Evaluation of Motexafin gadolinium (MGd) as a contrast agent for intraoperative MRI.

OBJECTIVE: The characteristics of an ideal contrast agent for use in the intraoperative MRI would be tumor-specificity and intracellular localization, combined with extended tumor enhancement, but with rapid elimination from the blood. The radiation sensitizing properties of Motexafin gadolinium (MGd) have been investigated in a number of clinical trials involving patients with brain metastases. These studies clearly show that MGd is detectable in magnetic resonance images many days following administration. The aim of this experimental study was to test whether Motexafin gadolinium (MGd) could serve as an efficient intraoperative contrast agent avoiding problems that arise with surgically induced intracranial enhancement. METHODS: F98 orthotopic brain tumors or surgical lesions were induced in Fisher rats. T1-weighted MRI studies were performed with either a single or multiple daily doses of MGd. The last contrast dose was administered either 7 or 24 hours prior to scanning in both tumor-bearing or surgically-treated animals. All scans were T1-weighted nce (TR=495 ms; TE=1 ms.) with a slice thickness of 1.0 mm. Three tubes containing 2.3, 0.23 and 0.023 mg/mL of MGd (in physiological saline) respectively, were used as standards to calibrate the scans. RESULTS: Animals receiving either 30 or 60 mg/kg MGd i.v. developed clinical signs of impaired motor activity, and increasing lethargy and were euthanized 48 hours after MGd administration due to their poor and deteriorating condition. MGd given i.p. was tolerated up to a dose of 140 mg/kg. Despite multiple dosages and several administration modes (i.p., i.v.) no significant enhancement was observed if the scans were performed 7 or 24 hours following the last MGd dose. Clear enhancement was seen though when the scans were performed 30 min following MGd administration, indicating that the agent was being taken up by the tumor. Scans of necrotic lesions though were positive though 7 hours following MGd injection. MGd scans had no significant enhancement following surgically-induced lesions while scans with conventional contrast agents showed both meningeal and intraparenchymal enhancement. CONCLUSION: This study suggests that MGd is not sequestered in viable tumor for the necessary time interval required to allow delayed imaging in this model. The agent does seem to remain in necrotic tissue for longer time intervals. MGd therefore would not be suitable as a contrast agent in intraoperative MRI for the detection of remaining tumor tissue during surgery.

Development of a novel indwelling balloon applicator for optimizing light delivery in photodynamic therapy.

BACKGROUND AND OBJECTIVE: A human glioma spheroid model is used to investigate the efficacy of different light delivery schemes in 5-aminolevulinic acid (ALA)--mediated photodynamic therapy (PDT). The results provide the rationale for the development of an indwelling balloon applicator for optimizing light delivery. STUDY DESIGN/MATERIALS AND METHODS: Human glioma spheroids were incubated in ALA (100 or 1000 microg /ml-1) for 4 hours and subjected to various light irradiation schemes. In one set of experiments, spheroid survival was monitored as a function of light fluence rate (5-200 mW cm-2). In all cases, spheroids were exposed to fluences of either 25 or 50 J cm-2. In a second study, the effects of repeated weekly PDT treatments, using sub-threshold fluences, were investigated. One group of spheroids was subjected to three treatments using fluences of 12, 12, and 25 J cm-2. Results were compared to spheroids receiving single treatments of either 12 or 25 J cm-2. A fluence rate of 25 mW cm-2 was used for all three groups of spheroids. In all cases, the effect of a given irradiation scheme was evaluated by monitoring spheroid growth. RESULTS: Low fluence rates produce greater cell kill than high fluence rates. The minimum effective fluence rate in human glioma spheroids is approximately 10 mW cm-2. Repeated weekly PDT treatments with sub-threshold fluences result in significant cell kill. In spheroids surviving the PDT treatments, growth is suppressed for the duration of the treatment period. CONCLUSION: The results of the in vitro studies support the development of an indwelling balloon applicator for the delivery of light doses in long term multi-fractionated PDT regimens.

Photodynamic therapy of human glioma spheroids using 5-aminolevulinic acid.

The response of human glioma spheroids to 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (< or = 50 J cm-2), lower fluence rates are more effective. At a fluence of 50 J cm-2, near-total spheroid kill is observed at fluence rates of as low as 10 mW cm-2. The fluence rate effect is not as pronounced at higher fluences (> 50 J cm-2), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.

Detection of low dose radiation induced DNA damage using temperature differential fluorescence assay.

A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures of between 0.004 and 1 Gy were measured with doses as low as 0.008 Gy yielding significant responses. The double-strand, sensitive dye PicoGreen was used as an indicator of DNA denaturation. Calibration plots indicate that fluorescence changes corresponding to amounts as low as 1 ng of double stranded DNA (10(6) copies for plasmid puc 19) are detected by this method.

A mathematical model for light dosimetry in photodynamic destruction of human endometrium.

We are involved in the development of photodynamic therapy (PDT) as a minimally invasive method for treating dysfunctional uterine bleeding, one of the primary clinical indications for hysterectomy. In this paper, we analyse light propagation through the uterus in order to specify the requirements for a light delivery system capable of effectively performing endometrial PDT. Our approach involves developing an analytical model based on diffusion theory to predict optical fluence rate distributions when cylindrical and spherical optical applicators are placed in the uterine cavity. We apply the results of our model calculations to estimate the thermal effects of optical irradiation and the effective photodynamic optical dose. Theoretical fluence rate calculations are compared to fluence rate measurements made in fresh, surgically removed human uteri. Our results show that a trifurcated cylindrical optical applicator inserted into the human uterus can provide a light dose that is sufficient to cause photodynamic destruction of the entire endometrium. When the optical power per unit length of each cylindrical applicator is 100 mW cm-1 (at 630 nm), a fluence rate of 40 mW cm-2 is delivered to the boundary layer between the endometrium and the myometrium (a depth of about 4-6 mm). The optical fluence delivered to the boundary layer after 20 min of exposure is 50 J cm-2, a level that is generally accepted to cause tissue damage throughout the endometrium in most patients.

Intrauterine light delivery for photodynamic therapy of the human endometrium.

Photodynamic therapy is currently being evaluated as a minimally invasive procedure for endometrial ablation not requiring anaesthesia. Light penetration depths at 630, 660 and 690 nm and the optimal configuration of intrauterine light-diffusing fibres were determined in 14 human uteri to assist in the design of a light intrauterine device. Post-menopausal ex-vivo uteri showed a significantly lower light penetration depth than pre-menopausal uteri. With a single central diffusing fibre inserted, the fluence rate measured in the uterine wall at the most remote point of the cavity decreased to 1.1 +/- 0.4% of that measured at closest proximity, whereas it decreased to only 40.0 +/- 9.0% with three fibres. Distension of the uterine cavity with 2 ml of an optically clear fluid increased the fluence rate at the fundus between the fibres at a depth of 2 mm by a factor of 4. We conclude that in normal-sized pre-menopausal uterine cavities, three diffusing fibres will deliver an optical dose above the photodynamic threshold level at a depth of 4 mm, even in the most remote areas, in < 30 min without causing thermal damage. For distorted and elongated cavities, either slight distension of the cavity or the insertion of a fourth diffusing fibre is required.

Determination of the optical properties of the human uterus using frequency-domain photon migration and steady-state techniques.

The optical properties (absorption and transport scattering coefficients) of freshly excised, bulk human uterine tissues were measured at 630 nm using frequency-domain and steady-state photon migration techniques. Measurements were made on both normal (pre- and post-menopausal) and non-neoplastic fibrotic tissues. The absorption coefficient of normal post-menopausal tissue (approximately 0.06 mm(-1)) was found to be significantly greater than that of normal pre-menopausal tissue (0.02-0.03 mm(-1)) and pre-menopausal fibrotic tissue (0.008 mm(-1)). The transport scattering coefficient was similar in all three tissue types considered (0.6-0.9 mm(-1)). From the preliminary results presented here, we conclude that optical properties can be reliably calculated either from the frequency-dependent behaviour of diffusely propagating photon density waves or by combining the frequency-independent photon density wave phase velocity with steady-state light penetration depth measurements. Instrument bandwidth and tissue absorption relaxation time ultimately determine the useful frequency range necessary for frequency-domain photon migration (FDPM) measurements. Based on the optical properties measured in this study, we estimate that non-invasive FDPM measurements of normal uterine tissue require modulation frequencies in excess of 350 MHz.

Portable, high-bandwidth frequency-domain photon migration instrument for tissue spectroscopy.

We describe a novel frequency-domain photon migration instrument employing direct diode laser modulation and avalanche photodiode detection, which is capable of noninvasively determinating the optical properties of biological tissues in near real time. An infinite medium diffusion model was used to extract absorption and transport scattering coefficients from 300-kHz to 800-MHz photon-density wave phase data. Optical properties measured in tissue-simulating solutions at 670 nm agreed to within 10% of those expected.

Experimental tests of a simple diffusion model for the estimation of scattering and absorption coefficients of turbid media from time-resolved diffuse reflectance measurements.

When a picosecond light pulse is incident upon a turbid medium such as tissue, the temporal distribution of diffusely reflected and transmitted photons depends on the optical absorption and scattering properties of the medium. From diffusion theory it is possible to derive analytic expressions for the pulse shape in terms of the optical interaction coefficients of a homogeneous semi-infinite medium. Experimental tests of this simple model in tissue-simulating liquid phantoms of different geometries are presented here. The results of these tests show that, in a semi-infinite phantom, the application of the diffusion model provides estimates of the absorption and transport-scattering coefficients that are accurate to better than 10%. Comparable accuracy was also obtained with this simple model for finite slab, cylindrical, and spherical volumes as long as the objects were of sufficient size. For smaller volumes the absorption coefficient was overestimated because of the significant loss of photons at the bounda ries of the object.

Experimental tests of the feasibility of singlet oxygen luminescence monitoring in vivo during photodynamic therapy.

Singlet oxygen (1O2) is thought to be the cytotoxic agent in photodynamic therapy (PDT) with current photosensitizers. Direct monitoring of 1O2 concentration in vivo would be a valuable tool in studying biological response. Attempts were made to measure 1O2 IR luminescence during PDT of cell suspensions and two murine tumour models using the photosensitizers Photofrin II and aluminium chlorosulphonated phthalocyanine. Instrumentation was virtually identical to that devised by Parker in the one positive report of in vivo luminescence detection in the literature. Despite the fact that our treatments caused cell killing and tissue necrosis, we were unable to observe 1O2 emission under any conditions. We attribute this negative result to a reduction in 1O2 lifetime in the cellular environment. Quantitative calibration of our system allowed us to estimate that the singlet oxygen lifetime in tissue is less than 0.5 microsecond. Some technical improvements are suggested which would improve detector performance and perhaps make such measurements feasible.