Photoirradiation therapy of experimental malignant glioma with 5-aminolevulinic acid

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Object. Accumulation of protoporphyrin IX (PPIX) in malignant gliomas is induced by 5-aminolevulinic acid (5-ALA). Because PPIX is a potent photosensitizer, the authors sought to discover whether its accumulation might be exploited for use in photoirradiation therapy of experimental brain tumors, without injuring normal or edematous brain.

Methods. Thirty rats underwent craniotomy and were randomized to the following groups: 1) photoirradiation of cortex (200 J/cm2, 635-nm argon-dye laser); 2) photoirradiation of cortex (200 J/cm2) 6 hours after intravenous administration of 5-ALA (100 mg/kg body weight); 3) cortical cold injury for edema induction; 4) cortical cold injury with simultaneous administration of 5-ALA (100 mg/kg body weight) and photoirradiation of cortex (200 J/cm2) 6 hours later; or 5) irradiation of cortex (200 J/cm2) 6 hours after intravenous administration of Photofrin II (5 mg/kg body weight). Tumors were induced by cortical inoculation of C6 cells and 9 days later, magnetic resonance (MR) images were obtained. On Day 10, animals were given 5-ALA (100 mg/kg body weight) and their brains were irradiated (100 J/cm2) 3 or 6 hours later. Seventy-two hours after irradiation, the brains were removed for histological examination.

Irradiation of brains after administration of 5-ALA resulted in superficial cortical damage, the effects of which were not different from those of the irradiation alone. Induction of cold injury in combination with 5-ALA and irradiation slightly increased the depth of damage. In the group that received irradiation after intravenous administration of Photofrin II the depth of damage inflicted was significantly greater. The extent of damage in response to 5-ALA and irradiation in brains harboring C6 tumors corresponded to the extent of tumor determined from pretreatment MR images.

Conclusions. Photoirradiation therapy in combination with 5-ALA appears to damage experimental brain tumors selectively, with negligible damage to normal or perifocal edematous tissue.

Abstract

Object. Accumulation of protoporphyrin IX (PPIX) in malignant gliomas is induced by 5-aminolevulinic acid (5-ALA). Because PPIX is a potent photosensitizer, the authors sought to discover whether its accumulation might be exploited for use in photoirradiation therapy of experimental brain tumors, without injuring normal or edematous brain.

Methods. Thirty rats underwent craniotomy and were randomized to the following groups: 1) photoirradiation of cortex (200 J/cm2, 635-nm argon-dye laser); 2) photoirradiation of cortex (200 J/cm2) 6 hours after intravenous administration of 5-ALA (100 mg/kg body weight); 3) cortical cold injury for edema induction; 4) cortical cold injury with simultaneous administration of 5-ALA (100 mg/kg body weight) and photoirradiation of cortex (200 J/cm2) 6 hours later; or 5) irradiation of cortex (200 J/cm2) 6 hours after intravenous administration of Photofrin II (5 mg/kg body weight). Tumors were induced by cortical inoculation of C6 cells and 9 days later, magnetic resonance (MR) images were obtained. On Day 10, animals were given 5-ALA (100 mg/kg body weight) and their brains were irradiated (100 J/cm2) 3 or 6 hours later. Seventy-two hours after irradiation, the brains were removed for histological examination.

Irradiation of brains after administration of 5-ALA resulted in superficial cortical damage, the effects of which were not different from those of the irradiation alone. Induction of cold injury in combination with 5-ALA and irradiation slightly increased the depth of damage. In the group that received irradiation after intravenous administration of Photofrin II the depth of damage inflicted was significantly greater. The extent of damage in response to 5-ALA and irradiation in brains harboring C6 tumors corresponded to the extent of tumor determined from pretreatment MR images.

Conclusions. Photoirradiation therapy in combination with 5-ALA appears to damage experimental brain tumors selectively, with negligible damage to normal or perifocal edematous tissue.

The median survival time in patients with malignant gliomas is limited to little more than 12 months32 with tumor progression usually occurring at the margins of the former resection cavity.1,2 Taking recurrence patterns into account, patients might benefit from more aggressive local therapy. Photodynamic therapy, a form of photoirradiation therapy, is a cancer treatment based on the apparently selective accumulation of photosensitizing drugs in malignant tissue. When activated by light of an appropriate wavelength, the photosensitizer exerts tumor-toxic properties. Photodynamic therapy has proven useful in the treatment of a number of different neoplastic lesions6 and has also been used for local adjuvant therapy of malignant gliomas.12,21,23,25,26,44

Currently, HpD, and its purified versions, porfimer sodium (Photofrin) and dihematoporphyrin ether (Photofrin II) are used for photoirradiation therapy in combination with laser light in the range of 630 to 635 nm. These sensitizers, however, have the profound disadvantage of causing prolonged skin sensitization. Patients treated with these drugs are required to stay out of direct and indirect sunlight for as long as 2 months. Furthermore, damage to normal brain tissue has been reported in preclinical studies3,5,44 and local treatment selectivity may be limited, because these sensitizers appear to participate in edema bulk flow and thus may be transported into brain regions devoid of tumor.34

Recently, we investigated a novel substance, 5-ALA, for use in intraoperative fluorescence-guided resection of malignant gliomas.36–39 The substance 5-ALA is a naturally occurring metabolite in the heme biosynthesis pathway. Excess exogenous 5-ALA leads to accumulation of highly fluorescent heme precursor porphyrins, such as PPIX, in a number of malignant tissues,16,24 including malignant gliomas.36,38,39 Accumulation is highly specific and does not appear to occur in normal brain; however, apart from their strong fluorescence, endogenous porphyrins such as PPIX are also efficient photosensitizers.16,20 In contrast to hematoprophyrin derivatives, side-effects of 5-ALA in patients are negligible and include mild elevation of liver enzymes and a period of skin sensitization limited to fewer than 48 hours.7,19,27,36,39 Consequently, photoirradiation therapy with 5-ALA—induced porphyrins may be a modality worth investigating, especially as an adjuvant treatment after 5-ALA fluorescence-guided resection of malignant gliomas and for direct treatment of residual tumor areas for which the use of conventional surgical techniques may prove too dangerous.

To qualify as a photosensitizer, 5-ALA must fulfill a number of prerequisites. Its accumulation should be specific for malignant glioma tissue. It should not propagate or diffuse with cerebral edema to sensitize adjacent normal brain tissue while exerting sufficient phototoxic effects on tumor tissue.

The present investigation seeks to elucidate whether the photosensitizing effects of 5-ALA—induced porphyrins are sufficient for photoirradiation therapy in an animal model of glioma. Magnetic resonance imaging was used to determine tumor size prior to therapy and to compare it with the extent of phototoxic tumor damage. To determine the degree of unwanted sensitization of perifocal edematous tissue, a brain edema model was used.17,34 This edema model allowed assessment of any damage related to edema alone, thus allowing us to rule out interference from sensitized infiltrating tumor cells. Finally, 5-ALA was compared with a traditional sensitizer, Photofrin II, with regard to unwanted sensitization of normal brain.

Materials and Methods

General Procedures

All procedures were performed in accordance with German animal protection laws after review of the experimental protocol by the Bavarian government. Spontaneously breathing male Wistar rats weighing 240 to 260 g were anesthetized with isoflurane (1–2%) in oxygen during tumor implantation, cold lesioning, and photoirradiation. The animals were maintained on temperature-controlled feedback heating pads at 37°C. Their heads were immobilized in a stereotactic head holder and a right parietal craniotomy (4 mm length × 3 mm breadth) was performed using a high-speed drill, bordering to the midline and the coronal suture for all irradiation experiments. Care was taken not to injure the dura mater to avoid cortical damage.

The 5-ALA (Medac GmbH, Hamburg, Germany) was obtained as a hydrochloride powder. For intravenous administration, 5-ALA was dissolved in phosphate-buffered saline at a concentration of 30 mg/ml immediately before use and adjusted to a pH of 6.5. Photofrin II (Lederle Parenterals, Puerto Rico, USA) was dissolved in a 5% glucose solution at a concentration of 2.5 mg/ml. All injections were performed by direct puncture of the exposed left femoral vein.

An argon-pumped rhodamine dye laser (Coherent INNOVA Sabre R DBW 15 dye laser: Coherent 599; Coherent, Darmstadt, Germany) was used for photoillumination at 635 nm of red light. The power density was adjusted to 100 mW/cm2 and an energy density of 200 J/cm2 was applied. This dose was considered the upper limit to be expected in the clinical setting. Prior to and after photoillumination the power density was checked using a power meter (Labmaster; Coherent). For monitoring of power density during photoirradiation, a beamsplitter deflected a constant portion of laser light onto a photodiode. Light density was manually readjusted when deviations of more than 5% occurred.

Subsequent to photoillumination during the different experiments, skin incisions were sutured. The brains were removed for histological analysis 72 hours later. For this purpose animals were deeply anesthetized with ether and perfusion-fixed by cardiac puncture and rinsing with phosphate-buffered paraformaldehyde (2%), pH 7.4, after rinsing with 0.9% saline. Paraffin-embedded brain tissue blocks were cut serially into 5µm-coronal slices and stained with hematoxylin and eosin. Lesion depths were determined by measuring the extent of morphological changes perpendicular to the brain surface, that is, in the direction of laser light propagation. For this purpose, videomicroscope images at × 20 magnification of the slice were obtained through the lesion demonstrating the deepest extent of damage. Gross tissue necrosis with loss of tissue coherence, loss of neurons and glia, regions of diffuse tissue hemorrhage, karyopyknosis of brain cells, and perinuclear vacuolization were considered to be indicators of phototoxic damage. The extent of more subtle injury, such as karyopyknosis and perinuclear vacuolization, was determined under × 80 magnification.

Effect of Photoirradiation on Normal Brain Tissue

To determine the impact of photoirradiation therapy on normal brain tissue after intravenous administration of 5-ALA, six rats were given 100 mg/kg of 5-ALA. A second group of animals received 5 mg/kg of Photofrin II to determine the effects of a conventional photosensitzer in a clinically relevant dosage.18 Six hours later, the exposed dura and underlying brain in both groups were irradiated at a total dose of 200 J/cm2. To assess the extent of thermal damage induced by solitary laser irradiation, the cortices of six control rats were treated without prior administration of a photosensitizer.

Effect of Photoirradiation on Edematous Brain Tissue

Systemic administration of 5-ALA leads to elevated serum porphyrins,30,42 which might participate in tumor edema propagation, thus unspecifically sensitizing edematous perifocal brain tissue. To test this hypothesis with regard to 5-ALA—induced serum porphyrins, the cold injury model17 was used, with modifications.34 Briefly, cold injury to the cortex was performed by placing a 1-mm-diameter copper stamp, cooled to −68°C by a mixture of dry ice and acetone, on the exposed dura for 15 seconds with the aid of a micromanipulator. In six animals, 5-ALA was administered intravenously at a dose of 100 mg/kg body weight 3 hours prior to cortical cold injury. Six hours later, that is, 3 hours after cold injury, the region of the lesion and adjacent exposed cortex were irradiated with 200 J/cm2. In six additional animals, cold injury was induced without subsequent irradiation to quantify the depth of tissue damage resulting from cold injury alone.

Effect of Photoirradiation on C6 Gliomas

For the present experiments, the C6 model of malignant glioma was used as previously described, with minor modifications.38 The C6 glioma cells were grown in monolayer tissue cultures in Dulbecco minimal essential medium supplemented with 10% fetal calf serum and sodium pyruvate (1 mM) at 37°C in a 5% carbon dioxide atmosphere. Penicillin G (300 IU/ml) and streptomycin (300 µg/ml) were added to the medium to prevent bacterial infection. Cultures between passages 72 and 88 were harvested by trypsinization, and the cells were suspended in saline at a concentration of 2 × 104 cells/µl. Five µl (105 cells) was stereotactically implanted using a 10-µl syringe via a small burr hole 2 mm lateral and 2 mm caudal to the bregma, that is, the region designated for later craniotomy. Cells were implanted at a depth of 2 mm from the skull surface, within the animals' cortices. A superficial location was chosen to ensure reliable access to evolving tumors for photoirradiation via the planned craniotomy and to prevent the syringe needle from traversing the ventricle, potentially resulting in disseminated intrathecal rather than circumscribed tumor growth. The location was also chosen to ensure the detection of even superficial phototoxic tumor lesions that might be missed if the tumor cells were implanted more deeply.

To examine the tumor configuration and accurately assess the extent of phototoxic tumor damage, MR images were obtained in all animals on Day 9 after tumor implantation. Animals received an intraperitoneal injection of 3.6% chloral hydrate anesthetic (1.2 ml/100 g body weight). All animals received 0.45 ml Gd—diethylenetriamine pentaacetic acid (Magnevist; Schering AG Pharma, Berlin, Germany) intravenously immediately prior to undergoing T1-weighted MR imaging (slice thickness 2 mm; TR 480 msec; TE 14 msec) for which a 1.5-tesla unit was used (Magnetom Vision; Siemens, Munich, Germany). Images were printed on Dry Star TM 1b films (Agfa, Cologne, Germany) for determining the depth of contrast-enhancing tumor and the coronal dimension of the brain.

Animals were reanesthetized the following day in preparation for craniotomy and photoirradiation therapy (100 J/cm2) either 3 or 6 hours after intravenous administration of 5-ALA 100 mg/kg body weight. The lower energy density in these experiments compared with the control experiments in animals without tumors was used to test for tumor toxicity at energy levels expected to be more commonly achieved clinically. The time points were chosen in accordance with previous experiments measuring porphyrin accumulation in this model.38 The brains were removed after perfusion-fixation 72 hours later, embedded, cut, and stained. Lesion depths perpendicular to the surface were measured, along with the coronal diameter of the brain in the histological section. To account for specimen shrinkage associated with the embedding procedure, this value was compared with the corresponding measurement available from the MR image. The ratio of histological neuroimaging to measurements for coronal brain diameters was then used to correct the value for the depth of the histological lesion.

To test the accuracy of predicting histological tumor size from tumor dimensions obtained on MR imaging, a separate group of five animals underwent implantation of C6 gliomas and were subjected to the same protocol; however, this group did not undergo photoirradiation therapy. In these animals, the size of contrast-enhancing tumor was unequivocally measured in two dimensions: its greatest diameter and the size perpendicular to its greatest diameter. These values were compared with the respective measurements obtained histologically, which were corrected for specimen shrinkage.

Statistical Analysis

All data are reported as the means ± SDs. Group differences were tested by analysis of variance with the post hoc Scheffé F-test.

Results
Photoirradiation Therapy of Normal and Edematous Brain Tissue

Each experimental group was composed of six animals. The results obtained in these groups were assessed for effects of laser irradiation of normal or edematous cortex with or without pretreatment with 5-ALA and for irradiation of normal cortex after Photofrin II pretreatment. Solitary laser irradiation of normal cortex without prior administration of 5-ALA resulted in a superficial layer of tissue changes characterized by karyopyknosis and perinuclear vacuolization at a mean depth of 0.44 ± 0.07 mm (Fig. 1). The same type and depth of damage was observed when 5-ALA was given 6 hours before laser irradiation (0.41 ± 0.08 mm). Cold lesioning alone resulted in a sharply demarcated area of coagulative necrosis with destruction of all cellular elements. The mean lesion depth was 0.44 ± 0.11 mm; however, subsequent photoirradiation therapy, after administration of 5-ALA, aggravated the existing damage (0.83 ± 0.31 mm). The resulting lesions exhibited superficial coagulative necrosis in the area closer to the light source, with selective neuronal damage beyond this area. Laser irradiation of normal cortex after pretreatment of animals with Photofrin II produced sharply demarcated coagulative necrosis with a mean depth of 1.77 ± 0.22 mm, which was significantly greater than the depth of damage observed in the other groups. Figure 2 shows an example of the different qualities of cortical damage found in an animal after simultaneous cold injury for induction of vasogenic edema and treatment with 5-ALA 6 hours before photoirradiation.

Fig. 1.
Fig. 1.

Graph showing mean depths ± SDs of cortical damage observed after solitary laser irradiation (200 J/cm)2. Laser only group: six rats; laser irradiation (200 J/cm2) after intravenous administration 100 mg/kg of 5-ALA 6 hours earlier (ALA + Laser group: six rats); cortical cold lesion (Injury only group: six rats); simultaneous cold lesioning and administration of 100 mg/kg 5-ALA laser irradiation (200 J/cm2) 6 hours later (Injury + ALA + Laser group: six rats); and laser irradiation of normal cortex 6 hours after intravenous administration of 5 mg/kg Photofrin II (PF II + Laser group: six rats, *p < 0.01 compared with others).

Fig. 2.
Fig. 2.

Histological section showing various types of cortical damage encountered in response to photoirradiation therapy, in this case after simultaneous cold lesioning and administration of 5-ALA (100 mg/kg body weight) with laser irradiation (200 J/cm2) 6 hours later. a: Superficial coagulative necrosis; b: region of selective neuronal damage and margin of edema; c: circumscribed hemorrhage; and d: total depth of phototoxic damage. H & E, bar = 2 mm.

Photoirradiation Therapy in C6 Gliomas
Comparison of Tumor Size

To determine whether it was possible to predict histological tumor size accurately based on MR imaging, the greatest tumor diameter and the respective diameter perpendicular to the greatest diameter were measured on the images (Fig. 3 upper right) and on the corresponding histological section (Fig. 3 upper left). The mean of the greatest diameters of tumors in the histological sections was 3.72 ± 2.31 mm; the corresponding extent of contrast enhancement on the MR image was 3.78 ± 2.38 mm (Fig. 3 lower). The respective perpendicular measurements were 1.95 ± 1.49 mm and 1.88 ± 1.54 mm, respectively. The mean differences between MR imaging—derived and histologically derived diameters were 0.06 ± 0.24 mm (p = 0.61) for the greatest diameter and 0.07 ± 0.29 mm (p = 0.63) for the perpendicular diameter. Histologically derived tumor diameters were accurately demonstrated on MR images.

Fig. 3.
Fig. 3.

Upper Left: Histological section of a typical C6 tumor with measurement lines used for unequivocally determining tumor size. H & E, bar = 10 mm. Upper Right: Corresponding coronal MR image with measurement lines for estimating tumor size. Lower: Graph showing mean tumor dimensions as determined based on MR images and histological sections (mean ± SD in six rats).

Effects of Photoirradiation Therapy on Tumors

Magnetic resonance images were obtained in two groups of rats that were to undergo photoirradiation therapy, either 3 or 6 hours after administration of 100 mg/kg of 5-ALA. The mean depth of the tumor, measured perpendicular to the brain surface, was 2.7 ± 0.76 mm in the 3-hour group and 2.6 ± 1.08 mm in the 6-hour group.

Seventy-two hours after the rats underwent photoirradiation, their brains were removed subsequent to perfusion-fixation for histological assessment of damage depths. Phototoxic damage was observed in all animals at the site of tumor growth previously demonstrated on MR imaging. Histologically, damage was characterized either by coagulative or hemorrhagic necrosis (Fig. 4), sometimes surrounded by a region of perilesional pallor. Phototoxic damage was found down to a mean depth of 2.8 ± 0.16 mm in tumors in the 3-hour group and down to 2.7 ± 0.87 mm in the 6-hour group (Fig. 5). The deepest extent of damage (3.7 mm) was measured in an animal from the 6-hour group; MR imaging in this animal demonstrated the depth of damage to be 4 mm. Phototoxic damage was not always homogeneously distributed throughout the tumors. In some cases, residual nests of tumor cells, which appeared viable, were contained within apparently damaged tumor tissue. In these cases, the deepest extent of necrosis was measured.

Fig. 4.
Fig. 4.

Photomicrograph of a representative example for hemorrhagic tumor damage caused by irradiation with 100 J/cm2 of 635 nm laser light 6 hours after administration of 100 mg/kg 5-ALA. Note perilesional pallor of tissue. H & E, bar = 2 mm. Inset: Pretreatment MR image in the same animal.

Fig. 5.
Fig. 5.

Graph showing tumor depths as determined based on pretreatment MR imaging and extent of histological damage as determined from hematoxylin-eosin—stained brain sections 72 hours after PDT (3 hours: PDT 3 hours after administration of 5-ALA, six rats in each group, means ± SDs).

Compared with the control groups, in which normal and edematous brain was irradiated after administration of 5-ALA, the depth of damage in tumor tissue was significantly greater (p < 0.05).

Discussion
Photoirradiation Therapy of Malignant Gliomas

Photodynamic therapy has been used as an adjuvant after surgery for malignant gliomas, with the aim of killing residual tumor cells and prolonging time to recurrence. It relies on preferential accumulation of photosensitizers in brain tumor tissue.10,15,44 Activation of the photosensitizer by light of an appropriate wavelength leads to the destruction of sensitized tissue. Thus, this mode of therapy may serve to extend the existing treatment armamentarium without adversely interacting with traditional treatment modalities (radio- and chemotherapy) and without causing additive side effects. Malignant gliomas have been shown to respond to PDT;12,22 however, randomized prospective studies proving the efficacy of PDT for prolonging patient survival are still lacking. The most common regimen for treating brain tumors by PDT at the moment consists of intravenous administration of a porphyrin sensitizer (HpD Photofrin II) prior to surgery and irradiation of the resection cavity by using laser light at a wavelength of 630 to 635 nm.12,21,25,44

Five-Aminolevulinic Acid as a Photosensitizer

Five-aminolevulinic acid is conceptually different from traditional sensitizers; as a precursor itself, it is converted into a highly fluorescent and photosensitizing heme metabolite, PPIX, within malignant glioma tissue.8,36,38,39 The ability of 5-ALA to label specifically malignant gliomas has been exploited for enhancing their resection.36,39 Nevertheless, complete resection of malignant glioma tissue is not always possible when tumor borders on or extends into eloquent brain regions; resection then carries the risk of neurological deficit. In these cases, photoactivation of porphyrins in fluorescing tumor remnants may enable further cytoreduction. In addition, adjuvant illumination of the tumor cavity may serve to destroy infiltrating cells harboring PPIX.

For a substance to qualify as a sensitizer for photoirradiation therapy of malignant gliomas, it must meet a number of criteria. Ideally, the substance should be nontoxic and rapidly eliminated to prevent prolonged side effects. It should accumulate selectively in tumor tissue and reach infiltrating glioma cells, ensuring selective phototoxicity. Furthermore, it should be activated by a long wavelength light for maximum tissue penetration (650–800 nm). Sensitization should be great enough to allow low light doses and deliverable within a reasonable period of time in the operating room to cause significant damage. It appears that 5-ALA fulfills a number of these requirements.

When administered at a dose of 20 mg/kg, 5-ALA is well tolerated, causing photosensitization of the skin for fewer than 48 hours and mild temporary elevations of liver enzymes. When doses have exceeded 40 mg/kg,9,43 mild cardiopulmonary reactions have been reported, which are easily counteracted. Previous experimental and clinical work has demonstrated a high degree of selectivity with respect to the accumulation of fluorescent PPIX in malignant glioma tissue.8,36,39 Experimentally38 and clinically,36 a cytoplasmatic pattern of PPIX accumulation has also been observed in parts of infiltrating tumor.

Accordingly, the present experiments demonstrate a slight degree of superficial damage to cortex in response to 200 J/cm2 of laser light, the highest possible dose expected to be used in the clinical setting. Damage was not aggravated by prior administration of 5-ALA. In contrast, irradiation of normal cortex after prior administration of Photofrin II resulted in cortical damage exceeding that from laser light alone. On the other hand, the use of a lower light dose (100 J/cm2), a dose that would be more common in clinical usage, resulted in damage to tumor bearing tissue. The extent of damage corresponded to tumor dimensions demonstrated on pretreatment MR images, confirming the ability of 5-ALA to sensitize experimental malignant glioma selectively. The perilesional pallor observed in some of the animals that received photoirradiation treatment of tumors has also been observed in response to photoirradiation therapy using Photofrin II.11 In these experiments, pallor was demonstrated to be the consequence of BBB disturbance with ensuing edema, detectable on Days 1 to 3 but not on Day 7 after photoirradiation therapy.

Penetration Depth

In the present experiments, maximum phototoxic tissue reactions were observed to a depth of 3.7 mm, corresponding to the depth of this particular tumor demonstrated on pretreatment MR images. This value, however, may not represent the limit of lesion depth achievable with 5-ALA. For activation of HpD or Photofrin, the optimal wavelength is 628 nm. With these sensitizers, experimental damage has been perceived down to 7 mm14 and to 18 mm in the clinical setting.13 The wavelength at which the most pronounced phototoxic effect has been observed with 5-ALA—induced PPIX is slightly greater at 635 nm.31,33,40 This wavelength was used in the present experiments. The maximum therapeutic depth achievable using 5-ALA should therefore be similar to traditional porphyrin sensitizers, provided that the photosensitizer is present in sufficient concentrations.

In the present experiments, interestingly, no differences in lesion depth were observed when comparing tumors irradiated 3 hours and 6 hours after administration of 5-ALA, despite markedly lower PPIX fluorescence intensities at 3 hours than at 6 hours, which were previously reported.38 This observation infers that even the low concentrations of PPIX available 3 hours after administration of 5-ALA sensitize tumor sufficiently. Alternately, lower 5-ALA doses might suffice to enable a significant phototoxic effect.

Influence of Vasogenic Edema

Tumor-associated edema, which is vasogenic in nature, is of special concern for photoirradiation therapy of malignant gliomas. Edema results from tumor vessels with wide junctional clefts and endothelial fenestrations, enabling edema fluid to leave the vascular system.4 Because of its clearance by bulk flow, tumor-associated edema can be found far beyond tumor borders.28,29 Intravenously administered photosensitizers, such as Photofrin II, propagate with vasogenic edema into perifocal edematous but otherwise normal brain tissue, resulting in nonspecific phototoxcity.34,35 Experimentally, 5-ALA—induced porphyrins have been observed outside tumor tissue in regions where there is tumor-associated edema,8,38 but only to a mean distance of 1.5 mm.38 The origin of these porphyrins has not yet been elucidated. They might represent extravasated plasma prophyrins induced by 5-ALA; they might originate in tumor washed out by edema into perifocal tissue; they might result from nonspecific porphyrin synthesis in normal brain tissue that is exposed to edema-borne 5-ALA. It is also unknown whether normal neuronal or glial cells are basically capable of porphyrin synthesis after exposure to 5-ALA. It has been shown, however, that the normal BBB is virtually impermeable to hydrophilic 5-ALA41 so that any 5-ALA entering the tumor or perifocal tissue must do so through the leaky BBB within the tumor.

To test whether the concentrations of edema-associated porphyrins were great enough to nonspecifically sensitize perifocal tissue after administration of 5-ALA, a cortical cold lesion was induced in rats without tumor. The cold lesion gives rise to vasogenic edema, which is somewhat similar to tumor-related edema and has been previously used to study porphyrin kinetics in perifocal edematous tissue.8,34 In the cold injury experiments, photoillumination of edematous tissue led to a significant enlargement of necrosis, but only to a depth of 0.9 mm. This was double the lesion depth caused by the cold injury itself but was less than the depth of damage arising from irradiation of normal cortex sensitized with Photofrin II. The small zone of additional damage resulting from edema-associated porphyrins in the present experimental glioma model should not pose a concern in the human setting, where nonspecific accumulation of fluorescent porphyrins in perifocal edematous brain tissue has basically been ruled out based on 264 biopsy samples obtained from the margins of malignant gliomas in humans.36

Conclusions

To summarize, the present experiments demonstrate selective damage to experimental glioma after sensitization with 5-ALA and appropriate photoirradiation. Normal brain tissue was not susceptible to photoirradiation therapy, whereas only mild sensitization was observed in tissue harboring perifocal edema in the cold lesion model. In light of the positive experience gained from the clinical use of 5-ALA fluorescence-guided resection with regard to substance toxicity and particularly specificity of accumulation, elucidation of photoirradiation therapy with 5-ALA in the clinical setting appears warranted. Phase I/II studies in patients harboring primary or recurrent malignant gliomas will ultimately define the clinical potential of this approach, either for adjuvant therapy of tumor cavities subsequent to surgery or for treating residual fluorescing tumor tissue not amenable to surgical resection.

Acknowledgments

We gratefully acknowledge ongoing technical support and advice from Reinhold Baumgartner, Ph.D., and Herbert Stepp, Ph.D., Laser Research Laboratory, Ludwig-Maximilians-University.

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  • 24.

    Peng QBerg KMoan Jet al: 5-aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem Photobiol 65:2352511997Photochem Photobiol 65:

  • 25.

    Perria CCarai MFalzoi Aet al: Photodynamic therapy of malignant brain tumors: clinical results of, difficulties with, questions about, and future prospects for the neurosurgical applications. Neurosurgery 23:5575631988Neurosurgery 23:

  • 26.

    Popovic EAKaye AHHill JS: Photodynamic therapy of brain tumors. J Clin Laser Med Surg 14:2512611996J Clin Laser Med Surg 14:

  • 27.

    Regula JMacRobert AJGorchein Aet al: Photosensitization and photodynamic therapy of oesophageal, duodenal, and colorectal tumors using 5-aminolaevulinic acid induced protoporphyrin IX—a pilot study. Gut 36:67751995Gut 36:

  • 28.

    Reulen HJGraham RSpatz Met al: Role of pressure gradients and bulk flow in dynamics of vasogenic brain edema. J Neurosurg 46:24351977J Neurosurg 46:

  • 29.

    Reulen HJTsuyumu MTack Aet al: Clearance of edema fluid into cerebrospinal fluid. A mechanism for resolution of vasogenic brain edema. J Neurosurg 48:7547641978J Neurosurg 48:

  • 30.

    Rick KSroka RStepp Het al: Pharmacokinetics of 5-aminolevulinic acid-induced protoporphyrin IX in skin and blood. J Photochem Photobiol B 40:3133191997J Photochem Photobiol B 40:

  • 31.

    Riesenberg RFuchs CKriegmair M: Photodynamic effect of 5-aminolevulinic acid-induced porphyrin on human bladder carcinoma cells in vitro. Eur J Cancer 32A:3283341996Eur J Cancer 32A:

  • 32.

    Shapiro WRGreen SBBurger PCet al: Randomized trial of three chemotherapy regimens and two radiotherapy regimens in postoperative treatment of malignant glioma. Brain Tumor Cooperative Group Trial 8001. J Neurosurg 71:191989J Neurosurg 71:

  • 33.

    Stocker SKnüchel RSroka Ret al: Wavelength dependent photodynamic effects on chemically induced rat bladder tumor following intravesical instillation of 5-aminolevulinic acid. J Urol 157:357361 1997J Urol 157:357–361 1997

  • 34.

    Stummer WGoetz CHassan Aet al: Kinetics of Photofrin II in perifocal brain edema. Neurosurgery 33:107510821993Neurosurgery 33:

  • 35.

    Stummer WHassan AKempski Oet al: Photodynamic therapy within edematous brain tissue: considerations on sensitizer dose and time point of laser irradiation. J Photochem Photobiol B 36:1791811996J Photochem Photobiol B 36:

  • 36.

    Stummer WNovotny AStepp Het al: Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg 93:100310132000J Neurosurg 93:

  • 37.

    Stummer WStepp HMoller Get al: Technical principles for protoporphyrin-IX-fluorescence guided microsurgical resection of malignant glioma tissue. Acta Neurochir 140:99510001998Acta Neurochir 140:

  • 38.

    Stummer WStocker SNovotny Aet al: In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid. J Photochem Photobiol B 45:1601691998J Photochem Photobiol B 45:

  • 39.

    Stummer WStocker SWagner Set al: Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery 42:5185261998Neurosurgery 42:

  • 40.

    Szeimies RMAbels CFritsch Cet al: Wavelength dependency of photodynamic effects after sensitization with 5-aminolevulinic acid in vitro and in vivo. J Invest Dermatol 105:6726771995J Invest Dermatol 105:

  • 41.

    Terr LWeiner LP: An autoradiographic study of delta-aminolevulinic acid uptake by mouse brain. Exp Neurol 79:5645681983Exp Neurol 79:

  • 42.

    Webber JKessel DFromm D: Plasma levels of protoporphyrin IX in humans after oral administration of 5-aminolevulinic acid. J Photochem Photobiol B 37:1511531997J Photochem Photobiol B 37:

  • 43.

    Webber JKessel DFromm D: Side effects and photosensitization of human tissues after aminolevulinic acid. J Surg Res 68:31371997J Surg Res 68:

  • 44.

    Whelan HTSchmidt MHSegura ADet al: The role of photodynamic therapy in posterior fossa brian tumors. A preclinical study in a canine glioma model. J Neurosurg 79:5625681993J Neurosurg 79:

This work was supported by grants from the Curt-Bohnewand-Fonds, the K.L. Weigand'sche Stiftung and the Gravenhorst estate to W. Stummer.

Article Information

Address reprint requests to: Walter Stummer, M.D., Department of Neurosurgery, Klinikum Grosshadern, Ludwig-Maximilians-University, 81366 Munich, Germany. email: wstummer@nc.med.uni-muenchen.de.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Graph showing mean depths ± SDs of cortical damage observed after solitary laser irradiation (200 J/cm)2. Laser only group: six rats; laser irradiation (200 J/cm2) after intravenous administration 100 mg/kg of 5-ALA 6 hours earlier (ALA + Laser group: six rats); cortical cold lesion (Injury only group: six rats); simultaneous cold lesioning and administration of 100 mg/kg 5-ALA laser irradiation (200 J/cm2) 6 hours later (Injury + ALA + Laser group: six rats); and laser irradiation of normal cortex 6 hours after intravenous administration of 5 mg/kg Photofrin II (PF II + Laser group: six rats, *p < 0.01 compared with others).

  • View in gallery

    Histological section showing various types of cortical damage encountered in response to photoirradiation therapy, in this case after simultaneous cold lesioning and administration of 5-ALA (100 mg/kg body weight) with laser irradiation (200 J/cm2) 6 hours later. a: Superficial coagulative necrosis; b: region of selective neuronal damage and margin of edema; c: circumscribed hemorrhage; and d: total depth of phototoxic damage. H & E, bar = 2 mm.

  • View in gallery

    Upper Left: Histological section of a typical C6 tumor with measurement lines used for unequivocally determining tumor size. H & E, bar = 10 mm. Upper Right: Corresponding coronal MR image with measurement lines for estimating tumor size. Lower: Graph showing mean tumor dimensions as determined based on MR images and histological sections (mean ± SD in six rats).

  • View in gallery

    Photomicrograph of a representative example for hemorrhagic tumor damage caused by irradiation with 100 J/cm2 of 635 nm laser light 6 hours after administration of 100 mg/kg 5-ALA. Note perilesional pallor of tissue. H & E, bar = 2 mm. Inset: Pretreatment MR image in the same animal.

  • View in gallery

    Graph showing tumor depths as determined based on pretreatment MR imaging and extent of histological damage as determined from hematoxylin-eosin—stained brain sections 72 hours after PDT (3 hours: PDT 3 hours after administration of 5-ALA, six rats in each group, means ± SDs).

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Peng QBerg KMoan Jet al: 5-aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem Photobiol 65:2352511997Photochem Photobiol 65:

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Perria CCarai MFalzoi Aet al: Photodynamic therapy of malignant brain tumors: clinical results of, difficulties with, questions about, and future prospects for the neurosurgical applications. Neurosurgery 23:5575631988Neurosurgery 23:

26.

Popovic EAKaye AHHill JS: Photodynamic therapy of brain tumors. J Clin Laser Med Surg 14:2512611996J Clin Laser Med Surg 14:

27.

Regula JMacRobert AJGorchein Aet al: Photosensitization and photodynamic therapy of oesophageal, duodenal, and colorectal tumors using 5-aminolaevulinic acid induced protoporphyrin IX—a pilot study. Gut 36:67751995Gut 36:

28.

Reulen HJGraham RSpatz Met al: Role of pressure gradients and bulk flow in dynamics of vasogenic brain edema. J Neurosurg 46:24351977J Neurosurg 46:

29.

Reulen HJTsuyumu MTack Aet al: Clearance of edema fluid into cerebrospinal fluid. A mechanism for resolution of vasogenic brain edema. J Neurosurg 48:7547641978J Neurosurg 48:

30.

Rick KSroka RStepp Het al: Pharmacokinetics of 5-aminolevulinic acid-induced protoporphyrin IX in skin and blood. J Photochem Photobiol B 40:3133191997J Photochem Photobiol B 40:

31.

Riesenberg RFuchs CKriegmair M: Photodynamic effect of 5-aminolevulinic acid-induced porphyrin on human bladder carcinoma cells in vitro. Eur J Cancer 32A:3283341996Eur J Cancer 32A:

32.

Shapiro WRGreen SBBurger PCet al: Randomized trial of three chemotherapy regimens and two radiotherapy regimens in postoperative treatment of malignant glioma. Brain Tumor Cooperative Group Trial 8001. J Neurosurg 71:191989J Neurosurg 71:

33.

Stocker SKnüchel RSroka Ret al: Wavelength dependent photodynamic effects on chemically induced rat bladder tumor following intravesical instillation of 5-aminolevulinic acid. J Urol 157:357361 1997J Urol 157:357–361 1997

34.

Stummer WGoetz CHassan Aet al: Kinetics of Photofrin II in perifocal brain edema. Neurosurgery 33:107510821993Neurosurgery 33:

35.

Stummer WHassan AKempski Oet al: Photodynamic therapy within edematous brain tissue: considerations on sensitizer dose and time point of laser irradiation. J Photochem Photobiol B 36:1791811996J Photochem Photobiol B 36:

36.

Stummer WNovotny AStepp Het al: Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg 93:100310132000J Neurosurg 93:

37.

Stummer WStepp HMoller Get al: Technical principles for protoporphyrin-IX-fluorescence guided microsurgical resection of malignant glioma tissue. Acta Neurochir 140:99510001998Acta Neurochir 140:

38.

Stummer WStocker SNovotny Aet al: In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid. J Photochem Photobiol B 45:1601691998J Photochem Photobiol B 45:

39.

Stummer WStocker SWagner Set al: Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery 42:5185261998Neurosurgery 42:

40.

Szeimies RMAbels CFritsch Cet al: Wavelength dependency of photodynamic effects after sensitization with 5-aminolevulinic acid in vitro and in vivo. J Invest Dermatol 105:6726771995J Invest Dermatol 105:

41.

Terr LWeiner LP: An autoradiographic study of delta-aminolevulinic acid uptake by mouse brain. Exp Neurol 79:5645681983Exp Neurol 79:

42.

Webber JKessel DFromm D: Plasma levels of protoporphyrin IX in humans after oral administration of 5-aminolevulinic acid. J Photochem Photobiol B 37:1511531997J Photochem Photobiol B 37:

43.

Webber JKessel DFromm D: Side effects and photosensitization of human tissues after aminolevulinic acid. J Surg Res 68:31371997J Surg Res 68:

44.

Whelan HTSchmidt MHSegura ADet al: The role of photodynamic therapy in posterior fossa brian tumors. A preclinical study in a canine glioma model. J Neurosurg 79:5625681993J Neurosurg 79:

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