Activation of NLRP3 inflammasome in lung epithelial cells triggers radiation-induced lung injury.

BACKGROUND: Radiation-induced lung injury (RILI) is the most common and serious complication of chest radiotherapy. However, reported radioprotective agents usually lead to radiation resistance in tumor cells. The key to solving this problem is to distinguish between the response of tumor cells and normal lung epithelial cells to radiation damage. METHODS: RNA-Seq was used to recognize potential target of alleviating the progression of RILI as well as inhibiting tumor growth. The activation of NLRP3 inflammasome in lung epithelial cells was screened by qRT-PCR, western blotting, immunofluorescence, and ELISA. An in vivo model of RILI and in vitro conditioned culture model were constructed to evaluate the effect of NLRP3/interleukin-1ß on fibroblasts activation. ROS, ATP, and (NADP)+/NADP(H) level in lung epithelial cells was detected to explore the mechanism of NLRP3 inflammasome activation. The lung macrophages of the mice were deleted to evaluate the role of lung epithelial cells in RILI. Moreover, primary cells were extracted to validate the results obtained from cell lines. RESULTS: NLRP3 activation in epithelial cells after radiation depends on glycolysis-related reactive oxygen species accumulation. DPYSL4 is activated and acts as a negative regulator of this process. The NLRP3 inflammasome triggers interleukin-1ß secretion, which directly affects fibroblast activation, proliferation, and migration, eventually leading to lung fibrosis. CONCLUSIONS: Our study suggests that NLRP3 inflammasome activation in lung epithelial cells is essential for radiation-induced lung injury. These data strongly indicate that targeting NLRP3 may be effective in reducing radiation-induced lung injury in clinical settings.

Immature Testicular Tissue Engineered from Weaned Mice to Adults for Prepubertal Fertility Preservation-An In Vivo Translational Study.

Male pediatric survivors of cancers and bone marrow transplantation often require adjuvant chemoradiation therapy that may be gonadotoxic. The optimal methods to preserve fertility in these prepubertal males are still under investigation. This manuscript presents an in vivo experiment which involved transplantation of immature testicular tissues (ITT) from transgenic donor, to wild-type recipient mice. Donors and recipients were age-mismatched (from 20-week-old donors to 3-week-old recipients, and vice versa) and the transplantation sites involved the abdomen, skin of the head, back muscle, and scrotum. The application of poly-l-lactic acid (PLLA) scaffold was also evaluated in age-matched donors and recipients (both 3-weeks-old). To quantitively evaluate the process of spermatogenesis after ITT transplantation and scaffold application, bioluminescence imaging (BLI) was employed. Our result showed that ITT from 3-week-old mice had the best potential for spermatogenesis, and the optimal transplantation site was in the scrotum. Spermatogenesis was observed in recipient mice up to 51 days after transplantation, and up to the 85th day if scaffold was used. The peak of spermatogenesis occurred between the 42nd and 55th days in the scaffold group. This animal model may serve as a framework for further studies in prepubertal male fertility preservation.

Rat Models of Partial-body Irradiation with Bone Marrow-sparing (Leg-out PBI) Designed for FDA Approval of Countermeasures for Mitigation of Acute and Delayed Injuries by Radiation.

ABSTRACT: The goal of this study was to develop rat models of partial body irradiation with bone-marrow sparing (leg-out PBI) to test medical countermeasures (MCM) of both acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE) under the FDA animal rule. The leg-out PBI models were developed in female and male WAG/RijCmcr rats at doses of 12.5-14.5 Gy. Rats received supportive care consisting of fluids and antibiotics. Gastrointestinal ARS (GI-ARS) was assessed by lethality to d 7 and diarrhea scoring to d 10. Differential blood counts were analyzed between d 1-42 for the natural history of hematopoietic ARS (H-ARS). Lethality and breathing intervals (BI) were measured between d 28-110 to assess delayed injury to the lung (L-DEARE). Kidney injury (K-DEARE) was evaluated by measuring elevation of blood urea nitrogen (BUN) between d 90-180. The LD50/30, including both lethality from GI-ARS and H-ARS, for female and male rats are 14.0 Gy and 13.5 Gy, respectively, while the LD50/7 for only GI-ARS are 14.3 Gy and 13.6 Gy, respectively. The all-cause mortalities, including ARS and L-DEARE, through 120 d (LD50/120) are 13.5 Gy and 12.9 Gy, respectively. Secondary end points confirmed occurrence of four distinct sequelae representing GI, hematopoietic, lung, and kidney toxicities after leg-out PBI. Adult rat models of leg-out PBI showed the acute and long-term sequelae of radiation damage that has been reported in human radiation exposure case studies. Sex-specific differences were observed in the DRR between females and males. These rat models are among the most useful for the development and approval of countermeasures for mitigation of radiation injuries under the FDA animal rule.

Irradiation Accelerates Plaque Formation and Cellular Senescence in Flow-Altered Carotid Arteries of Apolipoprotein E Knock-Out Mice.

Background Chronic inflammation through cellular senescence, known as the senescence-associated secretory phenotype, is a mechanism of various organ diseases, including atherosclerosis. Particularly, ionizing radiation (IR) contributes to cellular senescence by causing DNA damage. Although previous clinical studies have demonstrated that radiotherapy causes atherosclerosis as a long-term side effect, the detailed mechanism is unclear. This study was conducted to investigate the relationship between radiation-induced atherosclerosis and senescence-associated secretory phenotype in murine carotid arteries. Methods and Results Partial ligation of the left carotid artery branches in 9-week-old male apolipoprotein E-deficient mice was performed to induce atherosclerosis. The mice received total body irradiation at a dose of 6 Gy using gamma rays at 2 weeks post operation. We compared the samples collected 4 weeks after IR with unirradiated control samples. The IR and control groups presented pathologically progressive lesions in 90.9% and 72.3% of mice, respectively. Plaque volume, macrophage accumulation, and phenotype switching of vascular smooth muscle cells were advanced in the IR group. Irradiated samples showed increased persistent DNA damage response (53BP1 [p53 binding protein 1]), upregulated cyclin-dependent kinase inhibitors (p16INK4a and p21), and elevated inflammatory chemokines expression (monocyte chemotactic protein-1, keratinocyte-derived chemokine, and macrophage inflammatory protein 2). Conclusions IR promoted plaque growth in murine carotid arteries. Our findings support the possibility that senescence-associated secretory phenotype aggravates atherogenesis in irradiated artery. This mice model might contribute to mechanism elucidation of radiation-induced atherosclerosis.

PEG-G-CSF and L-Citrulline Combination Therapy for Mitigating Skin Wound Combined Radiation Injury in a Mouse Model.

Radiation combined injury (RCI, radiation exposure coupled with other forms of injury, such as burn, wound, hemorrhage, blast, trauma and/or sepsis) comprises approximately 65% of injuries from a nuclear explosion, and greatly increases the risk of morbidity and mortality when compared to that of radiation injury alone. To date, no U.S. Food and Drug Administration (FDA)-approved countermeasures are available for RCI. Currently, three leukocyte growth factors (Neupogen®, Neulasta® and Leukine®) have been approved by the FDA for mitigating the hematopoietic acute radiation syndrome. However these granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) products have failed to increase 30-day survival of mice after RCI, suggesting a more complicated biological mechanism is in play for RCI than for radiation injury. In the current study, the mitigative efficacy of combination therapy using pegylated (PEG)-G-CSF (Neulasta) and -citrulline was evaluated in an RCI mouse model. L-citrulline is a neutral alpha-amino acid shown to improve vascular endothelial function in cardiovascular diseases. Three doses of PEG-G-CSF at 1 mg/kg, subcutaneously administered on days 1, 8 and 15 postirradiation, were supplemented with oral -citrulline (1 g/kg), once daily from day 1 to day 21 postirradiation. The combination treatment significantly improved the 30-day survival of mice after RCI from 15% (vehicle-treated) to 42%, and extended the median survival time by 4 days, as compared to vehicle controls. In addition, the combination therapy significantly increased body weight and bone marrow stem and progenitor cell clonogenicity in RCI mice, and accelerated recovery from RCI-induced intestinal injury, compared to animals treated with vehicle. Treatment with -citrulline alone also accelerated skin wound healing after RCI. In conclusion, these data indicate that the PEG-G-CSF and -citrulline combination therapy is a potentially effective countermeasure for mitigating RCI, likely by enhancing survival of the hematopoietic stem/progenitor cells and accelerating recovery from the RCI-induced intestinal injury and skin wounds.

The Effect of Resveratrol on Radioiodine Therapy-Associated Lacrimal Gland Damage.

PURPOSE: We have evaluated the potential radioprotective, antioxidant and anti-apoptotic effects of resveratrol (RSV) against high-dose radioactive iodine (RAI) therapy associated damage of the lacrimal glands by biochemical, histopathological and immunohistochemical methods. MATERIALS AND METHODS: Thirty Wistar-albino rats were randomly divided into three groups; the control group received no treatment or medication, the RAI group received RAI but no medication and the RSV group received oral RAI and intraperitoneal RSV. RSV was started at day one, before RAI administration, and continued for 8 days. Bilateral intraorbital (IG), extraorbital (EG), and Harderian (HG) lacrimal glands were evaluated in all rats for histopathological, immunohistochemical, tissue cytokine and oxidant and antioxidant level assessment. RESULTS: RSV group restored inflammation, fibrosis, vacuolization, change in nucleus characteristics, lipofuscin-like accumulation and cellular morphologic patterns were statistically significant in all lacrimal gland types, compared to the RAI group (p < .05 for all variables). Similarly, elevated Caspase-3 and TUNEL levels in the RAI group were significantly alleviated in the RSV group in all lacrimal gland types (p < .05 for all variables). RAI administration significantly elevated TNF-α, IL-6, NF-кb levels, and decreased IL-10 levels (p < .05 for all parameters) whereas TOS levels significantly increased and TAS levels were significantly decreased. However, RSV significantly diminished TNF-α, IL-6, IL-4, and NF-кb levels. Furthermore, RSV significantly decreased TOS and increased TAS levels (p < .05 for all variables). CONCLUSIONS: We conclude that with its anti-cancer effect as well as its antioxidant effect RSV has protected the histopathological pattern of the lacrimal glands from the damage, decreased inflammation in histopathologic assessments, and decreased tissue cytokine levels, apoptosis and DNA fragmentation on the lacrimal glands after RAI.

Astragalus polysaccharide has a protective effect on hematopoiesis in an irradiated mouse model and decreases apoptosis in megakaryocytes.

Huangqi, the dried root of Radix Astragali, is an essential herb in Traditional Chinese Medicine and has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), the bioactive compound of Huangqi, serves a crucial role in hematopoiesis. The aim of the present study was to investigate the hematopoietic effects, in particular the thrombopoietic effects, and the molecular mechanisms of ASPS using an irradiation­induced myelosuppressive mouse model. Colony­forming unit assays, flow cytometric analysis of apoptosis, ELISAs, Giemsa staining and western blotting were performed to determine the hematopoietic and anti­apoptotic effects of ASPS. The results demonstrated that ASPS enhanced the recovery of red blood cells at day 21 following treatment, as well as platelets and white blood cells at day 14. In addition, ASPS promoted colony formation in all lineages (megakaryocytes, granulocyte monocytes, erythroid cells and fibroblasts). The morphological study of the bone marrow demonstrated that tri­lineage hematopoiesis was preserved in the ASPS­ and thrombopoietin (TPO)­treated groups compared with the control group. The overall cellularity (mean total cell count/area) of the ASPS­treated group was similar to that of the TPO­treated group. Additionally, in vitro experiments indicated that treatment with 100 µg/ml ASPS exhibited the maximum effect on colony formation. ASPS attenuated cell apoptosis in megakaryocytic cells via inhibiting the mitochondrial caspase­3 signaling pathway. In conclusion, ASPS promoted hematopoiesis in irradiated myelosuppressive mice possibly via enhancing hematopoietic stem/progenitor cell proliferation and inhibiting megakaryocytes apoptosis.

A Potential Role for Excess Tissue Iron in Development of Cardiovascular Delayed Effects of Acute Radiation Exposure.

Murine hematopoietic-acute radiation syndrome (H-ARS) survivors of total body radiation (TBI) have a significant loss of heart vessel endothelial cells, along with increased tissue iron, as early as 4 mo post-TBI. The goal of the current study was to determine the possible role for excess tissue iron in the loss of coronary artery endothelial cells. Experiments used the H-ARS mouse model with gamma radiation exposure of 853 cGy (LD50/30) and time points from 1 to 12 wk post-TBI. Serum iron was elevated at 1 wk post-TBI, peaked at 2 wk post-TBI, and returned to non-irradiated control values by 4 wk post-TBI. A similar trend was seen for transferrin saturation, and both results correlated inversely with red blood cell number. Perls' Prussian Blue staining, used to detect iron deposition in heart tissue sections, showed myocardial iron was present as early as 2 wk following irradiation. Pretreatment of mice with the iron chelator deferiprone decreased tissue iron but not serum iron at 2 wk. Coronary artery endothelial cell density was significantly decreased as early as 2 wk vs. non-irradiated controls (P<0.05), and the reduced density persisted to 12 wk after irradiation. Deferiprone treatment of irradiated mice prevented the decrease in endothelial cell density at 2 and 4 wk post-TBI compared to irradiated, non-treated mice (P<0.03). Taken together, the results suggest excess tissue iron contributes to endothelial cell loss early following TBI and may be a significant event impacting the development of delayed effects of acute radiation exposure.

A Mouse Model for Skeletal Structure and Function Changes Caused by Radiation Therapy and Estrogen Deficiency.

Radiation therapy and estrogen deficiency can damage healthy bone and lead to an increased fracture risk. The goal of this study is to develop a mouse model for radiation therapy using a fractionated biologically equivalent dose for cervical cancer treatment in both pre- and postmenopausal women. Thirty-two female C57BL/6 mice 13 weeks of age were divided into four groups: Sham + non-irradiated (SHAM + NR), Sham + irradiated (SHAM + IRR), ovariectomy + non-irradiated (OVX + NR) and ovariectomy + irradiated (OVX + IRR). The irradiated mice received a 6 Gy dose of X-rays to the hindlimbs at Day 2, Day 4 and Day 7 (18 Gy total). Tissues were collected at Day 35. DEXA, microCT analysis and FEA were used to quantify structural and functional changes at the proximal tibia, midshaft femur, proximal femur and L1 vertebra. There was a significant (p < 0.05) decline in proximal tibia trabecular BV/TV from (1) IRR compared to NR mice within Sham (- 46%) and OVX (- 41%); (2) OVX versus Sham within NR mice (- 36%) and IRR mice (- 30%). With homogenous material properties applied to the proximal tibia mesh using FEA, there was (1) an increase in whole bone (trabecular + cortical) structural stiffness from IRR compared to NR mice within Sham (+ 10%) and OVX (+ 15%); (2) a decrease in stiffness from OVX versus Sham within NR mice (- 18%) and IRR mice (- 14%). Fractionated irradiation and ovariectomy both had a negative effect on skeletal microarchitecture. Ovariectomy had a systemic effect, while skeletal radiation damage was largely specific to trabecular bone within the X-ray field.

Metformin modulates cardiac endothelial dysfunction, oxidative stress and inflammation in irradiated rats: A new perspective of an antidiabetic drug.

Cardiovascular disease is one of the most pivotal disorders after radiotherapy. The aim of this study investigates the possible protective effect of metformin against gamma radiation-induced heart damage in male rats. Group 1 (control) received saline, group 2 was whole body gamma-irradiated 5 Gy, group 3 was orally administered metformin 50 mg/kg/day for 2 weeks, group 4 received metformin 50 mg/kg/day for 1 week, then exposed to whole-body gamma radiation at a dose of 5 Gy and continued with metformin for further 1 week. The results revealed that the administration of metformin to irradiated rats significantly ameliorated the changes in cardiac biomarkers (LDH and CK-MB) compared with irradiated group. Heart catalase and SOD activities showed normal level when compared with the irradiated group. Also, NF-κB, IL-6 and TNF- α levels were markedly decreased compared with the corresponding values of irradiated group. Consequently, metformin reduced E-selectin as well ICAM and VCAM-1. These results confirmed by histopathological examination. In conclusion, concomitant administration of metformin during radiotherapy acts as a potent heart protector from oxidative stress, inflammatory mediators and endothelial dysfunction induced damages. Results thus hold a great promise for a new implication of an antidiabetic drug (metformin) as adjunct to radiotherapy.

BIO 300, a Nanosuspension of Genistein, Mitigates Radiation-Induced Erectile Dysfunction and Sensitizes Human Prostate Cancer Xenografts to Radiation Therapy.

PURPOSE: To assess whether BIO 300, a synthetic genistein nanosuspension, improves the therapeutic index in prostate cancer treatment by preventing radiation-induced erectile dysfunction (ED) without reducing tumor radiosensitivity. METHODS AND MATERIALS: Male Sprague-Dawley rats were exposed to 25 Gy of 220-kV prostate-confined x-rays. Animals were randomized to receive sham radiation therapy (RT), RT alone, RT with daily BIO 300 at 2 experimental dosing regimens, or RT with daily genistein. Erectile response was evaluated over time. Penile shaft tissue was harvested for histologic analyses. Murine xenograft studies using prostate cancer cell lines determined the effects of BIO 300 dosing on RT efficacy. RESULTS: Prostate-confined RT significantly decreased apomorphine-induced erectile response (P < .05 vs sham RT). Erection frequency in animals receiving prophylactic treatment with BIO 300 starting 3 days before RT was similar to sham controls after RT. Treatment with synthetic genistein did not mitigate loss in erectile frequency. At week 14, post-RT treatment with BIO 300 resulted in significantly higher quality of erectile function compared with both the RT arm and the RT arm receiving genistein starting 3 days before irradiation (P < .05). In hormone-sensitive and insensitive prostate tumor-bearing mice, BIO 300 administration did not negatively affect radiation-induced tumor growth delay. CONCLUSIONS: BIO 300 prevents radiation-induced ED, measured by erection frequency, erectile function, and erection quality, when administered 3 days before RT and continued daily for up to 14 weeks. Data also suggest that BIO 300 administered starting 2 hours after RT mitigates radiation-induced ED. Data provide strong nonclinical evidence to support clinical translation of BIO 300 for mitigation of ED while maintaining treatment response to RT.

dTMP-GH Fusion Protein Therapy Improves Survival after Radiation Injury Combined with Skin-Burn Trauma in Mice.

Exposure to ionizing radiation combined with traumatic tissue injury is an important life-threatening condition found in the civilian populations after nuclear and radiological events. The significance feature of radiation combined injury (RCI) is the severe combined effect, which makes the injury more complicated. At present, there are limited measures available to treat RCI. Here we show that a chimeric protein dTMP-GH, fusing human growth hormone (hGH) with a tandem dimer of thrombopoietin mimetic peptide (dTMP), could be an effective therapy agent for RCI in a mice model. In this study, using a RCI mouse model exposed to 60Co γ-ray photons (6.0 Gy, 0.3 Gy/min) followed by a 20% total-body-surface-area burns (henceforth called: RB-CI) was established. Administration of dTMP-GH (200 ug/kg) for 10 consecutive days beginning at 24 h after injury improved survival rate during a 30-day observation period compared with the control vehicle group. dTMP-GH treatment also showed enhanced bone marrow hematopoiesis recovery determined by peripheral blood analysis and bone marrow histopathology. Meanwhile, dTMP-GH treatment accelerated skin wound closure and mitigated ileum injury in the RCI model. These results suggest that dTMP-GH may prove to be an effective therapeutic drug for RCI.

Protective Effect of Selenium-L-methionine on Radiation-induced Acute Pneumonitis and Lung Fibrosis in Rat.

BACKGROUND: In this study, we aimed to detect the changes in the level of interleukin (IL)-4 and IL-13 cytokines and their downstream genes including interleukin-13 receptor subunit alpha-2 (IL13Ra2), interleukin-4 receptor subunit alpha-1 (IL4Ra1), dual oxidase 1 (DUOX1) and dual oxidase 2 (DUOX2). The protective effects of Selenium-L-methionine on radiation-induced histopathological damages and changes in the level of these cytokines and genes were detected. METHODS: Four groups of 20 rats (5 rats in each) namely, control; Selenium-L-methionine, radiation and radiation plus Selenium-L-methionine were used in this study. 4 mg/kg of Selenium-Lmethionine was administered 1 day before irradiation and five consecutive days after irradiation. Irradiation was done using a dose of 15 Gy 60Co gamma rays at 109 cGy/min. All rats were sacrificed 10 weeks after irradiation for detecting changes in IL-4 and IL-13 cytokines, the expressions of IL13Ra2, IL4Ra1, Duox1 and Duox2 and histopathological changes. RESULTS: The level of IL-4 but not IL-13 increased after irradiation. This was associated with increased expression of IL4Ra1, Duox1 and Duox2, in addition to changes in morphological properties. Selenium-L-methionine could attenuate all injury markers following lung irradiation. CONCLUSION: Selenium-L-methionine can protect lung tissues against toxic effects of ionizing radiation. It is possible that the modulation of immune responses and redox interactions are involved in the radioprotective effect of this agent.

Rebamipide alleviates radiation-induced colitis through improvement of goblet cell differentiation in mice.

BACKGROUND AND AIM: Radiation-induced colitis is a common clinical problem associated with radiotherapy and accidental exposure to ionizing radiation. Goblet cells play a pivotal role in the intestinal barrier against pathogenic bacteria. Rebamipide, an anti-gastric ulcer drug, has the effects to promote goblet cell proliferation. The aim of this study was to investigate whether radiation-induced colonic injury could be alleviated by rebamipide. METHODS: This study orally administered rebamipide for 6 days to mice, which were subjected to 13 Gy abdominal irradiation, to evaluate the therapeutic effects of rebamipide against radiation-induced colitis. To confirm the effects of rebamipide on irradiated colonic epithelial cells, this study used the HT29 cell line. RESULTS: Rebamipide clearly alleviated the acute radiation-induced colitis, as reflected by the histopathological data, and significantly increased the number of goblet cells. The drug also inhibited intestinal inflammation and protected from bacterial translocation during acute radiation-induced colitis. Furthermore, rebamipide significantly increased mucin 2 expression in both the irradiated mouse colon and human colonic epithelial cells. Additionally, rebamipide accelerated not only the recovery of defective tight junctions but also the differentiation of impaired goblet cells in an irradiated colonic epithelium, which indicates that rebamipide has beneficial effects on the colon. CONCLUSIONS: Rebamipide is a therapeutic candidate for radiation-induced colitis, owing to its ability to inhibit inflammation and protect the colonic epithelial barrier.

Cavernous Nerve Injury by Radiation Therapy May Potentiate Erectile Dysfunction in Rats.

PURPOSE/OBJECTIVES: Radiation-induced erectile-dysfunction (RiED) is one of the most common side effects of radiation therapy (RT) and significantly reduces the quality of life (QoL) of cancer patients. Approximately 50% of prostate cancer patients experience RiED within 3 to 5 years after completion of RT. A series of vascular, muscular, and neurogenic injuries after prostate RT lead to RiED; however, the precise role of RT-induced neurogenic injury in RiED has not been fully established. The cavernous nerves (CN) are postganglionic parasympathetic nerves located beside the prostate gland that assist in penile erection. This study was designed to investigate the role of CN injury, tissue damage, and altered signaling pathways in an RiED rat model. METHODS AND MATERIALS: Male rats were exposed to a single dose of 25 Gy prostate-confined RT. Erectile function was evaluated by intracavernous pressure (ICP) measurements conducted both 9 and 14 weeks after RT. Neuronal injury was evaluated in the CN using quantitative polymerase chain reaction, conduction studies, transmission electron microscopy, and immunoblotting. Masson trichrome staining was performed to elucidate fibrosis level in penile tissues. RESULTS: There were significant alterations in the ICP (P<.0001) of RT rats versus non-RT rats. TEM analysis showed decreased myelination, increased microvascular damage, and progressive axonal atrophy of the CN fibers after RT. Electrophysiologic analysis showed significant impairment of the CN conduction velocity after RT. RT also significantly increased RhoA/Rho-associated protein kinase 1 (ROCK1) mRNA and protein expression. In addition, penile tissue showed increased apoptosis and fibrosis 14 weeks after RT. CONCLUSIONS: RT-induced CN injury may contribute to RiED; this is therefore a rationale for developing novel therapeutic strategies to mitigate CN and tissue damage. Moreover, further investigation of the RhoA/ROCK pathway's role in mitigating RiED is necessary.

Hemorrhage enhances cytokine, complement component 3, and caspase-3, and regulates microRNAs associated with intestinal damage after whole-body gamma-irradiation in combined injury.

Hemorrhage following whole-body γ-irradiation in a combined injury (CI) model increases mortality compared to whole-body γ-irradiation alone (RI). The decreased survival in CI is accompanied by increased bone marrow injury, decreased hematocrit, and alterations of miRNA in the kidney. In this study, our aim was to examine cytokine homeostasis, susceptibility to systemic bacterial infection, and intestinal injury. More specifically, we evaluated the interleukin-6 (IL-6)-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), Flt-3 ligand, and corticosterone. CD2F1 male mice received 8.75 Gy 60Co gamma photons (0.6 Gy/min, bilateral) which was followed by a hemorrhage of 20% of the blood volume. In serum, RI caused an increase of IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17A, IL-18, G-CSF, CM-CSF, eotaxin, IFN-γ, MCP-1, MIP, RANTES, and TNF-α, which were all increased by hemorrhage alone, except IL-9, IL-17A, and MCP-1. Nevertheless, CI further elevated RI-induced increases of these cytokines except for G-CSF, IFN- γ and RANTES in serum. In the ileum, hemorrhage in the CI model significantly enhanced RI-induced IL-1ß, IL-3, IL-6, IL-10, IL-12p70, IL-13, IL-18, and TNF-α concentrations. In addition, Proteus mirabilis Gram(-) was found in only 1 of 6 surviving RI mice on Day 15, whereas Streptococcus sanguinis Gram(+) and Sphingomonas paucimobilis Gram(-) were detected in 2 of 3 surviving CI mice (with 3 CI mice diseased due to inflammation and infection before day 15) at the same time point. Hemorrhage in the CI model enhanced the RI-induced increases in C3 and decreases in CRP concentrations. However, hemorrhage alone did not alter the basal levels, but hemorrhage in the CI model displayed similar increases in Flt-3 ligand levels as RI did. Hemorrhage alone altered the basal levels of corticosterone early after injury, which then returned to the baseline, but in RI mice and CI mice the increased corticosterone concentration remained elevated throughout the 15 day study. CI increased 8 miRNAs and decreased 10 miRNAs in serum, and increased 16 miRNA and decreased 6 miRNAs in ileum tissue. Among the altered miRNAs, CI increased miR-34 in the serum and ileum which targeted an increased phosphorylation of ERK, p38, and increased NF-κB, thereby leading to increased iNOS expression and activation of caspase-3 in the ileum. Further, let-7g/miR-98 targeted the increased phosphorylation of STAT3 in the ileum, which is known to bind to the iNOS gene. These changes may correlate with cell death in the ileum of CI mice. The histopathology displayed blunted villi and villus edema in RI and CI mice. Based on the in silico analysis, miR-15, miR-99, and miR-100 were predicted to regulate IL-6 and TNF. These results suggest that CI-induced alterations of cytokines/chemokines, CRP, and C3 cause a homeostatic imbalance and may contribute to the pathophysiology of the gastrointestinal injury. Inhibitory intervention in these responses may prove therapeutic for CI and improve recovery of the ileal morphologic damage.

Cerebromicrovascular dysfunction predicts cognitive decline and gait abnormalities in a mouse model of whole brain irradiation-induced accelerated brain senescence.

Whole brain irradiation (WBI) is a mainstream therapy for patients with both identifiable brain metastases and prophylaxis for microscopic malignancies. However, it also promotes accelerated senescence in healthy tissues and leads to progressive cognitive dysfunction in up to 50% of tumor patients surviving long term after treatment, due to γ-irradiation-induced cerebromicrovascular injury. Moment-to-moment adjustment of cerebral blood flow (CBF) via neuronal activity-dependent cerebromicrovascular dilation (functional hyperemia) has a critical role in maintenance of healthy cognitive function. To determine whether cognitive decline induced by WBI associates with impaired cerebromicrovascular function, C56BL/6 mice (3 months) subjected to a clinically relevant protocol of fractionated WBI (5 Gy twice weekly for 4 weeks) and control mice were compared. Mice were tested for spatial memory performance (radial arm water maze), sensorimotor coordination (computerized gait analysis, CatWalk), and cerebromicrovascular function (whisker-stimulation-induced increases in CBF, measured by laser Doppler flowmetry) at 3 to 6 months post-irradiation. We found that mice with WBI exhibited impaired cerebromicrovascular function at 3 months post-irradiation, which was associated with impaired performance in the radial arm water maze. At 6 months, post-irradiation progressive impairment in gait coordination (including changes in the regularity index and phase dispersion) was also evident. Collectively, our findings provide evidence for early and persisting neurovascular impairment after a clinically relevant protocol of fractionated WBI, which predict early manifestations of cognitive impairment.

Radioprotective effect of ursolic acid in radiation-induced impairment of neurogenesis, learning and memory in adolescent BALB/c mouse.

The effect of acute irradiation with 5Gy or fractionated exposure with 0.5Gy continuously for 10days (a total dose of 5Gy) was evaluated in an immature BALB/c mouse model. Radioprotective effect of ursolic acid (at 25mg/kg/daily administered 1h after acute or each of fractionated irradiations, and continuously for 30days) was also investigated. We found that both acute and fractionated irradiation at a total dose of 5Gy did not induce any mortality within 30days after exposure to postnatal day 26 (P26) BALB/c mice, but reduced animal weigh gain in the first few weeks. At 90days after irradiation, the weight of animals with acute irradiation was still significantly lower than the control group; no significant difference though was observed for those fractionatedly exposed mice compared to the control group. Behavioral tests indicated that acute irradiation at 5Gy induced deficits in learning and memory in the contextual fear conditioning test. The memory for novel object recognition was also impaired. Similar changes were not observed in mice with fractionated irradiation. Immunohistochemical study demonstrated clearly that acute and fractionated irradiations induced impairment of neurogenesis in the subgranular zone (SGZ) of the dentate gyrus although fractionated exposure induced much lesser loss of newly generated neurons. Ursolic acid administered at 25mg/kg/daily for 30days after irradiation greatly improved acute irradiation-induced deficits in contextual learning and memory and in novel object recognition memory although it exacerbated radiation-induced reduction of neurogenesis in SGZ.

Exposure to subthreshold dose of UVR-B induces apoptosis in the lens epithelial cells and does not in the lens cortical fibre cells.

PURPOSE: The aim of this study was to investigate in which part of the lens in vivo exposure to subthreshold dose of UVR-B radiation induces apoptosis. METHODS: Twenty 6-week-old female albino Sprague-Dawley rats were exposed to subthreshold dose (1 kJ/m2 ) of UVR-B unilaterally and killed at 120 hr after exposure. Lenses were enucleated and dissected on three regions: the lens epithelium, the cortex and the nucleus. The lens nucleus then was removed. Apoptosis markers p53 and caspase 3 were used to study apoptosis in the lens regions. qRT-PCR and Western blot were utilized to analyse the lenses. RESULTS: TP53 and CASP3 mRNA expressions are increased in exposed lenses, both in the lens epithelium and in the cortex regions, in relation to non-exposed lenses. Expression of p53 protein is increased in exposed lens epithelium in relation to non-exposed lens epithelium. Caspase 3 protein is expressed in exposed lens epithelial cells, while it is not expressed in non-exposed lens epithelial cells. p53 and caspase 3 proteins are not expressed in either exposed nor non-exposed lens fibre cells. CONCLUSION: Exposure to UVR-B increases mRNA transcription of apoptosis marker p53 in vivo in both regions of the lens and of apoptosis marker caspase 3 in the lens cortex. Exposure to UVR-B increases p53 and caspase 3 proteins expression just in the lens epithelium. In vivo exposure to subthreshold dose of UVR-B induces apoptosis in the lens epithelial cells and does not in the lens fibre cells.

Ghrelin accelerates wound healing in combined radiation and wound injury in mice.

Impaired wound healing caused by radiation happens frequently in clinical practice, and the exact mechanisms remain partly unclear. Various countermeasures have been taken to tackle with this issue. Ghrelin was considered as a potent endogenous growth hormone-releasing peptide, and its role in enhancing wound repair and regeneration was firstly investigated in whole-body irradiated (γ-ray) mice in this study. Collagen deposition and neovascularization were mostly discussed. The results demonstrated that ghrelin administration promoted cutaneous wound healing in irradiated mice, followed with reduced average wound closure time, increased spleen index (SI) and improved haematopoiesis. After isolation and analysis of granulation tissues in combined radiation and wound injury (CRWI) mice treated with and without ghrelin, a phenomenon of increased DNA, hexosamine, nitrate and nitrite synthesis, elevated collagen content and enhanced neovascularization was observed after ghrelin treatment. Western blotting indicated that ghrelin also increased the expression of vascular endothelial growth factor (VEGF) and transforming growth factor-ß (TGF-ß), both responsible for wound healing. However, previous administration of growth hormone secretagogue receptor 1a (GHS-R1a) blocker blunted these therapeutic effects of ghrelin on CRWI mice. Our results identify ghrelin as a novel peptide that could be used for radiation-induced impaired wound healing.