Fluorescence real-time kinetics of protoporphyrin IX after 5-ALA administration in low-grade glioma.

OBJECTIVE: 5-Aminolevulinic acid (5-ALA) induces fluorescence in high-grade glioma (HGG), which is used for resection. However, the value of 5-ALA-induced fluorescence in low-grade glioma (LGG) is unclear. Time dependency and time kinetics have not yet been investigated. The purpose of this study was to investigate real-time kinetics of protoporphyrin IX (PpIX) in LGG based on hyperspectral fluorescence-based measurements and identify factors that predict fluorescence. METHODS: Patients with grade II gliomas and imaging from which HGGs could not be completely ruled out received 5-ALA at 20 mg/kg body weight 4 hours prior to surgery. Fluorescence intensity (FI) and PpIX concentration (CPpIX) were measured in tumor tissue utilizing a hyperspectral camera. Apparent diffusion coefficient (ADC)-based tumor cell density, Ki-67/MIB-1 index, chromosomal 1p/19q codeletion, and 18F-fluoroethyl-l-tyrosine (18F-FET) PET values and their role for predicting fluorescence were evaluated. RESULTS: Eighty-one biopsies from 25 patients were included. Tissues with fluorescence demonstrated FI and CPpIX maxima between 7 and 8 hours after administration. When visible fluorescence was observed, peaks of FI and CPpIX were observed within this 7- to 8-hour time frame, regardless of any MRI gadolinium contrast enhancement. Gadolinium enhancement (p = 0.008), Ki-67/MIB-1 index (p < 0.001), 18F-FET PET uptake ratio (p = 0.004), and ADC-based tumor cellularity (p = 0.017) significantly differed between fluorescing and nonfluorescing tissue, but not 1p/19q codeletions. Logistic regression demonstrated that 18F-FET PET uptake and Ki-67/MIB-1 index were independently related to fluorescence. CONCLUSIONS: This study reports a fluorescence-based assessment of CPpIX in human LGG tissues related to 18F-FET PET uptake and Ki-67/MIB-1. As in HGGs, fluorescence in LGGs peaked between 7 and 8 hours after 5-ALA application, which has consequences for the timing of administration.

5-ALA kinetics in meningiomas: analysis of tumor fluorescence and PpIX metabolism in vitro and comparative analyses with high-grade gliomas.

INTRODUCTION: Although the utility 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is increasingly discussed, data about the kinetics of protoporphyrin IX (PpIX) and tumor fluorescence are sparse. METHODS: PpIX kinetics after exposition to varying 5-ALA doses (12.5-150 µg/ml) was analyzed in two immortalized as well as primary WHO grade I and II meningioma and U87 high-grade glioma cell lines. Expression of FECH, ABCB6 and ABCG2 was investigated by quantitative real-time PCR. RESULTS: Fluorescence in Ben-Men 1 and primary WHO grade I/II meningioma increased with rising 5-ALA doses up to 100 µg/ml but then showed a saturation effect. However, decrease of fluorescence was slower after 150 than after 100 µg/ml 5-ALA. Fluorescence in U87 cells marginally increased with rising 5-ALA doses. Kinetics of the fluorescence in Ben-Men 1 cells did not differ from primary meningioma cells after 25-150 µg/ml 5-ALA (p > .05, each). No difference was found when comparing the fluorescence between primary grade I and II meningiomas after any 5-ALA dosage (p > .05, each). No relevant fluorescence was found in IOMM-Lee cells. Expression of FECH, ABCB6 and ABCG2 as well as PpIX export differed between all analyzed cell lines but were not connected to fluorescence. CONCLUSIONS: Eligibility of established meningioma cell lines for in-vitro analyzes of tumor fluorescence significantly differs. Fluorescence in Ben-Men 1 and primary meningioma cell lines but less in IOMM Lee cells is 5-ALA dose-dependent, encouraging in-situ trials to encounter currently discussed shortcomings of FGS in meningiomas. Fluorescence is not related to expression of FECH, ABCB6 and ABCG2.

Photodynamic and Sonodynamic Therapy with Protoporphyrin IX: In Vitro and In Vivo Studies.

Sono-photodynamic therapy is a promising anticancer technique based on the combination of sonodynamic and photodynamic therapy to improve the cancer treatment effectiveness. This study was aimed at analyzing the effects of the sono-photodynamic (SPD) activity on protoporphyrin IX (PpIX) solution and PpIX-loaded rat liver. In vitro, PpIX 5 µM solutions were irradiated with light (635 nm, 30-50 mW/cm2), ultrasound (1 MHz, 1-2 W/cm2) and both. The PpIX absorption spectra recorded over exposure time revealed that the PpIX decay rate induced by SPD activity (combined irradiation) was approximately the sum of those induced by photodynamic and sonodynamic activity. In vivo, rats were intraperitoneally injected with 5-aminolevulinic acid at the dose of 500 mg/kg weight. After 3 h of injection, the PpIX-loaded livers were irradiated with light (635 nm, 180 ± 9 J/cm2), ultrasound (1.0 MHz, 770 ± 40 J/cm2) and both using a single probe capable of illuminating and sonicating the liver simultaneously. After 30 h, the liver damage induced by each protocol was analyzed histologically. It was found that a greater necrosis depth was induced by the SPD activity. These results suggest that the SPD activity could improve the PpIX decay rate and have greater scope than photodynamic or sonodynamic activity. Further studies should be performed to gain a better understanding of this protocol.

Broaden sources and reduce expenditure: Tumor-specific transformable oxidative stress nanoamplifier enabling economized photodynamic therapy for reinforced oxidation therapy.

Cancer cells immersed in inherent oxidative stress are more vulnerable to exogenous oxidative damages than normal cells. Reactive oxygen species (ROS)-mediated oxidation therapy preferentially aggravating tumor oxidative stress to disrupt redox homeostasis, has emerged as an effective and specific anticancer treatment. Herein, following an ingenious strategy of "broaden sources and reduce expenditure", we designed a versatile tumor-specific oxidative stress nanoamplifier enabling economized photodynamic therapy (PDT), to achieve synergistic oxidative stress explosion for superior oxidation therapy. Methods: Cinnamaldehyde (CA) as a therapeutic ROS generator was first conjugated to hyaluronic acid (HA) through acid-labile hydrazone bond to synthesize tailored amphiphilic HA@CA conjugates, which could surprisingly self-assemble into uniform nanofibers in aqueous media. Photosensitizer protoporphyrin (PpIX) was efficiently encapsulated into HA@CA nanofibers and transformed HA@CA nanofibers to final spherical HA@CAP. Results: With beneficial pH-responsiveness and morphology transformation, improved bioavailability and selective tumor accumulation, HA@CAP combining ROS-based dual chemo/photodynamic treatment modalities could induce cytotoxic ROS generation in a two-pronged approach to amplify tumor oxidative stress, termed "broaden sources". Moreover, utilizing CA-induced H2O2 production and cascaded Fenton reaction in mitochondria to consume intracellular overloaded Fe(II), HA@CAP could skillfully block endogenic heme biosynthesis pathway on site to restrain undesired elimination of PpIX for economized PDT, termed "reduce expenditure". Both in vitro and in vivo results demonstrated the superior antitumor performance of HA@CAP. Conclusion: This study offered an inspiring strategy of "broaden sources and reduce expenditure" to specifically boost tumor oxidative stress for reinforced oxidation therapy.

Evaluation of the effect of 5-aminolevulinic acid hexyl ester (H-ALA) PDT on EBV LMP1 protein expression in human nasopharyngeal cells.

Nasopharyngeal carcinoma (NPC) is of high prevalence in Hong Kong and southern China. The pathogenesis of NPC is closely associated with Epstein-Barr virus (EBV) infection via regulation of viral oncoprotein latent membrane protein 1 (LMP1). The conventional treatment for NPC is chemo-radiotherapy, but the prognosis remains poor for advanced stage, recurrent and metastatic NPC. Photodynamic therapy (PDT) is a therapeutic approach to combat tumors. PDT effectiveness depends on the interaction of photosensitizers, light and molecular oxygen. 5- aminolevulinic acid hexyl derivative (H-ALA) is one of the photosensitizers derived from 5-ALA. H-ALA with improved lipophilic properties by adding a long lipophilic chain (hexyl group) to 5-ALA, resulted in better penetration into cell cytoplasm. In this study, the effect of H-ALA-PDT on NPC cells (EBV positive C666-1 and EBV negative CNE2) was investigated. The H-ALA mediated cellular uptake and cytotoxicity was revealed via flow cytometry analysis and MTT assay respectively. H-ALA PDT mediated protein modulation was analysed by western blot analysis. Our finding reported that the cellular uptake of H-ALA in C666-1 and CNE2 cells was in a time dependent manner. H-ALA PDT was effective to C666-1 and CNE2 cells. EBV LMP1 proteins was expressed in C666-1 cells only and its expression was responsive to H-ALA PDT in a dose dependent manner. This work revealed the potential of H-ALA PDT as a treatment regiment for EBV positive NPC cells. Understanding the mechanism of H-ALA mediated PDT could develop improved strategies for the treatment of NPC.

Real-time in vivo kinetics of protoporphyrin IX after administration of 5-aminolevulinic acid in meningiomas and comparative analyses with glioblastomas.

BACKGROUND: The usefulness of 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is intensely discussed. However, data about kinetics of 5-ALA and protoporphyrin (Pp) IX in meningiomas are lacking. METHODS: As the first study so far, we performed longitudinal intraoperative real-time ex situ measurements of fluorescence intensity and PpIX concentrations during FGS of ten benign and two atypical meningiomas. Kinetics were subsequently compared with data from 229 glioblastomas. RESULTS: Spectroscopy revealed fluorescence (median 2945.65 a.u.) and PpIX accumulation (median 18.31 µg/ml) in all 43 analyzed samples. Fluorescence intensity (2961.50 a.u. vs 118.41 a.u.; p < .001) and PpIX concentrations (18.72 µg/ml vs .98 µg/ml; p < .001) were higher in samples with (N = 30) than without (N = 2) visible intraoperative tumor fluorescence. ROC curve analyses revealed a PpIX cut-off concentration of 3.85 µg/ml (AUC = .992, p = .005) and a quantitative fluorescence cut-off intensity of 286.73 a.u. (AUC = .983, p = .006) for intraoperative visible tumor fluorescence. Neither fluorescence intensity (p = .356) nor PpIX (p = .631) differed between atypical and benign meningiomas. Fluorescence and PpIX peaked 7-8 h following administration of 5-ALA. Meningiomas displayed a higher fluorescence intensity (p = .012) and PpIX concentration (p = .005) than glioblastomas 5-6 h after administration of 5-ALA. Although fluorescence was basically maintained, PpIX appeared to be cleared faster in meningiomas than in glioblastomas. CONCLUSIONS: Kinetics of PpIX and fluorescence intensity differ between meningiomas and glioblastomas in the early phase after 5-ALA administration. Modification of the timing of drug administration might impact visibility of intraoperative fluorescence and helpfulness of FGS and should be investigated in future analyses.

Temperature effect on the PpIX production during the use of topical precursors.

Decreasing incubation time, as well as enhanced PpIX production, are present challenges for topical photodynamic therapy (PDT). There are reports concerning the existence of a strong relationship between site temperature and porphyrin synthesis in biological tissue, that suggest temperature increase in the tissue can improve the formation of PpIX. The main objective of this study is to determine whether the temperature change of the tissue favors the production of PpIX. Creams containing aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) were topically administered for 30 min on healthy skin of rats' back and the formation of PpIX was evaluated for 180 min. The animals were divided into 5 groups: cooling tissue to 20 °C or heating tissue to 40 °C (either before or after incubation of the cream) and control group (unchanged temperature). The tissue temperature was evaluated by thermography. The influence of temperature was evaluated both concerning cream penetration and the production of PpIX. It was found that both ALA and MAL led to an increase of about 20% PpIX production when the tissue was warmed before incubation of the cream, suggesting that the penetration improved. When the thermal change was promoted after incubation of the cream, the production of PpIX decreased both by heating and cooling, probably related to enzyme modification. The results found in this study suggest that the increase of tissue temperature before the cream incubation can improve the clinical protocols of topical PDT using ALA or MAL, improving the efficiency of the procedure by increasing the production of PpIX and allowing the decrease of the incubation period.

Imaging of hypoxia, oxygen consumption and recovery in vivo during ALA-photodynamic therapy using delayed fluorescence of Protoporphyrin IX.

BACKGROUND: Hypoxic lesions often respond poorly to cancer therapies. Particularly, photodynamic therapy (PDT) consumes oxygen in treated tissues, which in turn lowers its efficacy. Tools for online monitoring of intracellular pO2 are desirable. METHODS: The pO2 changes were tracked during photodynamic therapy (PDT) with δ-aminolevulinic acid (ALA) in mouse skin, xenograft tumors, and human skin. ALA was applied either topically as Ameluz cream or systemically by injection. Mitochondrial pO2 was quantified by time-gated lifetime-based imaging of delayed fluorescence (DF) of protoporphyrin IX (PpIX). RESULTS: pO2-weighted images were obtained with capture-times of several seconds, radiant exposures near 10 mJ/cm2, spatial resolution of 0.3 mm, and a broad dynamic range 1-50 mmHg, corresponding to DF lifetimes ≈20-2000 µs. The dose-rate effect on oxygen consumption was investigated in mouse skin. A fluence rate of 1.2 mW/cm2 did not cause any appreciable oxygen depletion, whereas 6 mW/cm2 and 12 mW/cm2 caused severe oxygen depletion after radiant exposures of only 0.4-0.8 J/cm2 and <0.2 J/cm2, respectively. Reoxygenation after PDT was studied too. With a 5 J/cm2 radiant exposure, the recovery times were 10-60 min, whereas with 2 J/cm2 they were only 1-6 min. pO2 distribution was spatially non-uniform at (sub)-millimeter scale, which underlines the necessity of tracking pO2 changes by imaging rather than point-detection. CONCLUSIONS: Time-gated imaging of PpIX DF seems to be a unique tool for direct online monitoring of pO2 changes during PDT with a promising potential for research purposes as well as for comparatively easy clinical translation to improve efficacy in PDT treatment.

Energy analysis of PDT using thermography during the treatment of basal cell carcinoma.

The changes in tissue temperature of basal cell carcinoma lesions were investigated during photodynamic therapy in order to better understand the effects and mechanisms of PDT in tissue. In this study, the monitoring of 40 lesions of basal cell carcinoma was performed during photodynamic therapy. The lesion region becomes thermally evident throughout the procedure, and there is an improved contrast of the lesion edges after the end of the irradiation. The comparison between thermal and fluorescence images showed a correlation between the PpIX evidenced through widefield fluorescence and the temperature gradient of the thermal images after the procedure, indicating that thermography is a potential diagnostic tool to evaluate the selective response of PDT. A model was created to calculate the amount of light energy converted to heat, tissue damage, and other energy transfer processes involved in the PDT. Using this model, it was shown that most of the energy conversion was in photodynamic action (48.7% and 48.3%, in first and second session, respectively), followed by the energy ratio attributable to blood perfusion (37.2%). This is evidence that photodynamic therapy does not generate a significant thermal component, an important aspect of the study of its mechanisms.

Three-in-One Functional Silica Nanocarrier with Singlet Oxygen Generation, Storage/Release, and Self-Monitoring for Enhanced Fractional Photodynamic Therapy.

At present, the intermittent photodynamic therapy (fractional PDT) for overcoming tumor hypoxia still have their own defects, such as irradiation-dependence and rapid metabolism of organic photosensitizers. Therefore, it is still a really formidable challenge to achieve efficient fractional PDT. Herein, a three-in-one functional silica nanocarrier (FSNC) with singlet oxygen (1O2) generating unit (protoporphyrin IX derivative), 1O2 storage/release unit (2-pyridone derivative), and 1O2 self-monitoring unit (cyanine derivative) was prepared by reverse microemulsion method. Also, it could be efficiently internalized in the HeLa cells because of an appropriate particle size (∼44.8 nm). In the presence of light, the endoperoxide is formed to achieve 1O2 storage together with 1O2 generated by 1O2 generating unit for traditional PDT. In the absence of light, the endoperoxide produces 1O2 through cycloreversion for continuous PDT. As a result, the fractional PDT process of the FSNC on the HeLa cells performed a higher phototoxicity than traditional photosensitizer protoporphyrin IX. Furthermore, this real-time release behavior of 1O2 can be visually captured by confocal laser scanning microscope via monitoring fluorescent bleaching of 1O2 self-monitoring unit. Therefore, this fluorescent imaging-guided fractional PDT process could effectively enhance the PDT effect compared with traditional PDT.

Fluorescence-Based Measurement of Real-Time Kinetics of Protoporphyrin IX After 5-Aminolevulinic Acid Administration in Human In Situ Malignant Gliomas.

BACKGROUND: Five-aminolevulinic acid (5-ALA) is well established for fluorescence-guided resections of malignant gliomas by eliciting the accumulation of fluorescent protoporphyrin IX (PpIX) in tumors. Because of the assumed time point of peak fluorescence, 5-ALA is recommended to be administered 3 h before surgery. However, the actual time dependency of tumor fluorescence has not yet been evaluated in humans and may have important implications. OBJECTIVE: To investigate the time dependency of PpIX by measuring fluorescence intensities in tumors at various time points during surgery. METHODS: Patients received 5-ALA (20 mg/kg b.w.) 3 to 4 h before surgery. Fluorescence intensities (FI) and estimated tumor PpIX concentrations (CPPIX) were measured in the tumors over time with a hyperspectral camera. CPPIX was assessed using hyperspectral imaging and by evaluating fluorescence phantoms with known CPPIX. RESULTS: A total of 201 samples from 68 patients were included in this study. On average, maximum values of calculated FI and CPPIX were observed between 7 and 8 h after 5-ALA administration. FI and CPPIX both reliably distinguished central strong and marginal weak fluorescence, and grade III compared to grade IV gliomas. Interestingly, marginal (weak) fluorescence was observed to peak later than strong fluorescence (8-9 vs 7-8 h). CONCLUSION: In human in Situ brain tumor tissue, we determined fluorescence after 5-ALA administration to be maximal later than previously thought. In consequence, 5-ALA should be administered 4 to 5 h before surgery, with timing adjusted to internal logistical circumstances and factors related to approaching the tumor.

Mitochondria and plasma membrane dual-targeted chimeric peptide for single-agent synergistic photodynamic therapy.

Mitochondria and cell membrane play important roles in maintaining cellular activity and stability. Here, a single-agent self-delivery chimeric peptide based nanoparticle (designated as M-ChiP) was developed for mitochondria and plasma membrane dual-targeted photodynamic tumor therapy. Without additional carrier, M-ChiP possessed high drug loading efficacy as well as the excellent ability of producing reactive oxygen species (ROS). Moreover, the dual-targeting property facilitated the effective subcellular localization of photosensitizer protoporphyrin IX (PpIX) to generate ROS in situ for enhanced photodynamic therapy (PDT). Notably, plasma membrane-targeted PDT would enhance the membrane permeability to improve the cellular delivery of M-ChiP, and even directly disrupt the cell membrane to induce cell necrosis. Additionally, mitochondria-targeted PDT would decrease mitochondrial membrane potential and significantly promote the cell apoptosis. Both in vitro and in vivo investigations indicated that this combinatorial PDT in mitochondria and plasma membrane could achieve the therapeutic effect maximization with reduced side effects. The single-agent self-delivery system with dual-targeting strategy was demonstrated to be a promising nanoplatform for synergistic tumor therapy.

Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model?

OBJECTIVE: To study the curative effect of red-light 5-Aminolevulinic photodynamic therapy(ALA-PDT) to port wine stains(PWS) on comb animal model. METHODS: 160 male cocks were randomly divided into 16 groups. The ALA only group was given ALA only topical or systemic application. Light only groups were only given 630 nm red light irradiation with different light density. ALA-PDT groups were given red light after the application of topical or systemic ALA. PDL group was given PDL irradiation. The distribution of fluorescence in tissue after topical or systemic application of ALA was detected. The morphological changes, the pathological changes and the capillary reduction rate of the comb were observed after treatment for 0, 1, 3, 5, 7, 14 days. RESULTS: The PpIX fluorescence generated after topical and systemic application of ALA. In the topical ALA-PDT group at low light density 80 J/cm2, the morphology and the histopathology had no obvious change. While under 160 J/cm2 and 200 J/cm2 light density, severe erosion and thick scab appeared. The histopathology showed epidermal necrosis and loss. The immunohistochemistry showed that there was no significant change in the number of capillaries under different light density (P > 0.05). In the systemic ALA-PDT group under low light density 80 J/cm2, only partial erosion and thin scab was observed on the treatment side. With the increase of light density, thick charred crust and even scar was observed. The histopathology showed that there were different degrees of damage to dermal and epidermal tissues. And the immunohistochemistry showed the capillary reduced significantly in the treatment side (P < 0.01). In control group, the comb is ruddy and plump. CONCLUSION: These results suggest that either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.

Protoporphyrin IX formation after application of methyl aminolevulinate on the face and scalp with and without prior curettage.

BACKGROUND: Pulse photodynamic therapy (PDT) is carried out as classical PDT except that methyl aminolaevulinate (MAL) is removed after 30 min. Protoporphyrin IX (PpIX) accumulation becomes lower after 3 h in pulse-PDT than in classical PDT, but pulse-PDT still has full treatment effect on actinic keratosis (AK). The present study aimed to investigate when PpIX formation in classical PDT without curettage reaches the PpIX level in pulse-PDT with curettage in the skin of the face and scalp. METHODS: Fourteen patients with AKs in the face and scalp were included in this pilot study. Each patient had two fields randomly allocated to two different regimes: (i) continuing MAL incubation without prior curettage (-Cur) and (ii) 30 min MAL incubation after curettage (+Cur). The PpIX level in each field was measured with a hand-held fluorescence photometer every 30 min for 6 h after MAL application. No illumination was performed. RESULTS: The median PpIX level in the +Cur field after 3 h was 22 arbitrary units (AU), which represents the amount of PpIX that could have been photobleached after pulse-PDT with maximal treatment effect. After 3 h the median PpIX level in the -Cur field was 20 AU, which was not significantly different from the level in the +Cur field (p = 0.9). CONCLUSIONS: We propose PDT without curettage as follows: 3 h of MAL incubation and subsequently sufficient illumination. The treatment effect of this regime should be tested to verify if curettage of the face and scalp area may be omitted in PDT.

Enhanced Immunotherapy Based on Photodynamic Therapy for Both Primary and Lung Metastasis Tumor Eradication.

Metastasis and recurrence are two unavoidable and intractable problems in cancer therapy, despite various robust therapeutic approaches. Currently, it seems that immunotherapy is an effective approach to solve these problems, but the high heterogeneity of tumor tissue, inefficient presentation of tumor antigen, and deficient targeting ability of therapy usually blunt the efficacy of immunotherapy and hinder its clinical application. Herein, an approach based on combining photodynamic and immunological therapy was designed and developed. We synthesized a chimeric peptide, PpIX-1MT, which integrates photosensitizer PpIX with immune checkpoint inhibitor 1MT via a caspase-responsive peptide sequence, Asp-Glu-Val-Asp (DEVD), to realize a cascaded synergistic effect. The PpIX-1MT peptide could form nanoparticles in PBS and accumulate in tumor areas via the enhanced penetration retention effect. Upon 630 nm light irradiation, the PpIX-1MT nanoparticles produced reactive oxygen species, induced apoptosis of cancer cells, and thus facilitated the expression of caspase-3 and the production of tumor antigens, which could trigger an intense immune response. The subsequently released 1MT upon caspase-3 cleavage could further strengthen the immune system and help to activate CD8+ T cells effectively. This cascaded synergistic effect could inhibit both primary and lung metastasis tumor effectively, which may provide the solution for solving tumor recurrence and metastasis clinically.

Visualizing protoporphyrin IX formation in the dura tail of meningiomas by mass spectrometry imaging.

BACKGROUND: The advantages of 5-aminolevulinacid (5-ALA)-induced fluorescence-guided surgery in meningiomas are increasingly discussed. In this context, despite detectable tumor tissue in histopathologial analyses, no fluorescence was shown at the dura tail using the standard operating microscope. Thus, 5-ALA metabolism in this surgically important site remains unknown but needs to be elucidated when further evaluating indications of fluorescence-guided surgery in meningiomas. METHOD: We here present the spatially resolved identification of protoporphyrin IX (PpIX) in sphenoid ridge meningioma cryosections from a patient who underwent fluorescence-guided microsurgery using molecular imaging analysis by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). RESULTS: Despite a strong fluorescence of the main tumor, no fluorescence could be detected at the dura tail using the standard operating microscope (blue-light, 405 nm). However, histopathological analyses clearly showed meningioma tissue. Remarkably, MALDI-MS/MS analysis revealed PpIX formation also at the non-fluorescing dura tail. However, no PpIX was detected in the tumor free dura mater. CONCLUSION: MALDI-MS/MS visualized a selective accumulation of PpIX within the tumor tissue including the dura tail. Thus, absence of fluorescence in the dura tail as visualized by the operating microscope is not caused by the lack of PpIX formation.

5-Aminolevulinic acid-based photodynamic therapy of chordoma: In vitro experiments on a human tumor cell line.

BACKGROUND: Chordomas are very rare tumors of the skull base and the sacrum. They show infiltrating and destructive growth and are known to be chemo- and radio-resistant. After surgical resection, the recurrence rate is high and overall survival limited. As current adjuvant treatments are ineffective, new treatment concepts are urgently needed. 5-aminolevulinic acid-based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. However, it is unknown so far, whether chordomas accumulate protoporphyrin IX (PPIX) after application of 5-ALA and whether they are sensitive to subsequent 5-ALA based PDT. METHODS: The immortalized human chordoma cells U-CH2 were used as in vitro model. After incubation for 4h or 6h with different 5-ALA concentrations, PPIX accumulation was determined by flow cytometry. To assess sensitivity to PDT, chordoma cells were incubated at 30.000cells/well (high cell density) or 15.000cells/well (low cell density) with graded doses of 5-ALA (0-50µg/ml) in 96-well plates and subsequently exposed to laser light of 635nm wavelength (18.75J/cm2). Cell survival was measured 24h after exposure to laser light using the WST-1 assay. RESULTS: U-CH2 cells dose-dependently accumulated PPIX (ANOVA; p<0.0001). PPIX fluorescence was significantly higher, when cells were incubated with 5-ALA for 6h compared to 4h at higher 5-ALA concentrations (ANOVA/Bonferroni; p≤0.05 for≥30µg/ml 5-ALA). For both cell densities, a 5-ALA dose-dependent decline in viability was observed (ANOVA; p<0.0001). Viability was significantly lower at higher 5-ALA concentrations, when 30.000 cells/wells were treated compared to 15.000cells/well (ANOVA/Bonferroni; p≤0.001 for≥30µg/ml 5-ALA). LD50 was 30.25µg/ml 5-ALA. CONCLUSION: The human UCH-2 cell line was a very useful in vitro model to study different effects of 5-ALA based PDT. For the first time, it could be shown that human chordoma cells may be destroyed by 5-ALA/PDT.

Neurosurgical microscopic solid laser-based light inhibits photobleaching during fluorescence-guided brain tumor removal with 5-aminolevulinic acid.

BACKGROUND: Fluorescence image guided surgery (FIGS) with 5-aminolevulinic acid for malignant gliomas improves surgical outcome. One of the problems during FIGS is photobleaching under surgical microscopic white light. A solid laser-based white light source for neurosurgery that we developed does not include light with a wavelength of around 405nm, which is strongly absorbed by protoporphyrin IX. In the present study, we examined the efficacy of this light source to prevent the photobleaching of protoporphyrin IX-induced fluorescence. METHODS: Filter papers transfused with protoporphyrin IX solution and a coronally sectioned F98 glioma rat model pretreated with 50mg/kg 5-aminolevulinic acid were continuously exposed to white light. One group was exposed to conventional xenon-based white light and another group was exposed to laser-based white light. Fluorescence at a wavelength of 635nm was measured with a radiospectrometer (in vitro study) and the relative fluorescence brightness was also measured in digital images (in vivo study) under excitation from violet blue light emitted from diodes every 5min. RESULTS AND CONCLUSION: Estimated time for 50% photobleaching was prolonged about two times in the laser-based white light exposure group compared with that in the xenon-based white light exposure group (9.1/18.7min). In the brain tumor rat model, it was also prolonged about 2.7 times (15.1/40.7min). A laser-based white light source may inhibit photobleaching during FIGS for malignant gliomas. This light source for neurosurgical microscopy has the potential to prolong the prognosis of malignant glioma patients.

Efficient fluorescence detection of protoporphyrin IX in metastatic lymph nodes of murine colorectal cancer stained with indigo carmine.

Protoporphyrin IX (PpIX), a biochemical converted from 5-aminolevulinc acid (5-ALA) in living cells, is useful for intraoperative fluorescent detection of cancer metastasis in lymph nodes (LNs). However, unknown is whether the fluorescence of PpIX can be detected in the LNs when they coexist with indigo carmine, a blue dye commonly used for identification of sentinel LNs during surgery. To address this issue, we sought to evaluate the diagnostic usefulness of PpIX fluorescence in the presence of indigo carmine in a mouse LN metastasis model of rectal cancer after administration of 5-ALA. Spectral analysis of pure chemicals revealed that the absorption spectrum of indigo carmine widely overlapped with the fluorescence spectrum of PpIX specifically at the peak of 632nm, a common emission wavelength for detecting PpIX, but not at the other peak of 700nm. Due to such spectral overlap, the PpIX fluorescence intensity was significantly attenuated by mixture with indigo carmine at 632nm, but not at 700nm. Accordingly, fluorescent measurements of the mouse metastatic LN revealed more intense presentation of PpIX at 700nm than at 632nm, indicating that the diagnostic usefulness is greater at 700nm than at 632nm for the indigo carmine-dyed LNs after administration of 5-ALA. From these observations, we propose that the fluorescence measurement is more efficient at 700nm than at 632nm for detection of PpIX in metastatic LNs stained with indigo carmine.

Anti-inflammatory and anti-nociceptive effects of strontium ranelate on the zymosan-induced temporomandibular joint inflammatory hypernociception in rats depend on TNF-α inhibition.

BACKGROUND: Temporomandibular joint (TMJ) disorders show inflammatory components, heavily impacting on quality of life. Strontium ranelate has previously shown anti-inflammatory and antinociceptive effects on other experimental inflammatory pain models. Thus, we aim to investigate the strontium ranelate efficacy in reducing the zymosan-induced inflammatory hypernociception in the TMJ of rats by evaluating the TNF-α, IL-1ß, and hemeoxygenase-1 (HO-1) involvement. METHODS: Wistar rats were treated with strontium ranelate (0.5, 5 or 50 mg/kg, per os) 1 h before zymosan injection (iart). Mechanical threshold was assessed by Von Frey test and synovial lavage was collected for leukocyte counting and myeloperoxidase measurement, joint tissue and trigeminal ganglion were excised for histopathological analysis (H&E) and TNF-α/IL-1ß levels dosage (ELISA). Moreover, rats were pre-treated with ZnPP-IX (3 mg/kg, sc), a specific HO-1 inhibitor, before strontium ranelate administration (0.5 mg/kg, per os), and Evans Blue (5 mg/kg, iv) was administered to assess plasma extravasation. Pre-treatment with indomethacin (5 mg/kg, sc) was used as positive control while the sham group received 0.9% sterile saline (per os and iart). RESULTS: Strontium ranelate did not reduce leukocyte counting, myeloperoxidase activity, Evans Blue extravasation, IL-1ß levels, and TNF-α/IL-1ß immunolabeling; but it increased the nociceptive threshold and reduced TNF-α levels. Additionally, HO-1 inhibition did not change the strontium ranelate effects. CONCLUSION: Strontium ranelate may achieve its antinociceptive effects through the reduction of TNF-α levels in the trigeminal ganglion, but not suppressing IL-1ß expression nor inducing the HO-1 pathway.