Non-irradiated Control Group Research Articles

Effects of pentoxifylline and tocopherol on an osteoradionecrosis animal model

PurposeOsteoradionecrosis (ORN) is known to be a refractory disease in the oral and maxillofacial field. The purpose of this study was to examine the effects of pentoxifylline (PTX) and tocopherol (TP) on an ORN animal model focused on bone healing. Materials and methodsA total of 48 Sprague–Dawley rats were used: 40 received a single irradiation dose of 35 Gy on the left mandible, and eight were used as the nonirradiated control group. The rats received PTX (T1, C1), TP (T2, C2), a combination of PTX and TP (T3, C3), or normal saline (T4, C4). Three weeks after irradiation, the mandibular posterior teeth were extracted. The rats were sacrificed 4 weeks after extraction. ResultsIn the T3 group, bone volume/tissue volume was 19.62 ± 16.03 (%), bone mineral density was as 0.31 ± 0.16 (g/cm3) in the micro-CT analysis, which were higher than that of other groups (p = 0.025, p = 0.012, respectively). In the histological analysis, bone regeneration was the most prominent in the T3 group. The ratio of empty lacunae was the highest in the T4 group, 68.77 ± 15.47 (%, p = 0.004). Immunohistochemistry showed that the expression of TNF-α was relatively lower in the T3 than in the T4 or T2 groups. The RT-qPCR showed the expression level of PECAM, VEGF-A, and osteocalcin was more than twofold as high as in the T3 group compared to the other groups. ConclusionThe combination of PTX and TP appears to promote angiogenesis and osteogenesis in a rat ORN model. Therefore, PTX and TP might be useful in the treatment and prevention of ORN.

The effects of experimentally irradiated pituitary gland on the growth of rat’s tibia, skull, maxilla and mandible

Background/Aim. The reaction of an organism to radiation depends on the level of irradiation and the sensitivity of the affected tissue cells. The biological effects on the cells and tissues are proportional to the absorbed radiation energy. The aim of our study was to examine the effects of hypofunction of the pituitary gland, previously irradiated with x-rays, on the growth of rat?s craniofacial and stomatognathic system as well as rat?s tibia. Methods. A total dose of x-rays of 27.92 Gy was applied in eight sessions in the period from 8 to 63 days of animal age on their heads and the effects of irradiation-induced hypofunction of the pituitary gland on the growth of rat?s craniofacial and stomatognathic system and tibia were examined. In order to differentiate the effects of the irradiated pituitary gland from the direct effects of x-rays on the head, the experiment was set as a double study. One group of animals had the pituitary gland protected with a lead plate set beneath the projection of the gland, while the second group was irradiated with the same dose, but without the protection. The control group of animals were non-irradiated. Results. Growth measurements of numerous parameters of the craniofacial and stomatognathic system as well as tibia in rats with the entire locally irradiated head showed a statistically significant delay, compared to both the non-irradiated control group and the group with the protected pituitary gland. Conclusion. The damage of the stomatognathic and craniofacial system was greater in animals with irradiated head without pituitary gland protection compared to those with the gland protection whereby the growth of the craniofacial system was most affected. Irraduition-induced pituitary gland hypofunction and irradiation itself showed a stronger effect on the lower jaw growth rate impairment compared to the upper jaw. Similarly, the delay in tibia growth was more pronounced in animals without pituitary gland protection during irradiation of their heads compared to non-protected ones.

Increasing Fat Graft Retention in Irradiated Tissue after Preconditioning with External Volume Expansion.

Fat grafting is an adjuvant that may improve the quality of radiation-damaged tissue. However, fat grafting for volume restoration in irradiated sites may be less effective because of a poorly vascularized fibrotic recipient bed. External volume expansion has emerged as a potential technique to prepare the recipient sites for improved survival of grafted fat. The authors previously demonstrated increased vasculature with external volume expansion stimulation of irradiated tissues. The authors now hypothesize that external volume expansion's improvements in recipient-site vascularity will increase the volume retention and quality of fat grafts in fibrotic irradiated sites. Athymic mice were irradiated until development of chronic radiation injury. Then, the irradiated site was stimulated by external volume expansion (external volume expansion group), followed by subcutaneous fat grafting. Grafts in an irradiated site without external volume expansion stimulation (irradiated control group) and grafts in a healthy nonirradiated (nonirradiated control group) site were used as controls. All grafts were monitored for 8 weeks and evaluated both histologically and by micro-computed tomography for analysis of volume retention. Hyperspectral imaging confirmed a 25 percent decrease in vascularity of irradiated tissue (irradiated control group) compared with nonirradiated tissue (nonirradiated control group). Grafts in the irradiated control group retained 11 percent less volume than grafts in the nonirradiated control group. The experimental external volume expansion group achieved a 20 percent (p = 0.01) increase in retained graft volume compared with the irradiated control group. External volume expansion stimulation can mitigate the effects of irradiation at the recipient site and in turn help preserve fat graft volume retention. Possible mechanisms include increased vascularity, adipogenic conversion, and increased compliance of a fibrotic recipient site.

Wavelength Effect in Laser Therapy of Diabetic Rats on Oxidants: AGEs, AOPP, ox-LDL Levels

In Low-Level Laser Therapy (LLLT) choosing the proper laser wavelength is extremely important. In this article, we have investigated the effects of four different laser wavelengths on oxidant parameters (AGE, AOPP, and ox-LDL) levels in diabetic Wistar rats experimentally. At first, 24 rats were divided into six equal groups. Except for the none diabetic control group, the other five groups received streptozotocin (STZ) injection to induce diabetes. Four groups of diabetic rats were then irradiated by four different laser wavelengths IR (808nm), Red (638nm), Green (532nm) and Blue (450nm). The last group which did not receive any irradiation is named non-irradiated diabetic control group. Laser therapies were performed Intravenously through an animal's caudal vein by a fiber Optics. Finally, the levels of oxidant parameters in rat's blood samples of each group were discussed. Results show a decrease in oxidants levels in all four irradiated groups of rats relative to the non-irradiated diabetic control group. More importantly, shorter wavelengths affect more efficient than longer wavelengths on reducing the oxidants levels with constant Laser energy. As a result, we conclude that laser with shorter wavelength e.g. Blue is more effective than longer wavelengths e.g. IR or Red, in reducing the oxidant parameters (AGE, AOPP, and ox-LDL) levels in Intravenous LLLT.

Abstract 1944: Detection of metabolic change in glioblastoma after radiotherapy using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model

Abstract OBJECTIVES: Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation therapy due to its different metabolic profile, investigation of the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after irradiation between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. A microarray analysis was also performed to compare to the imaging study. METHODS: DNP-MRI A 3T MRI scanner along with a custom-made head coil was used. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by an acquisition of 13C-two-dimensional MR-spectroscopy to measure the pyruvate-to-lactate ratios (Lac/Pyr) in the tumor region and the contralateral normal brain separately. Cell lines A human glioblastoma cell line, U251 and a glioma CSC line, NSC11 were cultured for stereotactic intracranial injection. Animal model Orthotopic brain tumor model was developed in athymic nude mice by intracranial implantation. When the tumor volume reached 50 +/- 10 mm3 on T2-weighted MRI, a sequence of DNP-MRI scans was performed 24 hours before and 6, 16 and 24 after 6-Gy whole-brain irradiation (N = 4 each with a non-irradiated control group). Independently, a microarray analysis using the tumor tissue samples excised 6 and 24 hours after irradiation was performed on Affymetrix Human Genome U133 Plus 2.0 array. RESULTS: DNP-MRI showed a decrease in Lac/Pyr 6 and 16 hours after irradiation compared to the control group in U251 whereas no significant change was observed in the NSC11 tumor. The microarray analysis showed a significant down-regulation of lactate dehydrogenase A (LDH-A) expression in U251 6 and 24 hours after irradiation, consistent with the finding of DNP-MRI. In the microarray analysis, SIRT2 and KLF4 expression also changed in the U251 tumor, suggested to regulate the LDH-A activity/expression. CONCLUSION: DNP-MRI demonstrated the difference in radiation response in glycolytic metabolism between the glioblastoma cell line and the CSC-derived glioma. This technique might be a feasible diagnostic method evaluating metabolic profiles of the brain tumors in the clinical settings of pretreatment and post-treatment. Citation Format: Tatsuya Kawai, Murali C. Krishna, Tamalee Kramp, Jeffery R. Brender, Philip Tofilon, Kevin A. Camphausen. Detection of metabolic change in glioblastoma after radiotherapy using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1944.

Treatment With Topical Deferoxamine Improves Cutaneous Vascularity and Tissue Pliability in an Irradiated Animal Model of Tissue Expander-Based Breast Reconstruction.

Postmastectomy radiation therapy is an important component of the multimodality approach to later-stage breast cancers. Unfortunately, despite its proven survival benefits, postmastectomy radiation therapy is deleterious to the skin and soft tissue, causing increased complications and worse aesthetic outcomes after breast reconstruction.There is currently no effective pharmaceutical agent to mitigate the soft tissue fibrosis and hypovascularity associated with soft tissue radiation. We hypothesized that a novel topical formulation of deferoxamine (DFX) will result in improved cutaneous vascularity and soft tissue pliability in an animal model of irradiated tissue expander-based breast reconstruction. This study consisted of 16 hairless rats divided into 4 equal groups: a control group (expander only), a tissue expanded and irradiated group, a tissue expanded + DFX group, and a tissue expanded/irradiated/DFX group. A novel topical formulation of DFX consisted of reconstituted drug dissolved in agents designed to enhance dermal penetrance. Vessels per high-power field (vHPF) were quantified histologically; micro-computed tomography angiography was used to assess vessel volume fraction (VVF) and vessel length density. Irradiated skin had less vascularity compared with control (3.81 vHPF vs 8.25 vHPF, P = 0.03; 0.79% VVF vs 1.53% VVF, P = 0.06). Treatment of irradiated skin with topical DFX reversed these effects, resulting in vascular findings similar to the control group histologically (7.94 vHPF vs 8.25 HPF, P = 0.985) and via micro-computed tomography angiography (1.05% VVF vs 1.53% VVF, P = 0.272). Similarly, radiation resulted in less volume expansion compared with controls (0.72 vs 0.8 mL, P = 0.04), whereas treatment with topical DFX reversed this effect, allowing for an expansion volume similar to the control group (0.81 vs 0.80 mL, P = 0.999). In an animal model of irradiated tissue expander-based breast reconstruction, treatment with topical DFX improved the cutaneous vascularity and tissue pliability, resulting in vascular density and final tissue expansion volumes similar to those found in the nonirradiated control group. Topical DFX may be an effective agent for the treatment of soft tissue radiation injury; future studies are indicated to further characterize this novel drug formulation.

Synergistic effects of Ferula gummosa and radiotherapy on induction of cytotoxicity in HeLa cell line.

Cervical cancer is the second most common type of cancer among women, worldwide; and for treatment of this type of cancer radiotherapy is commonly used. Ferula gummosa Boiss ("Barije" in Persian, from the family Apiaceae), (F. gummosa), is an extremely precious medicinal plant which naturally grows throughout the Mediterranean and Central Asia and is a native plant in Iran. The present study examined the cytotoxic effects of F. gummosa in terms of induction of apoptosis and radiosensitivity in HeLa cells. In order to determine F. gummosa cytotoxicity in HeLa cells, the cells were incubated with different concentrations of the plant resin (0-1000 µg/ml) for 24, 48 and 72 hr. Cytotoxicity was determined by MTT assay. The role of apoptosis in F. gummosa cytotoxicity was investigated using flow cytometry following propidium iodide (PI) staining of DNA. For radiosensitivity assessment, F. gummosa-treated cells were exposed to 2 Gy γ-rays, and cytotoxicity was determined in irradiated and non-irradiated (control) groups by MTT and the synergism factor was calculated. F. gummosa decreased cell viability in HeLa cells in a concentration- and time-dependent manner. Flow cytometry analysis indicated that apoptosis is involved in F. gummosa-induced cytotoxicity. Co-administration of F. gummosa and radiotherapy, showed that this plant at non-toxic low doses, could result in almost 5-fold increment in sensitization of cells towards radiation-induced toxicity. The concurrent use of F. gummosa and radiation increases radiosensitivity and cell death. Therefore, F. gummosa can be considered as a potential radiosensitizer agent against cervical cancer.

Topical emulsion containing pyrrolidine dithiocarbamate: effectiveness against ultraviolet B irradiation-induced injury of hairless mouse skin.

To evaluate the effects of a topical emulsion containing pyrrolidine dithiocarbamate (PDTC) (EcPDTC) in skin oxidative stress and inflammation triggered by ultraviolet B (UVB) irradiation (dose of 4.14J/cm2 ). Hairless mouse received treatment with 0.5g of EcPDTC or control emulsion (CTRLE) on the dorsal surface skin 12h, 6h and 5min before and 6h after the irradiation. Oxidative stress was evaluated by ferric reducing antioxidant power (FRAP), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS) scavenging capacity, reduced glutathione quantitation, catalase activity, superoxide anion production and lipid peroxidation products. Inflammation parameters were as follows: skin oedema, myeloperoxidase activity (neutrophil marker), matrix metalloproteinase-9 activity, collagen fibre damage, mast cell and sunburn cell counts, and cytokine production. Topical treatment with EcPDTC protected from UVB-induced skin injury by maintaining the antioxidant capacity levels similar to non-irradiated control group. Furthermore, EcPDTC inhibited UVB irradiation-induced superoxide anion production, lipid peroxidation and reduced skin inflammation by inhibiting skin oedema, neutrophil recruitment, metalloproteinase-9 activity, collagen fibre damage, mast cell and sunburn cell counts, and cytokine (TNF-α and IL-1β) production. Topical treatment with EcPDTC improves antioxidant systems and inhibits inflammation, protecting the skin from the damaging effects of UVB irradiation.

Blue light emitting diodes irradiation causes cell death in colorectal cancer by inducing ROS production and DNA damage.

The light emitting diodes (LEDs) irradiation has been demonstrated to be potential therapeutic strategies for several diseases. However, the blue LED effects remain largely unknown in colorectal cancer (CRC), which is a major cause of morbidity and mortality throughout the world. In this study, we determined the effects of blue LED irradiation, the maximal light emission at 470 nm in wavelength, in human CRC cell lines SW620 and HT29. The cells were irradiated with blue LED light for 0 J/cm2, 72 J/cm2, 144 J/cm2, 216 J/cm2 and 288 J/cm2 respectively. We found that irradiation with blue LED light induced a marked decrease of live cells and an increase of dead cells. Additionally, lower cell proliferation and a remarkably increase of cell apoptosis were observed in blue LED-irradiated cells as compared with non-irradiated control group. The cell migration was significantly inhibited by blue LED irradiation 24, 48 and 72 h later compared with non-treated group. Blue LED-treated CRC cells further displayed a remarkably inhibition of EMT process in CRC cells. Finally, we found the accumulation of ROS production and DNA damage were induced by blue LED irradiation. These results indicated that blue LED irradiation inhibits CRC cell proliferation, migration and EMT process as well as induces cell apoptosis, which may result from increased ROS accumulation and induction of DNA damage.

Abstract 72: Radiation induces LAP, latency-associated peptide of TGF-beta, on the surface of lymphoid cells in the tumor microenvironment

Abstract LAP, latency-associated peptide of TGFβ, is a protein that is co-synthesized with TGFβ and serves to effectively cage the cytokine, holding it in a latent state in the extracellular matrix and on the surface of immunosuppressive cell types. In the tumor microenvironment, activation of the LAP-TGFβ complex by integrins or proteases releases mature TGFβ, producing an immunosuppressive environment and fostering EMT. Anti-LAP antibodies have been shown to be effective at slowing tumor growth in mouse models (Gabriely et al., 2017) and offer a promising new approach to treating cancer. Anti-LAP antibodies are thought to mediate anti-tumor activity both by inhibiting the release of active TGFβ and through reducing the number of LAP+ inhibitory cells. Radiation is used as a first line therapy for many forms of cancer and is known to be a potent inducer of TGFβ production. This study was designed to determine the effects of radiation on the expression of LAP in the tumor microenvironment. 30 Balb/c mice were inoculated sc in the right flank with 106 CT26 colorectal cancer cells. When the mean tumor volume was approximately 300 mm3, half of the mice received a single dose of 20 Gy of targeted ionizing radiation. Groups of 3 mice from each cohort were sacrificed 1, 2, 3, 7 and 14 days after radiation and spleen and TILs collected for analysis by flow cytometry. As expected, radiation reduced tumor growth, with mean tumor volumes of 250 mm3 at day 14 vs 3600 mm3 in the non-irradiated control group. This was accompanied by a marked increase in CD45+ cells in the tumor which started on day 2 post-radiation and peaked on day 7, when 84% of live cells recovered from tumors were CD45+ (vs 25% in the non-irradiated controls). Radiation induced a significant (p<0.001) increase in CD8+ T cell numbers in the tumor, and prevented the increase in M2 macrophage numbers that occurred in the non-irradiated tumors. Radiation increased LAP expression on multiple cell types. Most notably, expression of LAP on DC and M2 macrophages increased steadily in response to radiation, with 33% of DC and 64% of M2 macrophages expressing LAP, significantly more than in control animals (10%, p=0.007 and 25%, p=0.01, respectively). In addition, 30% of M-MDSC from irradiated tumors expressed LAP, compared to 6% of non-irradiated tumors (p=0.03); expression on M-MDSC peaked at day 7. These data are the first demonstration that radiation increases cell-associated latent TGFβ in the tumor microenvironment. Radiation significantly altered both the distribution of immune cells in the tumor microenvironment and the expression of LAP on the surface of those cells. The increased LAP/TGFβ expression is anticipated to offset the effects of increased CD8+ effector cells. These observations suggest testing the use of an anti-LAP antibody in combination with radiation therapy to enhance effective immune responses against the tumor. Citation Format: Stavros Kopsiaftis, Xiufeng Song, Patricia E. Rao, Barbara S. Fox. Radiation induces LAP, latency-associated peptide of TGF-beta, on the surface of lymphoid cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 72.

Radiation-induced carotid artery lesions.

To review the current aspects of knowledge related to the risk of cerebrovascular events in patients receiving head and neck radiotherapy. Aliterature search was performed in PubMed. Papers meeting selection criteria were reviewed. We provide an update on the problem by identifying key studies that have contributed to our current understanding of the epidemiology, radiologic features, pathogenesis, and treatment of the disease. The incidence of carotid artery stenosis ranged from 18 to 38% in patients who underwent radiotherapy for head and neck cancer versus from 0 to 9.2% among the nonirradiated patients. Neck irradiation increases the intima-media thickness of the carotid artery wall. These changes are the earliest visible alteration in the carotid wall and are also detected with color Doppler ultrasonography. Endovascular treatment with acarotid angioplasty and stenting is the first-line treatment for most symptomatic patients. Radiation-induced atherosclerosis is adifferent and accelerated form of atherosclerosis, which implies amore aggressive disease with adifferent biologic behavior. The disease is characterized by ahigh rate of carotid artery stenosis compared to those observed in nonirradiated control group patients. To prevent the risk of stroke, surveillance and imaging with ultrasonography should enable detection of severe stenosis. Endovascular treatment with acarotid angioplasty and stenting has been proposed as an attractive and minimally invasive alternative for some radiation-induced stenoses.

MRI Study on the Changes of Bone Marrow Microvascular Permeability and Fat Content after Total-Body X-Ray Irradiation.

In this study, we investigated microvascular perfusion status, changes to fat content and fatty acid composition in the bone marrow of rat femurs after total-body irradiation by quantitative permeability parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and ex vivo high-resolution magic angle spinning (HRMAS) 1H nuclear magnetic resonance spectroscopy (NMRS). Thirty-six Sprague-Dawley rats were randomly assigned to either an irradiated or nonirradiated control group. Permeability imaging using DCE-MRI and HRMAS 1H NMRS was performed before irradiation, as well as at days 4 and 7 postirradiation. The volume transfer constant (Ktrans) values increased to 2.219 ± 0.418/min ( P < 0.01) at day 4 and to 2.760 ± 0.217/min at day 7 ( P < 0.01) postirradiation. The plasma fraction (vp) values gradually decreased. The proportion of (n-6) polyunsaturated fatty acids (PUFA) gradually reached a peak at day 7, the proportion of (n-3) PUFA gradually decreased and the proportion of saturated fatty acids gradually increased. After irradiation, Ktrans at different times showed significant negative correlation with (n-3) PUFA ( r = -0.6393, P < 0.01) and significant positive correlation with (n-6) PUFA ( r = 0.6841, P < 0.05). These findings indicate that bone marrow microcirculation perfusion and vascular permeability correlated with fat content at an early time point after irradiation. A pathophysiological mechanism may exist based on fat-vascular permeability in the case of injury to bone marrow microcirculation.

Er,Cr:YSGG Laser Energy Delivery: Pulse and Power Effects on Enamel Surface and Erosive Resistance.

High power lasers have been suggested as a useful tool for dental caries and erosion prevention due to the increase of enamel acid resistance. to evaluate the effect of Er,Cr:YSGG (erbium,chromium:yttrium, scandium, gallium, garnet) laser irradiation pulse frequency and power on enamel surface and acid erosion resistance. By combining pulse frequency (5-75 Hz) and power settings (0.10-1.00 W), 20 irradiated groups and one nonirradiated control group were tested. A total of 63 bovine enamel blocks (n = 3/group) were prepared for surface hardness and roughness evaluation, performed in three phases: baseline, after irradiation, and after erosive challenge. Enamel blocks were irradiated with Er,Cr:YSGG laser with MZ8 tip (iPlus; Waterlase, Biolase, CA) for 30 sec according to experimental group and submitted. Erosive challenge consisted of four cycles alternating immersion in 0.01 M HCl (5 mL/mm2; 2 min; at 37°C) and immersion in artificial saliva for 3 h. Analysis of variance (three-way ANOVA), Tukey's test, and Pearson correlation were performed for the statistical analysis (p < 0.05). After irradiation, groups irradiated with pulse frequency of 10 and 15 Hz showed a decrease in surface hardness. After erosive challenge, 5 and 75 W groups showed increase in surface hardness; 0.25, 0.5, 0.75, and 1 W groups showed minor alterations in surface roughness. the irradiation of Er,Cr:YSGG laser with different parameters of power and pulse frequency settings may alter enamel surface and erosive resistance differently. Pulse frequency of 30 Hz and power of 0.50 W was considered the best parameter to prevent enamel acid erosion.

Localized WR-1065 Delivery to Murine Salivary Glands has Radioprotective Effect and Hemodynamic Benefit Over Systemic Administration

There are over 50,000 cases of head and neck cancer (HNC) diagnosed each year in the United States. Approximately 75% of these patients will undergo radiation at some point during their treatment, and of those patients over 50% will go on to develop permanent xerostomia. Amifostine is the only FDA approved drug for use in xerostomia radioprophylaxis, but suffers from a narrow therapeutic window and systemic toxicities including severe nausea and hypotension that severely limit its clinical utility. To overcome challenges of systemic administration and potentially increase effectiveness, local delivery via Wharton’s duct of WR-1065 (the active metabolite of Amifostine) was utilized. Our hypothesis is that retroductal delivery of WR-1065 to the murine submandibular gland (SMG) will improve salivary gland function while reducing drug side effects such as hypotension at 90 days following a single xerogenic dose (15.0 Gy) of irradiation. All studies used n = 5 C57BL/6 female mice per treatment group. To assess blood pressure response, anesthetized mice underwent bilateral femoral catheterization and aortic placement of a pressure transducer. Mice received either IV or retroductal doses of Amifostine or WR-1065. Baroreflex was subsequently measured by bolus administration of phenylephrine. For radiation studies, mice received unilateral SMG injection with WR-1065 or saline. A single dose of 15.0 Gy was then delivered to the submandibular glands using a Cs137 irradiator with a slit collimator. Study endpoints include volume of stimulated saliva secretion 90 days post irradiation, SMG histology, and immunohistochemical staining compared to irradiated and nonirradiated control groups. Hemodynamic data show retained baroreflex sensitivity and mean arterial pressure following retrograde WR-1065 injection. Furthermore, mice receiving retrograde WR-1065 show significantly higher volumes of stimulated saliva secretion (61±29 μL) compared to irradiated (14±8 μL) and saline treated controls (19 ±7 μL) at 90 days (p<0.05). Furthermore, no significant difference exists in gland secretion between irradiated WR-1065 treated mice and non-irradiated mice. Preliminary data shows improvement in stimulated saliva secretion and gland histology in WR-1065 retroductally treated mice 90 days post irradiation. Hemodynamic data further supports development of this drug delivery modality for SMG radioprotection, as blood pressure and baroreflex sensitivity are retained following retrograde WR-1065 injection.

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408nm) to induce the native fluorescence that is emitted in the visible range. Under 408nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668nm for irradiated and control groups. A relative increase of the fluorescence at 633nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633nm emission are needed to confirm this hypothesis.

Porphyra-334, a mycosporine-like amino acid, attenuates UV-induced apoptosis in HaCaT cells.

The main aim of the current research was to study the effect of porphyra-334, one of mycosporine-like amino acids (MAAs), well known as UV-absorbing compounds, on UVinduced apoptosis in human immortalized keratinocyte (HaCaT) cells. Due to their UV-screening capacity and ability to prevent UV-induced DNA damage, MAAs have recently attracted considerable attention in both industry and research in pharmacology. Herein, human HaCaT cells were used to determine the biological activities of porphyra- 334 by various in vitro assays, including proliferation, apoptosis and Western blot assays. The proliferation rate of UV-irradiated HaCaT cells was significantly decreased compared to the control group. Pretreatment with porphyra- 334 markedly attenuated the inhibitory effect of UV and induced a dramatic decrease in the apoptotic rate. Expression of active caspase-3 protein was increased in response to UV irradiation, while caspase-3 levels were similar between cells treated with porphyra-334 and the non-irradiated control group. Taken together, our data suggest that porphyra-334 inhibits UV-induced apoptosis in HaCaT cells through attenuation of the caspase pathway.

Effects of Medical Diagnostic Low-dose X Rays on Human Lymphocytes: Mitochondrial Membrane Potential, Apoptosis and Cell Cycle.

Low-dose radiation is widely used across the world for the diagnosis of many diseases by means of a variety of imaging technologies. However, the harmful effects of exposure to low-dose radiation during medical examination remain controversial. The authors studied the effects of medical diagnostic low-dose x rays (i.e., 0.03, 0.05, or 0.1 mGy) after an in vitro exposure of human lymphocytes. Cells with no irradiation served as the non-irradiated control group. Three biological indicators were used to determine the effects of medical diagnostic low-dose x rays at 4, 8, 24, 48, and 72 h post-irradiation. These biological endpoints were mitochondrial membrane potential (ΔΨm), cell cycle, and apoptosis. Results indicated no changes in the ΔΨm, number of apoptotic cells, and cell cycle in lymphocytes exposed to these low doses of radiation, as compared to the corresponding non-irradiated lymphocytes at all harvest time-points. These results suggested that there were no harmful effects of the diagnostic low-dose x rays when human lymphocytes were exposed in an in vitro condition.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

Origanum vulgare leaf extract protects mice bone marrow cells against ionizing radiation.

Ionizing radiation produces free radicals which induce DNA damage and cell death. Origanum vulgare leaf extract (OVLE) is a natural compound and its capability of scavenging free radicals and its antioxidant activity have been demonstrated by many researchers. In this study, using micronucleus assay, radioprotective effect of OVLE against clastogenic and cytotoxic effect of gamma irradiation has been investigated in mice bone marrow cells. OVLE was injected intraperitoneally to the BALB/c mice 1hr prior to gamma irradiation (3Gy) at the doses of 100 and 200 mg/kg. Twenty four hours after irradiation or treatment, animals were killed and smears were prepared from the bone marrow cells. The slides were stained with May Grunwald-Giemsa method and analyzed microscopically. The frequency of micronucleated polychromatic erythrocytes (MnPCEs), micronucleated normochromatic erythrocyte (MnNCEs) and cell proliferation ratio PCE/PCE+NCE (polychromatic erythrocyte/polychromatic erythrocyte + normochromatic erythrocyte) were calculated. The results showed that gamma irradiation (3Gy) increased the frequency of MnPCEs, MnNCEs and reduced the PCE/PCE+NCE ratio in mice bone marrow compared to the non-irradiated control group (p<0.0001). Injection of OVLE significantly reduced the frequency of MnPCEs (p<0.0001) and MnNCEs (p<0.05) and increased the PCE/PCE+NCE ratio as compared to the irradiated control group (p<0.05). It seems that OVLE with its antioxidant properties and its capability of scavenging free radicals and reactive oxygen species can reduce the cytotoxic effects of gamma irradiation in mice bone marrow cells.

An investigation into corneal enzymatic resistance following epithelium-off and epithelium-on corneal cross-linking protocols

The aim of this study was to investigate corneal enzymatic resistance following epithelium off and on riboflavin/UVA cross-linking (CXL). One hundred and fourteen porcine eyes were divided into four non-irradiated control groups and seven CXL groups. The latter comprised; (i) epithelium-off, 0.1% iso-osmolar riboflavin, 9 mW UVA irradiation for 10 min, (ii) disrupted epithelium, 0.1% hypo-osmolar riboflavin, 9 mW UVA for 10 min, (iii) epithelium-on, 0.25% hypo-osmolar riboflavin with 0.01% benzylalkonium chloride (BACS), 9 mW UVA for 10 min, (iv) epithelium-on, 5 min iontophoresis at 0.1 mA for 5 min with 0.1% riboflavin solution, 9 mW UVA for 10 min or (v) 12.5 min, (vi) epithelium-on, prolonged iontophoresis protocol of 25 min with 1.0 mA for 5 min and 0.5 mA for 5 min with 0.25% riboflavin with 0.01% BACS, 9 mW UVA for 10 min or (vii) 12.5 min. Enzymatic resistance was assessed by daily measurement of a corneal button placed in pepsin solution and measurement of corneal button dry weight after 11 days of digestion. This study revealed that the enzymatic resistance was greater in CXL corneas than non-irradiated corneas (p < 0.0001). Epithelium-off CXL showed the greatest enzymatic resistance (p < 0.0001). The prolonged iontophoresis protocol was found to be superior to all other trans-epithelial protocols (p < 0.0001). A 25% increase in UVA radiance significantly increased corneal enzymatic resistance (p < 0.0001). In conclusion, although epithelium-on CXL appears to be inferior to epithelium-off CXL in terms of enzymatic resistance to pepsin digestion, the outcome of epithelium-on CXL may be significantly improved through the use of higher concentrations of riboflavin solution, a longer duration of iontophoresis and an increase in UVA radiance.