 hours post-PDT and 15 minutes following i.p. contrast injection. (c, d) Coronal H&E sections from the brains of animals corresponding to a and b taken 14 days post-treatment. In the area exposed to the highest fluence level (5–15 μm from the fiber) no significant pathology was observed following delivery of 9 J (c). At a fluence level of 17 J, extensive infiltration of lymphocytes and macrophages (some loaded with hemosiderin) was apparent as denoted by the arrow in (d).

Fig. 3

Average contrast volume measured from MRI scans performed 24 hours post-treatment for three light fluences (fluence rate = 10 mW). A significant light dose response was apparent, with increasing light fluence resulting in increased contrast. A fluence of 26 J, in the absence of ALA (light-only control), resulted in minimal contrast enhancement suggesting an intact BBB.

Fig. 4

a: Average contrast intensity measured from T1-weighed MR images performed 24 hours post-treatment for three light fluences. Images were obtained 15 minutes. post-contrast injection. b: Concentration of contrast medium calculated from the calibration curve shown in Figure 1. Contrast concentrations between 125 and 710 μg/ml were obtained for the three fluence levels examined.

Fig. 5

T1-weighted MRI contrast enhanced images showing focal contrast enhancement as evidence of BBB disruption. The effects of fluence rate on BBB disruption are clearly evident. Increased fluence rates resulted not only in increased contrast volume but increased signal intensity as well, indicating an increased contrast concentration.

Fig. 6

T1-weighted MRI contrast enhanced images showing focal contrast enhancment at 2 (a), 24 (b) and 72 (c) hours post-treatment, respectively. PDT treatments using 9 J (a,b,c) or 17 J (d,e,f) were performed at a fluence rate of 10 mW. All animals were scanned 15 minutes, following i.p. contrast injection. A localized contrast enhancement is clearly seen 2 and 24 hours following light treatment (a,b,d,e) for both 9 and 17 J. Images taken 72 hours following treatment with 9 J showed no enhancing regions (c) while reduced enhancement persisted up to day 7 following PDT with 17 J (f).

Fig. 7

T1 signal intensity with time post-contrast injection on days 1 and 7 following light treatment. The signal intensity 24 hours following PDT treatment and 60 minutes post-contrast injection was considered as the 100% point. The rate of flow was clearly biphasic on day 1 with a rapid increase in volume during the initial 10 minutes, following contrast injection and decreasing to a much lower rate following the initial contrast influx. A greatly reduced and linear increase in average intensity with time was observed on day 7.

Fig. 8

Time course of BBB closing. Scanning was performed 4 hours and 1,3,7, and 17 days post-PDT treatment to determine changes in the BBB with time. Contrast flow rate, based on increasing volume of T1 signal intensity, on each of these days was calculated by rescanning the rats at several intervals following contrast injection. The rate of flow fell rapidly from 6.7 to 1.3 μl/minutes (80% decrease) from day 1 to day 3. PDT treatment was performed 4 hours following i.p. injection of 125 mg/kg ALA. All animals were subjected to light fluence and fluence rates of 17 J and 10 mW, respectively.