Chorioallantoic membrane (CAM) assay to study treatment effects in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor. While there are a number of in vivo rodent models for evaluating tumor biology and response to therapy, these models require significant time and resources. Here, we established the chick-embryo chorioallantoic (CAM) assay as an affordable and time efficient xenograft model for testing a variety of treatment approaches for DIPG. We found that patient-derived DIPG tumors develop in the CAM and maintain the same genetic and epigenetic characteristics of native DIPG tumors. We monitored tumor response to pharmaco- and radiation therapy by 3-D ultrasound volumetric and vasculature analysis. In this study, we established and validated the CAM model as a potential intermediate xenograft model for DIPG and its use for testing novel treatment approaches that include pharmacotherapy or radiation.

Effects of 445-nm Laser on Vessels of Chick Chorioallantoic Membrane with Implications to Microlaryngeal Laser Surgery.

OBJECTIVE: Previous research has shown that effective application of angiolytic lasers in microlaryngeal surgery is determined by wavelength, pulse width (PW), and fluence. Recently, a 445-nm (blue) laser (BL) has been developed with a potentially greater hemoglobin absorption than previous lasers. The chick chorioallantoic membrane (CAM) represents a suitable model for testing various settings to find out the most optimal settings of this laser. This study used the CAM model to examine whether successful photoangiolytic effects could be obtained using BL. METHODS: Seven hundred and ninety three third-order vascular segments of viable CAM were irradiated using BL via 400-µm diameter fiber, 1 pulse/second, with PW and power varied systematically at standardized fiber-to-vessel distances of 1 and 3 mm. Outcome measures including vessel ablation rate (AR), rupture rate (RR), and visible tissue effects were analyzed using Chi-square test. RESULTS: Energy levels of 400, 540, and 600 mJ (per pulse) were most effective for vessel ablation. A working distance of 3 mm resulted in higher ablation and less vessel rupture compared with 1 mm at these optimal energy levels. At 3 mm, a longer PW resulted in higher AR. At 1 mm, AR increased with shorter PW and higher power. The 1-mm working distance resulted in lower tissue effects than 3 mm. CONCLUSION: Findings in this study showed that BL was effective in vessel ablation using relevant combination of working distance, PW, and energy levels. To obtain high AR, longer working distance plus longer PW was required and if working distance was reduced, shorter PW should be set. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E1950-E1956, 2021.

Comparison of cytokine expression and ultrastructural alterations in fresh-frozen and dried electron beam-irradiated human amniotic membrane and chorion.

The purpose of the current study was to compare the effects of drying and fresh-freezing on human amniotic membrane (HAM) and amnion/chorion membrane (HACM) in terms of histological and structural characteristics and cytokine levels. HAM and HACM samples, obtained from six placentae, were investigated. HAM and HACM were dried, electron beam-irradiated (dehydration group; d-HAM/d-HACM), or fresh-frozen (freezing group; f-HAM/f-HACM). Luminex assay was used to assay the levels of 15 cytokines. The ultrastructural characteristics of HAM and HACM were evaluated using light and transmission electron microscopies. Total cytokine contents did not show the statistical difference between dehydration and fresh-freezing process. Significantly higher levels of total cytokines were observed in HACM than in HAM. Epidermal growth factor (EGF) level was significantly higher in d-HAM than in the other samples. The levels of most of the other growth factors were higher in HACM than in HAM, but there was no statistical difference between the dehydration process and the fresh-freezing process. The levels of the cytokines, other than the growth factors, were higher in HACM than in HAM, and higher concentrations of cytokines were observed in the freezing group than in the dehydration group. Histological examination revealed that the dehydration group had thinner tissues than the freezing group, but the structural stability, including the basement membrane, did not differ between the two groups. Microscopic structures such as microvilli and nuclei were well-preserved in the freezing group, based on the results of the transmission electron microscopy. Our dehydration process maintained the histological structure of HAM/HACM and a variety of growth factors and cytokines were identified. Especially, the HAM, processed with the dehydration method, had a higher EGF level than that processed with the fresh-freezing method. Therefore, dehydration method can be used to effectively promote wound repair.

Effect of cell-phone radiofrequency on angiogenesis and cell invasion in human head and neck cancer cells.

BACKGROUND: Today, the cell phone is the most widespread technology globally. However, the outcome of cell-phone radiofrequency on head and neck cancer progression has not yet been explored. METHODS: The chorioallantoic membrane (CAM) and human head and neck cancer cell lines, FaDu and SCC25, were used to explore the outcome of cell-phone radiofrequency on angiogenesis, cell invasion, and colony formation of head and neck cancer cells, respectively. Western blot analysis was used to investigate the impact of the cell phone on the regulation of E-cadherin and Erk1/Erk2 genes. RESULTS: Our data revealed that cell-phone radiofrequency promotes angiogenesis of the CAM. In addition, the cell phone enhances cell invasion and colony formation of human head and neck cancer cells; this is accompanied by a downregulation of E-cadherin expression. More significantly, we found that the cell phone can activate Erk1/Erk2 in our experimental models. CONCLUSION: Our investigation reveals that cell-phone radiofrequency could enhance head and neck cancer by stimulating angiogenesis and cell invasion via Erk1/Erk2 activation.

Activation of tumor cell integrin αvß3 by radiation and reversal of activation by chemically modified tetraiodothyroacetic acid (tetrac).

PURPOSE: Integrin αvß3 is an important structural and signaling protein of the plasma membrane of cancer cells and dividing blood vessel cells. The plastic extracellular domain of the protein binds to extracellular matrix proteins and plasma membrane proteins, changing cell-cell interactions and generating intracellular signals that influence cell behavior. αvß3 also contains a receptor for thyroid hormone and derivatives, including tetraiodothyroacetic acid (tetrac). MATERIALS AND METHODS: Human prostate cancer (PC3) cells were engrafted in the chicken chorioallantoic membrane model. The well-vascularized spheroidal xenografts were exposed to X-radiation in varying dosages (1-10 Gy) and in the presence and absence of an antibody that recognizes unliganded human ß3 integrin monomer in the extended or open (activated) configuration. RESULTS: Radiation significantly increased activated ß3 within 1 h (P < .001), a radiation response not previously reported. Incubation of cells with unmodified tetrac or tetrac covalently linked to a nanoparticle (Nanotetrac, NDAT) did not change basal activation state of the integrin monomer, but prevented radiation-induced activation of ß3. CONCLUSIONS: Activation of the integrin in response to radiation is interpreted as a defensive response, perhaps leading to increased intercellular affinity and inhibition of cell division, a radioresistant state. Action of NDAT indicates that pharmacologic interventions in the radiation response of integrin ß3 monomer and therefore of αvß3 are feasible.

Photostimulation effects on chicken egg development: Perspectives on human newborn treatment.

It is well known that, under exposure to bright light, eggs tend to hatch earlier than control, without any damage to the birds. This report aims to systematically show the effect and establishes a proposal for a possible application to accelerate chicken egg formation, which could be extrapolated or adapted as a great advance in premature human newborns. Comparing several protocols, the experiments show that lower doses of light slowly delivered for 24 h promote higher efficiency in embryo development, increasing on average 25% of its size and more than 70% in weight when compared to the control. This weight difference shows promising results compared to rates of up to 17% found in the literature. These results can be a first step to reduce the stay of premature human infants in hospitals because light, when applied in very low doses, can accelerate the natural biological processes without risks.

Evaluation of vascular effect of Photodynamic Therapy in chorioallantoic membrane using different photosensitizers.

Photodynamic Therapy (PDT) is a local treatment that requires a photosensitizing agent, light and molecular oxygen. With appropriate illumination, the photosensitizer is excited and produces singlet oxygen that is highly reactive and cytotoxic. Tumor vascular network is essential for the tumor growth and the understanding of vascular response mechanisms enables an improvement in the PDT protocol for cancer treatment. Compounds of porphyrin (Photogem®) and chlorin (Photodithazine®) were the photosensitizers tested. The incubation times varied from 20 to 80 min and the concentration ranged between 0.1 and 100 µg/cm(2). Different light doses were used between 4.8 and 40 J/cm(2) with irradiance varying between 80 and 100 mW/cm(2). The light dose of 30 J/cm(2) was used in the intravenous photosensitizer application. The membrane images were made from 0 to 300 min after treatment. The vascular response was evaluated by the average vessel area. Different responses was observed depending on the photosensitizer concentration and administration form. Intravenous application has been more efficient to produce vessel constriction and the most pronounced effect was observed for the chlorin.

Microbeam radiation-induced tissue damage depends on the stage of vascular maturation.

PURPOSE: To explore the effects of microbeam radiation (MR) on vascular biology, we used the chick chorioallantoic membrane (CAM) model of an almost pure vascular system with immature vessels (lacking periendothelial coverage) at Day 8 and mature vessels (with coverage) at Day 12 of development. METHODS AND MATERIALS: CAMs were irradiated with microplanar beams (width, ∼25 µm; interbeam spacing, ∼200 µm) at entrance doses of 200 or 300 Gy and, for comparison, with a broad beam (seamless radiation [SLR]), with entrance doses of 5 to 40 Gy. RESULTS: In vivo monitoring of Day-8 CAM vasculature 6 h after 200 Gy MR revealed a near total destruction of the immature capillary plexus. Conversely, 200 Gy MR barely affected Day-12 CAM mature microvasculature. Morphological evaluation of Day-12 CAMs after the dose was increased to 300 Gy revealed opened interendothelial junctions, which could explain the transient mesenchymal edema immediately after irradiation. Electron micrographs revealed cytoplasmic vacuolization of endothelial cells in the beam path, with disrupted luminal surfaces; often the lumen was engorged with erythrocytes and leukocytes. After 30 min, the capillary plexus adopted a striated metronomic pattern, with alternating destroyed and intact zones, corresponding to the beam and the interbeam paths within the array. SLR at a dose of 10 Gy caused growth retardation, resulting in a remarkable reduction in the vascular endpoint density 24 h postirradiation. A dose of 40 Gy damaged the entire CAM vasculature. CONCLUSIONS: The effects of MR are mediated by capillary damage, with tissue injury caused by insufficient blood supply. Vascular toxicity and physiological effects of MR depend on the stage of capillary maturation and appear in the first 15 to 60 min after irradiation. Conversely, the effects of SLR, due to the arrest of cell proliferation, persist for a longer time.

In vitro and in vivo photocytotoxicity of boron dipyrromethene derivatives for photodynamic therapy.

To understand the effects of substitution patterns on photosensitizing the ability of boron dipyrromethene (BODIPY), two structural variations that either investigate the effectiveness of various iodinated derivatives to maximize the "heavy atom effect" or focus on the effect of extended conjugation at the 4-pyrrolic position to red-shift their activation wavelengths were investigated. Compounds with conjugation at the 4-pyrrolic position were less photocytotoxic than the parent unconjugated compound, while those with an iodinated BODIPY core presented better photocytotoxicity than compounds with iodoaryl groups at the meso-positions. The potency of the derivatives generally correlated well with their singlet oxygen generation level. Further studies of compound 5 on HSC-2 cells showed almost exclusive localization to mitochondria, induction of G(2)/M-phase cell cycle block, and onset of apoptosis. Compound 5 also extensively occluded the vasculature of the chick chorioallantoic membrane. Iodinated BODIPY structures such as compound 5 may have potential as new photodynamic therapy agents for cancer.

Nitric oxide and the collagenous protein biosynthesis of irradiated chick chorioallantoic membrane.

PURPOSE: To study the effect of irradiation doses (2 Gy and 5 Gy) on the rate of collagenous protein biosynthesis (CPB) of chick embryo chorioallantoic membrane (CAM) and to investigate the possible role of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), its inactive enantiomer D-NAME and the NO donor sodium nitroprusside (SNP) in this effect. MATERIALS AND METHODS: The originally established CAM angiogenesis model was used with few modifications described earlier. The CAM areas were irradiated on the 10th or the 14th day of embryo development with a single dose of 2 or 5 Gy and the CPB was determined 6 h later. L-NAME, D-NAME and SNP were added to the CAM after the irradiation. RESULTS: The experiments didn't show any significant differences in almost all of the treatment courses. In the 10-day CAM preparations the irradiation not significantly (NS) inhibited the CAM CPB, whereas L-NAME abolished this effect only in the case of 2 Gy dose (NS). The addition of SNP or D-NAME in the post-irradiated 10-day CAM exerted radiosensitization that was significant only in the case of the combination of D-NAME with the 5 Gy dose. There was no significant effect on the respective treatments of the 14-day CAM. The CPB of the 14-day CAM was significantly lower with regard to the values of the 10-day CAM. CONCLUSION: The biochemical evaluation of the CAM (CPB method) seems to be not suitable for radiobiological studies. Nevertheless, the implication of NO in the X-ray induced antiangiogenicity cannot be excluded.

Predicting clinical efficacy of photoangiolytic and cutting/ablating lasers using the chick chorioallantoic membrane model: implications for endoscopic voice surgery.

OBJECTIVES/HYPOTHESIS: The optimal balance between a laser's clinical efficacy and collateral thermal damage is the major determinant for selection of a particular laser in endolaryngeal surgery. The chick chorioallantoic membrane (CAM) simulates the microvasculature of the human vocal fold and is, therefore, useful for testing effects of laser settings, mode of delivery, active cooling, and wavelength. Such information is essential for optimizing the effectiveness of lasers in treating laryngeal pathology while preserving vocal function. STUDY DESIGN AND METHODS: The thermal and coagulative effects of four lasers (585 nm PDL, 532 nm KTP, 2.01ìm Thulium, 10.6ìm CO2) were quantified at selected (and clinically relevant) energy settings before and after tissue cooling using the CAM model. Measures included imaging real-time vascular reactions in the CAM model (i.e., vessel coagulation and/or rupture), and post-procedure histologic analysis of CAM tissue. In each experiment, laser energy was applied to the CAM in a controlled manner. Cooling was done using a dermatological cold-air device, and temperatures were measured with a thermistor. Lasers tested included the photoangiolytic pulsed-dye (PDL) and KTP, as well as the ablative/cutting CO2 and thulium lasers. The vessel rupture/coagulation and thermal effects of various energy-delivery parameters on the CAM, with and without cooling, were assessed. After removal of the CAM, specimens were stained as whole-mounts, photographed at 4X magnification, and evaluated by two independent, blinded surgeon reviewers. The efficacy of increased pulse-width (KTP laser) on treating larger vessels (>0.5 mm) and the effects of extravasated blood on photoangiolysis were also evaluated. RESULTS: Photoangiolytic lasers: Vessel coagulation/rupture rates showed that the PDL caused more frequent vessel rupture than the KTP laser. For both lasers, cooling the CAM by approximately 20 degrees C resulted in 30% - 60% reduction in the thermal-damage zone (P < .05). Cooling reduced the efficacy of coagulation with the PDL but not with the KTP laser. The clinically observed phenomenon that laser heating of extravasated blood increases thermal damage and decreases efficacy of coagulation was clearly evident in the CAM model. Ablative lasers: The thermal-damage zone of the CO2 laser (0.3 mm spot size) was not significantly different with or without cooling (0.32 mm2 and 0.34 mm2, respectively) (P = .30). However, when the spot size was defocused to 1 mm, the thermal-damage zone was over 2x greater when the tissue was not cooled (0.74 mm2 vs 0.35 mm2) (P < .002). The thermal-damage zone of the Thulium laser was reduced by an average of 58% for the three power settings tested when the CAM was air-cooled (P < .05). CONCLUSIONS: The CAM was an excellent model for studying the effects of photoangiolytic lasers, for which optimal pulse-widths exist for vessel coagulation. Smaller vessels coagulated reliably at pulse widths >15 msec, and larger vessels required pulse widths >35 msec for optimal coagulation. Cooling the target tissue decreased the thermal-damage zone created by photoangiolytic lasers. While cooling had no effect on the efficacy of coagulation with longer pulse widths (KTP), tissue cooling decreased the coagulation rate at shorter pulse widths (PDL). The thermal effects of cutting/ablating lasers can be reduced with cooling, but the CAM was not a good model with which to study coagulation/rupture rates in cutting/ablating lasers.

Tissue damage by pulsed electrical stimulation.

Repeated pulsed electrical stimulation is used in a multitude of neural interfaces; damage resulting from such stimulation was studied as a function of pulse duration, electrode size, and number of pulses using a fluorescent assay on chick chorioallontoic membrane (CAM) in vivo and chick retina in vitro. Data from the chick model were verified by repeating some measurements on porcine retina in-vitro. The electrode size varied from 100 microm to 1 mm, pulse duration from 6 micros to 6 ms, and the number of pulses from 1 to 7500. The threshold current density for damage was independent of electrode size for diameters greater than 300 microm, and scaled as 1/r2 for electrodes smaller than 200 microm. Damage threshold decreased with the number of pulses, dropping by a factor of 14 on the CAM and 7 on the retina as the number of pulses increased from 1 to 50, and remained constant for a higher numbers of pulses. The damage threshold current density on large electrodes scaled with pulse duration as approximately 1/t0.5, characteristic of electroporation. The threshold current density for repeated exposure on the retina varied between 0.061 A/cm2 at 6 ms to 1.3 A/cm2 at 6 micros. The highest ratio of the damage threshold to the stimulation threshold in retinal ganglion cells occurred at pulse durations near chronaxie-around 1.3 ms.

Complete blood vessel occlusion in the chick chorioallantoic membrane using two-photon excitation photodynamic therapy: implications for treatment of wet age-related macular degeneration.

Complete blood vessel occlusion is required for the treatment of age-related macular degeneration (AMD). AMD is the leading cause of blindness in developed countries and current treatment regimes have potential to cause collateral damage, or do not remove pre-existing unwanted vasculature. It has been proposed that two-photon excitation (TPE) photodynamic therapy (PDT) can be applied to cause local blood vessel occlusion without damaging surrounding retinal tissues. The in ovo chicken chorioallantoic membrane (CAM) is used as the model for vascularization in the wet form of AMD; novel techniques for the utilization of the CAM are reported. Complete occlusion of CAM vessels approximately 15 microm in diameter is achieved using the clinically approved photosensitizer Verteporfin (Visudyne, QLT, Incorporated, Vancouver, British Columbia, Canada) and TPE activation. The average and peak irradiances used for treatment are 3.3x10(6) Wcm(2) and 3.7x10(11) Wcm(2), respectively. A total fluence of 1.1x10(8) Jcm(2) is the dosage required for successful occlusion, and it is expected that for optimal conditions it will be much less. These results are the first proof-of-principle evidence in the literature that indicate TPE-PDT can be used to occlude small blood vessels. Further investigation will help determine the utility of TPE-PDT for treating wet AMD, perhaps through targeting feeder vessels.

Effect of nanoparticle size on the extravasation and the photothrombic activity of meso(p-tetracarboxyphenyl)porphyrin.

Particle size should be optimized to achieve targeted and extended drug delivery to the affected tissues. We describe here the effects of the mean particle size on the pharmacokinetics and photothrombic activity of meso-tetra(carboxyphenyl)porphyrin (TCPP), which is encapsulated into biodegradable nanoparticles based on poly(d,l-lactic acid). Four batches of nanoparticles with different mean sizes ranging from 121 to 343 nm, were prepared using the emulsification-diffusion technique. The extravasations of each TCPP-loaded nanoparticle formulation from blood vessels were measured, as well as the extent of photochemically induced vascular occlusion. These preclinical tests were carried out in the chorioallantoic membrane (CAM) of the chicken's embryo. Fluorescence microscopy showed that both the effective leakage of TCPP from the CAM blood vessels and its photothrombic efficiency were dependent on the size of the nanoparticle drug carrier. Indeed, the TCPP fluorescence contrast between the blood vessels and the surrounding tissue increased at the applied conditions, when the particle size decreased. This suggests that large nanoparticles are more rapidly eliminated from the bloodstream. In addition, after injection of a drug dose of 1mg/kg body weight and a drug-light application interval of 1 min, irradiation with a fluence of 10J/cm(2) showed that the extent of vascular damage gradually decreased when the particle size increased. The highest photothrombic efficiency was observed when using the TCPP-loaded nanoparticles batch with a mean diameter of 121 nm. Thus, in this range of applied conditions, for the treatment of for instance a disease like choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD), these experiments suggest that the smallest nanoparticles may be considered as the optimal formulation since they exhibited the greatest extent of vascular thrombosis as well as the lowest extravasation.

Enhanced photodynamic activity of hypericin by penetration enhancer N-methyl pyrrolidone formulations in the chick chorioallantoic membrane model.

Hypericin (HY) was examined for photodynamic therapy (PDT)-induced vascular damage using the chick chorioallantoic membrane (CAM) model. Clinically, plasma protein was used to solubilize HY. Upon binding to albumin, free HY available to be transported through the membrane may be limited. Hence, formulations containing a biocompatible solvent, N-Methyl pyrrolidone (NMP), have the potential to enhance HY delivery into solid tumors. At suitable concentrations, NMP and/or light irradiation did not produce antivascular damage. Hypericin-PDT effects showed to be HY and NMP concentrations-dependent. These findings indicate that NMP is a promising solvent and penetration enhancer for HY-PDT clinical applications.

Preclinical evaluation of a novel water-soluble chlorin E6 derivative (BLC 1010) as photosensitizer for the closure of the neovessels.

In the present study, photodynamic activity of a novel photosensitizer (PS), Chlorin e(6)-2.5 N-methyl-d-glucamine (BLC 1010), was evaluated using the chorioallantoic membrane (CAM) as an in vivo model. After intravenous (i.v.) injection of BLC 1010 into the CAM vasculature, the applicability of this drug for photodynamic therapy (PDT) was assessed in terms of fluorescence pharmacokinetics, i.e. leakage from the CAM vessels, and photothrombic activity. The influence of different PDT parameters including drug and light doses on the photodynamic activity of BLC 1010 has been investigated. It was found that, irrespective of drug dose, an identical continuous decrease in fluorescence contrast between the drug inside and outside the blood vessels was observed. The optimal treatment conditions leading to desired vascular damage were obtained by varying drug and light doses. Indeed, observable damage was achieved when irradiation was performed at light doses up to 5 J/cm(2) 1 min after i.v. injection of drug doses up to 0.5 mg/kg body weight(b.w.). However, when irradiation with light doses of more than 10 J/cm(2) was performed 1 min after injection of drug doses up to 2 mg/kg body weight, this led to occlusion of large blood vessels. It has been demonstrated that it is possible to obtain the desired vascular occlusion and stasis with BLC 1010 for different combinations of drug and/or light doses.

X-rays affect the expression of genes involved in angiogenesis.

BACKGROUND: We have previously shown, using the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis, that X-rays act on the extracellular matrix and enhance normal and tumor-induced angiogenesis. In the present work, we studied the effect of X-rays on the gene expression of three proteins that are important regulators of angiogenesis: vascular endothelial growth factor (VEGF), heparin affin regulatory peptide (HARP) and inducible nitric oxide synthase (iNOS). MATERIALS AND METHODS: An area of 1 cm2 of the CAM, restricted by a plastic ring was irradiated at room temperature. The expression of the genes was studied using RT-PCR and the amounts of the mRNAs were quantified using image analysis of the corresponding agarose gels of the RT-PCR products. RESULTS: VEGF mRNA was decreased 6 h after irradiation. However, at later time points, VEGF expression was significantly increased compared with the nonirradiated tissue. Similarly, X-rays down-regulated both HARP and iNOS expression 6 h after irradiation and the effect was reversed at later time points, similarly to the effect of X-rays on VEGF. CONCLUSION: These data support the notion that X-rays increase the expression of genes that favor angiogenesis.