Biomarkers to Predict Lethal Radiation Injury to the Rat Lung.

Currently, there are no biomarkers to predict lethal lung injury by radiation. Since it is not ethical to irradiate humans, animal models must be used to identify biomarkers. Injury to the female WAG/RijCmcr rat has been well-characterized after exposure to eight doses of whole thorax irradiation: 0-, 5-, 10-, 11-, 12-, 13-, 14- and 15-Gy. End points such as SPECT imaging of the lung using molecular probes, measurement of circulating blood cells and specific miRNA have been shown to change after radiation. Our goal was to use these changes to predict lethal lung injury in the rat model, 2 weeks post-irradiation, before any symptoms manifest and after which a countermeasure can be given to enhance survival. SPECT imaging with 99mTc-MAA identified a decrease in perfusion in the lung after irradiation. A decrease in circulating white blood cells and an increase in five specific miRNAs in whole blood were also tested. Univariate analyses were then conducted on the combined dataset. The results indicated that a combination of percent change in lymphocytes and monocytes, as well as pulmonary perfusion volume could predict survival from radiation to the lungs with 88.5% accuracy (95% confidence intervals of 77.8, 95.3) with a p-value of < 0.0001 versus no information rate. This study is one of the first to report a set of minimally invasive endpoints to predict lethal radiation injury in female rats. Lung-specific injury can be visualized by 99mTc-MAA as early as 2 weeks after radiation.

Protection From Radiation-Induced Neuroanatomic Deficits by CCL2 Deficiency Is Dependent on Sex.

PURPOSE: Cranial radiation therapy for the treatment of pediatric brain tumors results in changes to brain development that are detectable with magnetic resonance imaging. We have previously demonstrated similar structural changes in both humans and mice. The goal of the current study was to examine the role of inflammation in this response. Because neuroanatomic volume deficits in pediatric survivors are more pronounced in female patients, we also evaluated possible dependence on sex. METHODS AND MATERIALS: Other studies have shown that male mice deficient in the C-C chemokine ligand 2 gene (Ccl2; previously Mcp-1) have a muted neuroinflammatory response after irradiation. We irradiated Ccl2-/- (HOM; female = 12, male = 13), Ccl2-/+ (HET; female = 13, male = 16), and Ccl2+/+ (WT; female = 11, male = 13) mice with a whole brain dose of 7 Gy during infancy. Control mice (with approximately equal group sizes) were anesthetized but not irradiated. In vivo magnetic resonance images were acquired at 4 time points up to 3 months after irradiation, and deformation-based morphometry was used to identify volume differences. RESULTS: Irradiation of WT mice resulted in a deficit in neuroanatomic growth with limited sex dependence. HOM and HET male mice were significantly protected from this radiation-induced damage, whereas HOM and HET female mice were not. CONCLUSIONS: Interventions aimed at mitigating the effects of cranial radiation therapy in pediatric cancer survivors by modulating inflammatory response will need to consider patient sex.

An in vivo model of focal light emitting diode-induced cone photoreceptor phototoxicity in adult pigmented mice: Protection with bFGF.

PURPOSE: To develop a model of focal injury by blue light-emitting diode (LED)-induced phototoxicity (LIP) in pigmented mouse retinas and to study the effects on cone, Iba-1+ cells and retinal pigment epithelium (RPE) cell populations after administration of basic fibroblast growth factor (bFGF) and minocycline, alone or combined. METHODS: In anesthetized dark-adapted adult female pigmented C57BL/6 mice, left pupils were dilated and the eye exposed to LIP (500 lux, 45 s). The retina was monitored longitudinally in vivo with SD-OCT for 7 days (d). Ex vivo, the effects of LIP and its protection with bFGF (0.5 µg) administered alone or combined with minocycline (45 mg/kg) were studied in immunolabeled arrestin-cone outer segments (a+OS) and quantified within a predetermined fixed-size circular area (PCA) centered on the lesion in flattened retinas at 1, 3, 5 or 7d. Moreover, Iba-1+ cells and RPE cell morphology were analysed with Iba-1 and ZO-1 antibodies, respectively. RESULTS: LIP caused a focal lesion within the superior-temporal retina with retinal thinning, particularly the outer retinal layers (116.5 ± 2.9 µm to 36.8 ± 6.3 µm at 7d), and with progressive diminution of a+OS within the PCA reaching minimum values at 7d (6218 ± 342 to 3966 ± 311). Administration of bFGF alone (4519 ± 320) or in combination with minocycline (4882 ± 446) had a significant effect on a+OS survival at 7d and Iba-1+ cell activation was attenuated in the groups treated with minocycline. In parallel, the RPE cell integrity was progressively altered after LIP and administration of neuroprotective components had no restorative effect at 7d. CONCLUSIONS: LIP resulted in progressive outer retinal damage affecting the OS cone population and RPE. Administration of bFGF increased a+OS survival but did not prevent RPE deterioration.

Clinicopathologic and Transcriptomic Analysis of Radiation-Induced Lung Injury in Nonhuman Primates.

PURPOSE: Radiation-induced lung injury (RILI) is a progressive condition with an early phase (radiation pneumonitis) and a late phase (lung fibrosis). RILI may occur after partial-body ionizing radiation exposures or internal radioisotope exposure, with wide individual variability in timing and extent of lung injury. This study aimed to provide new insights into the pathogenesis and progression of RILI in the nonhuman primate (NHP) rhesus macaque model. METHODS AND MATERIALS: We used an integrative approach to understand RILI and its evolution at clinical and molecular levels in 17 NHPs exposed to 10 Gy of whole-thorax irradiation in comparison with 3 sham-irradiated control NHPs. Clinically, we monitored respiratory rates, computed tomography (CT) scans, plasma cytokine levels, and bronchoalveolar lavage (BAL) over 8 months and lung samples collected at necropsy for molecular and histopathologic analyses using RNA sequencing and immunohistochemistry. RESULTS: Elevated respiratory rates, greater CT density, and more severe pneumonitis with increased macrophage content were associated with early mortality. Radiation-induced lung fibrosis included polarization of macrophages toward the M2-like phenotype, TGF-ß signaling, expression of CDKN1A/p21 in epithelial cells, and expression of α-SMA in lung stroma. RNA sequencing analysis of lung tissue revealed SERPINA3, ATP12A, GJB2, CLDN10, TOX3, and LPA as top dysregulated transcripts in irradiated animals. In addition to transcriptomic data, we observed increased protein expression of SERPINA3, TGF-ß1, CCL2, and CCL11 in BAL and plasma samples. CONCLUSIONS: Our combined clinical, imaging, histologic, and transcriptomic analysis provides new insights into the early and late phases of RILI and highlights possible biomarkers and potential therapeutic targets of RILI. Activation of TGF-ß and macrophage polarization appear to be key mechanisms involved in RILI.

A rapid dynamic in vivo near-infrared fluorescence imaging assay to track lung vascular permeability after acute radiation injury.

To develop a dynamic in vivo near-infrared (NIR) fluorescence imaging assay to quantify sequential changes in lung vascular permeability-surface area product (PS) in rodents. Dynamic NIR imaging methods for determining lung vascular permeability-surface area product were developed and tested on non-irradiated and 13 Gy irradiated rats with/without treatment with lisinopril, a radiation mitigator. A physiologically-based pharmacokinetic (PBPK) model of indocyanine green (ICG) pulmonary disposition was applied to in vivo imaging data and PS was estimated. In vivo results were validated by five accepted assays: ex vivo perfused lung imaging, endothelial filtration coefficient (Kf) measurement, pulmonary vascular resistance measurement, Evan's blue dye uptake, and histopathology. A PBPK model-derived measure of lung vascular permeability-surface area product increased from 2.60 ± 0.40 [CL: 2.42-2.78] mL/min in the non-irradiated group to 6.94 ± 8.25 [CL: 3.56-10.31] mL/min in 13 Gy group after 42 days. Lisinopril treatment lowered PS in the 13 Gy group to 4.76 ± 6.17 [CL: 2.12-7.40] mL/min. A much higher up to 5× change in PS values was observed in rats exhibiting severe radiation injury. Ex vivo Kf (mL/min/cm H2O/g dry lung weight), a measure of pulmonary vascular permeability, showed similar trends in lungs of irradiated rats (0.164 ± 0.081 [CL: 0.11-0.22]) as compared to non-irradiated controls (0.022 ± 0.003 [CL: 0.019-0.025]), with reduction to 0.070 ± 0.035 [CL: 0.045-0.096] for irradiated rats treated with lisinopril. Similar trends were observed for ex vivo pulmonary vascular resistance, Evan's blue uptake, and histopathology. Our results suggest that whole body dynamic NIR fluorescence imaging can replace current assays, which are all terminal. The imaging accurately tracks changes in PS and changes in lung interstitial transport in vivo in response to radiation injury.

Ionising radiation causes vision impairment in neonatal B6C3F1 mice.

Ionising radiation interacts with lenses and retinae differently. In human lenses, posterior subcapsular cataracts are the predominant observation, whereas retinae of adults are comparably resistant to even relatively high doses. In this study, we demonstrate the effects of 2 Gy of low linear energy transfer ionising radiation on eyes of B6C3F1 mice aged postnatal day 2. Optical coherence tomography and Scheimpflug imaging were utilised for the first time to monitor murine lenses and retinae in vivo. The visual acuity of the mice was determined and histological analysis was conducted. Our results demonstrated that visual acuity was reduced by as much as 50 % approximately 9 months after irradiation in irradiated mice. Vision impairment was caused by retinal atrophy and inner cortical cataracts. These results help to further our understanding of the risk of ionising radiation for human foeti (∼ 8 mo), which follow the same eye development stages as neonatal mice.

A non-invasive ultrasound imaging method to measure acute radiation-induced bladder wall thickening in rats.

BACKGROUND: Methods for the non-invasive quantification of changes in bladder wall thickness as potential predictors of radiation cystitis in pre-clinical research would be desirable. The use of ultrasound for this aim seems promising, but is still relatively unexplored. A method using ultrasound for bladder wall thickness quantification in rats was developed and applied to measure early radiation-induced bladder wall thickness changes. METHODS: Two groups (n = 9 each) of female Fischer rats were treated with a single radiation dose of 25-30 and 35-40 Gy respectively, using an image-guided micro-irradiator; six untreated rats were monitored as a control group. Empty, half-filled and fully-filled bladder volumes were determined for four non-irradiated rats by measuring axes from ultrasound 3D-images and applying the ellipsoid formula. Mean bladder wall thickness was estimated for both ventral and dorsal bladder sides through the measurement of the bladder wall area along a segment of 4 mm in the central sagittal scan, in order to minimize operator-dependence on the measurement position. Ultrasound acquisitions of all fully-filled rat bladders were also acquired immediately before, and 4 and 28 days after irradiation. Mean bladder wall thickness normalized to the baseline value and corrected for filling were then used to evaluate acute bladder wall thickening and to quantify the dose-effect. RESULTS: The relationship between mean bladder wall thickness and volume in unirradiated rats showed that for a bladder volume > 1.5 mL the bladder wall thickness is almost constant and equal to 0.30 mm with variations within ± 15%. The average ratios between post and pre irradiation showed a dose-effect relationship. Bladder wall thickening was observed for the 25-30 Gy and 35-40 Gy groups in 2/9 (22%) and 5/9 (56%) cases at day 4 and in 4/9 (44%) and 8/9 (89%) cases at day 28, respectively. The two groups showed significantly different bladder wall thickness both relative to the control group (p < 0.0001) and between them (p = 0.022). The bladder wall thickness increment was on average 1.32 ± 0.41, and was 1.30 ± 0.21 after 25-30 Gy and 1.47 ± 0.29 and 1.90 ± 0.83 after 35-40 Gy at days 4 and 28 respectively. CONCLUSIONS: The feasibility of using ultrasound on a preclinical rat model to detect bladder wall thickness changes after bladder irradiation was demonstrated, and a clear dose-effect relationship was quantified. Although preliminary, these results are promising in addressing the potential role of this non-invasive approach in quantifying radiation cystitis.

Non-Human Primates Receiving High-Dose Total-Body Irradiation are at Risk of Developing Cerebrovascular Injury Years Postirradiation.

Nuclear accidents and acts of terrorism have the potential to expose thousands of people to high-dose total-body iradiation (TBI). Those who survive the acute radiation syndrome are at risk of developing chronic, degenerative radiation-induced injuries [delayed effects of acute radiation (DEARE)] that may negatively affect quality of life. A growing body of literature suggests that the brain may be vulnerable to radiation injury at survivable doses, yet the long-term consequences of high-dose TBI on the adult brain are unclear. Herein we report the occurrence of lesions consistent with cerebrovascular injury, detected by susceptibility-weighted magnetic resonance imaging (MRI), in a cohort of non-human primate [(NHP); rhesus macaque, Macaca mulatta] long-term survivors of high-dose TBI (1.1-8.5 Gy). Animals were monitored longitudinally with brain MRI (approximately once every three years). Susceptibility-weighted images (SWI) were reviewed for hypointensities (cerebral microbleeds and/or focal necrosis). SWI hypointensities were noted in 13% of irradiated NHP; lesions were not observed in control animals. A prior history of exposure was correlated with an increased risk of developing a lesion detectable by MRI (P = 0.003). Twelve of 16 animals had at least one brain lesion present at the time of the first MRI evaluation; a subset of animals (n = 7) developed new lesions during the surveillance period (3.7-11.3 years postirradiation). Lesions occurred with a predilection for white matter and the gray-white matter junction. The majority of animals with lesions had one to three SWI hypointensities, but some animals had multifocal disease (n = 2). Histopathologic evaluation of deceased animals within the cohort (n = 3) revealed malformation of the cerebral vasculature and remodeling of the blood vessel walls. There was no association between comorbid diabetes mellitus or hypertension with SWI lesion status. These data suggest that long-term TBI survivors may be at risk of developing cerebrovascular injury years after irradiation.

Ameliorating effect of CpG-ODN (oligodeoxynucleotide) against radiation-induced lung injury in mice.

While radiation-induced lung injury (RILI) is known to be progressed by Th2 skewed, pro-inflammatory immune response, there have been few therapeutic attempts through Th1 immune modulation. We investigated whether the immunostimulant CpG-oligodeoxynucleotide (CpG-ODN) would be effective against RILI by way of measuring reactive oxygen species (ROS) and nitric oxides (NO), histopathology, micro-three-dimensional computer tomography (CT), and cytokine profiling. We found that KSK CpG-ODN (K-CpG) significantly reduced histopathological fibrosis when compared to the positive control (PC) group (p < 0.01). The levels of ROS production in serum and splenocyte of PC group were significantly higher than that of K-CpG group (p < 0.01). The production of nitric oxide (NO) in CpG-ODNs group was higher than that of PC group. Last, cytokine profiling illustrated that the protein concentrations of Th1-type cytokines such as IL-12 and TNF-α as well as Th2-type cytokine IL-5 in K-CpG group inclined to be significantly (p < 0.001 or p < 0.01) higher than those of in PC group. Collectively, our study clearly indicates that K-CpG is effective against RILI in mice by modulating the innate immune response. To our knowledge, this is the first note on anti-RILI effect of human type, K-CpG, clinically implying the potential of immunotherapy for RILI control.

Brain irradiation leads to persistent neuroinflammation and long-term neurocognitive dysfunction in a region-specific manner.

Long-term cognitive deficits are observed after treatment of brain tumors or metastases by radiotherapy. Treatment optimization thus requires a better understanding of the effects of radiotherapy on specific brain regions, according to their sensitivity and interconnectivity. In the present study, behavioral tests supported by immunohistology and magnetic resonance imaging provided a consistent picture of the persistent neurocognitive decline and neuroinflammation after the onset of irradiation-induced necrosis in the right primary somatosensory cortex of Fischer rats. Necrosis surrounded by neovascularization was first detected 54 days after irradiation and then spread to 110 days in the primary motor cortex, primary somatosensory region, striatum and right ventricle, resulting in fiber bundle disruption and demyelination in the corpus callosum of the right hemisphere. These structural damages translated into selective behavioral changes including spatial memory loss, disinhibition of anxiety-like behaviors, hyperactivity and pain hypersensitivity, but no significant alteration in motor coordination and grip strength abilities. Concomitantly, activated microglia and reactive astrocytes, accompanied by infiltration of leukocytes (CD45+) and T-cells (CD3+) cooperated to shape the neuroinflammation response. Overall, our study suggests that the slow and gradual onset of cellular damage would allow adaptation in brain regions that are susceptible to neuronal plasticity; while other cerebral structures that do not have this capacity would be more affected. The planning of radiotherapy, adjusted to the sensitivity and adaptability of brain structures, could therefore preserve certain neurocognitive functions; while higher doses of radiation could be delivered to brain areas that can better adapt to this treatment. In addition, strategies to block early post-radiation events need to be explored to prevent the development of long-term cognitive dysfunction.

Hippocampal changes in inflammasomes, apoptosis, and MEMRI after radiation-induced brain injury in juvenile rats.

PURPOSE: The aim of this study was to characterize changes in hippocampal inflammasomes, pyroptosis and apoptosis in juvenile rats after brain irradiation and to assess whether manganese-enhanced magnetic resonance imaging (MEMRI) reflected those changes. MATERIALS AND METHODS: Four-week-old male Sprague-Dawley rats received a whole-brain radiation dose of 15 Gy or 25 Gy. Hippocampal inflammasomes and apoptosis were measured using Western blot analysis at 4 days and 8 weeks after irradiation. MEMRI and magnetic resonance spectroscopy (MRS) were performed at the same time points. RESULTS: Neither the 15 Gy nor 25 Gy group showed changes in the expression of inflammasome proteins absent in melanoma 2 (AIM2), gasdermin-D (GSDMD), nucleotide oligomerization domain-like receptor protein 1 (NLRP1) and NLRP3 at 4 days or 8 weeks after radiation injury (P > 0.05). Furthermore, the expression levels of the inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 were not significantly different among the groups (P > 0.05). The expression levels of cleaved caspase-1 and -3, indicators of apoptosis, were higher in the irradiation groups than in the control group at 4 days post irradiation, especially for caspase-3 (P < 0.05), but this increase was slightly attenuated at 8 weeks after radiation injury. Four days post irradiation, the MEMRI signal intensity (SI) in the irradiation groups, especially the 25 Gy group, was significantly lower than that in the control group (P < 0.05). Eight weeks after radiation injury, the SI of the 15 Gy group and the 25 Gy group recovered by different degrees, but the SI of the 25 Gy group was still significantly lower than that of the control group (P < 0.05). On day 4 post irradiation, the metabolic ratio of N-acetylaspartate (NAA) to creatine (Cr) in the 15 Gy group and 25 Gy group was significantly lower than that in the control group (P < 0.05). The NAA/Cr ratio in the 15 Gy group recovered to control levels at 8 weeks (P > 0.05), but the NAA/Cr ratio in the 25 Gy group remained significantly lower than that in the control group (P < 0.05). CONCLUSION: Radiation-induced brain injury is dose-dependently associated with apoptosis but not inflammasomes or pyroptosis, and the change in apoptosis can be detected by MEMRI.

Effects of pentoxifylline and tocopherol on a rat-irradiated jaw model using micro-CT cortical bone analysis.

PURPOSE: A combination of pentoxifylline (PTX) and tocopherol (TP) is believed to reduce chronic fibrosis and induce bone healing in osteoradionecrosis (ORN) of the mandible, but evidence of its therapeutic effectiveness for cortical bone is lacking. This study was designed to determine the effect of combined PTX and TP (PTX + TP) on mandibular cortical bone remodeling in a rat model of ORN, using micro-CT and histological analysis. METHODS: Forty-eight 8-week-old male Sprague-Dawley rats were randomly divided into irradiated (n = 40) and non-irradiated (n = 8) groups. Animals in the irradiated group were divided into four sub-groups, including PTX, TP, PTX + TP, and normal saline. Three weeks after irradiation, mandibular posterior tooth extraction was performed, and animals were sacrificed 7 weeks after irradiation. The mandibles were analyzed using micro-CT and histological evaluation. RESULTS: The alveolar bone height, cortical bone thickness, cortical bone volume, and total cortical bone surface of the PTX + TP group were significantly greater than those of other irradiated groups (p < 0.05). In 3D reconstructed images, the residual volumes of cortical and cancellous bone were inadequate in the irradiated groups. CONCLUSION: We found that a combination of PTX and TP improved quality and quantity of cortical bone in irradiated rat mandibles, thus providing supporting evidence of its utility as a treatment and prophylactic agent in ORN. We observed inadequate volumes of cortical and cancellous bone in ORN mandibles, suggesting that cortical bone could play an important role in further ORN studies.

Bone Regeneration Effect of Hyperbaric Oxygen Therapy Duration on Calvarial Defects in Irradiated Rats.

OBJECTIVE: In this study, we evaluated changes in bone remodeling in an irradiated rat calvarial defect model according to duration of hyperbaric oxygen therapy. MATERIALS AND METHODS: The 28 rats were divided into four groups. Radiation of 12 Gy was applied to the skull, and 5-mm critical size defects were formed on both sides of the skull. Bone grafts were applied to one side of formed defects. From the day after surgery, HBO was applied for 0, 1, and 3 weeks. At 6 weeks after bone graft, experimental sites were removed and analyzed for radiography, histology, and histomorphometry. RESULTS: Micro-CT analysis showed a significant increase in new bone volume in the HBO-3 group, with or without bone graft. When bone grafting was performed, BV, BS, and BS/TV all significantly increased. Histomorphometric analysis showed significant increases in %NBA and %BVN in the HBO-1 and HBO-3 groups, regardless of bone graft. CONCLUSION: Hyperbaric oxygen therapy was effective for bone regeneration with only 1 week of treatment.

Optical coherence tomography to detect acute esophageal radiation-induced damage in mice: A validation study.

Radiation therapy for patients with non-small-cell lung cancer is hampered by acute radiation-induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 µm), is able to visualize and monitor acute radiation-induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard. Irradiated mice receive a single dose of 40 Gy at proximal and distal spots of the esophagus of 10.0 mm in diameter. We scan mice using OCT at two, three, and seven days post-irradiation. In OCT analysis, we define ARIED as a presence of distorted esophageal layering, change in backscattering signal properties, or change in the esophageal wall thickness. The average esophageal wall thickness is 0.53 mm larger on OCT when ARIED is present based on histopathology. The overall sensitivity and specificity of OCT to detect ARIED compared to histopathology are 94% and 47%, respectively. However, the overall sensitivity of OCT to assess ARIED is 100% seven days post-irradiation. We validate the capability of OCT to detect ARIED induced by high doses in mice. Nevertheless, clinical studies are required to assess the potential role of OCT to visualize ARIED in humans.

Optical Coherence Tomography Angiography in Mice: Quantitative Analysis After Experimental Models of Retinal Damage.

Purpose: We implemented optical coherence tomography angiography (OCT-A) in mice to: (1) develop quantitative parameters from OCT-A images, (2) measure the reproducibility of the parameters, and (3) determine the impact of experimental models of inner and outer retinal damage on OCT-A findings. Methods: OCT-A images were acquired with a customized system (Spectralis Multiline OCT2). To assess reproducibility, imaging was performed five times over 1 month. Inner retinal damage was induced with optic nerve transection, crush, or intravitreal N-methyl-d-aspartic acid injection in transgenic mice with fluorescently labeled retinal ganglion cells (RGCs). Light-induced retinal damage was induced in albino mice. Mice were imaged at baseline and serially post injury. Perfusion density, vessel length, and branch points were computed from OCT-A images of the superficial, intermediate, and deep vascular plexuses. Results: The range of relative differences measured between sessions across the vascular plexuses were: perfusion density (2.8%-7.0%), vessel length (1.9%-4.1%), and branch points (1.9%-5.0%). In mice with progressive RGC loss, imaged serially and culminating in around 70% loss in the fluorescence signal and 18% loss in inner retinal thickness, there were no measurable changes in any OCT-A parameter up to 4 months post injury that exceeded measurement variability. However, light-induced retinal damage elicited a progressive loss of the deep vascular plexus signal, starting as early as 3 days post injury. Conclusions: Vessel length and branch points were generally the most reproducible among the parameters. Injury causing RGC loss in mice did not elicit an early change in the OCT-A signal.

Outer Retinal Oxidative Stress Measured In Vivo Using QUEnch-assiSTed (QUEST) OCT.

Purpose: To test the hypothesis that oxidative stress in the outer retina (OR = distance from external limiting membrane to the retinal pigment epithelium-choroid boundary) can be detected by using antioxidants (AOs) to correct an impaired light-evoked response as measured by optical coherence tomography (OCT). Methods: C57BL/6J mice were maintained in the dark for ∼20 hours and studied by OCT before and after 1 hour of light exposure. OR thickness in dark or light was measured, and the light-dark difference (i.e., the photoresponse) was calculated. Subgroups of mice were given either saline or d-cis-diltiazem (an inducer of transient and nondamaging OR oxidative stress) ± methylene blue (24 hours before examination) and α-lipoic acid (1 hour before examination); one group was kept only in the dark and given only AOs. Results: In uninjected or saline-injected control mice, the OR showed a similar and reproducible light-induced expansion; dark-adapted mice given AOs did not increase dark-adapted OR thickness. The d-cis-diltiazem-treated mice had no photoresponse (P > 0.05). The d-cis-diltiazem-treated mice given AOs corrected (P < 0.05) the suppressed OR photoresponse, indicating the presence of oxidative stress. Conclusions: QUEnch-assiSTed (QUEST) OCT reproduced results from previous gold standard assays, showing that oxidative stress impairs the OR photoresponse and that d-cis-diltiazem produces OR oxidative stress. We envision future applications of QUEST OCT in a range of oxidative stress-based retinopathies.

Monomethyl Fumarate Protects the Retina From Light-Induced Retinopathy.

Purpose: We determine if monomethyl fumarate (MMF) can protect the retina in mice subjected to light-induced retinopathy (LIR). Methods: Albino BALB/c mice were intraperitoneally injected with 50 to 100 mg/kg MMF before or after exposure to bright white light (10,000 lux) for 1 hour. Seven days after light exposure, retinal structure and function were evaluated by optical coherence tomography (OCT) and electroretinography (ERG), respectively. Retinal histology also was performed to evaluate photoreceptor loss. Expression levels of Hcar2 and markers of microglia activation were measured by quantitative PCR (qPCR) in the neural retina with and without microglia depletion. At 24 hours after light exposure, retinal sections and whole mount retinas were stained with Iba1 to evaluate microglia status. The effect of MMF on the nuclear factor kB subunit 1 (NF-kB) and Nrf2 pathways was measured by qPCR and Western blot. Results: MMF administered before light exposure mediated dose-dependent neuroprotection in a mouse model of LIR. A single dose of 100 mg/kg MMF fully protected retinal structure and function without side effects. Expression of the Hcar2 receptor and the microglia marker Cd14 were upregulated by LIR, but suppressed by MMF. Depleting microglia reduced Hcar2 expression and its upregulation by LIR. Microglial activation, upregulation of proinflammatory genes (Nlrp3, Caspase1, Il-1ß, Tnf-α), and upregulation of antioxidative stress genes (Hmox1) associated with LIR were mitigated by MMF treatment. Conclusions: MMF can completely protect the retina from LIR in BALB/c mice. Expression of Hcar2, the receptor of MMF, is microglia-dependent in the neural retina. MMF-mediated neuroprotection was associated with attenuation of microglia activation, inflammation and oxidative stress in the retina.

Influence of Dose Uniformity when Replicating a Gamma Knife Mouse Model of Radiation Necrosis with a Preclinical Irradiator.

A common mouse model used for studying radiation necrosis is generated with the gamma knife, which has a non-uniform dose distribution. The goal of this study was to determine whether the lesion growth observed in this mouse model is a function of non-uniform dose distribution and/or lesion progression. Here, a model similar to the gamma knife mouse model was generated; using a preclinical irradiator, mice received single-fraction doses from 50 to 100 Gy to a sub-hemispheric portion of the brain. The development of necrosis was tracked for up to 26 weeks with a 7T Bruker magnetic resonance imaging (MRI) scanner using T2 and post-contrast T1 imaging. MRI findings were validated with histology, specifically H&E staining. Single small beam 50 Gy irradiations failed to produce necrosis in a 26-week span, while doses from 60 to 100 Gy produced necrosis in a timeframe ranging from 16 weeks to 2 weeks, respectively. Postmortem histology confirmed pathological development in regions corresponding with those that showed abnormal signal on MRI. The growth of the necrotic lesion observed in this gamma knife model was due in part to a non-uniform dose distribution rather than to the increased severity of the lesion. Interpretation of results from the gamma knife model must take into consideration the potential effect of nonuniform dose distribution, particularly with regards to the timing of interventions. There are time points in this model at which pre-onset, onset and post-onset of radiation necrosis are all represented in the irradiated field.

A Comparative Dose-response Relationship Between Sexes for Mortality and Morbidity of Radiation-induced Lung Injury in the Rhesus Macaque.

Radiation-induced lung injury is a characteristic, dose- and time-dependent sequela of potentially lethal, delayed effects of acute radiation exposure. Understanding of these delayed effects to include development of medical countermeasures requires well-characterized and validated animal models that mimic the human response to acute radiation and adhere to the criteria of the US Food and Drug Administration Animal Rule. The objective herein was to establish a nonhuman primate model of whole-thorax lung irradiation in female rhesus macaques. Definition of the dose-response relationship to include key signs of morbidity and mortality in the female macaque served to independently validate the recent model performed with male macaques and importantly, to establish the lack of sex and institutional bias across the dose-response relationship for radiation-induced lung injury. The study design was similar to that described previously, with the exception that female rhesus macaques were utilized. In brief, a computed tomography scan was conducted prior to irradiation and used for treatment planning. Animals in 5 cohorts (n = 8 per cohort) were exposed to a single 6-MV photon exposure focused on the lung as determined by the computed tomography scan and treatment planning at a dose of 9.5, 10, 10.5, 11, or 11.5 Gy. Subject-based supportive care, including administration of dexamethasone, was based on trigger-to-treat criteria. Clearly defined euthanasia criteria were used to determine a moribund condition over the 180-day study duration post-whole-thorax lung irradiation. Percent mortality per radiation dose was 12.5% at 9.5 Gy, 25% at 10 Gy, 62.5% at 10.5 Gy, 87.5% at 11 Gy, and 100% at 11.5 Gy. The resulting probit plot for the whole-thorax lung irradiation model estimated an LD50/180 of 10.28 Gy, which was not significantly different from the published estimate of 10.27 Gy for the male rhesus. The key parameters of morbidity and mortality support the conclusion that there is an absence of a sex influence on the radiation dose-response relationship for whole-thorax lung irradiation in the rhesus macaque. This work also provides a significant interlaboratory validation of the previously published model.

The Time Course of Radiation-induced Lung Injury in a Nonhuman Primate Model of Partial-body Irradiation With Minimal Bone Marrow Sparing: Clinical and Radiographic Evidence and the Effect of Neupogen Administration.

The primary objectives of two companion manuscripts were to assess the natural history of delayed radiation-induced lung injury in a nonhuman primate model of acute high-dose, partial-body irradiation with 5% bone marrow sparing, to include the clinical, radiographic, and histopathological evidence and the effect of Neupogen administration on the morbidity and mortality. Nonhuman primates were exposed to 10.0 or 11.0 Gy with 6 MV linac-derived photons at approximately 0.80 Gy min. All nonhuman primates received subject-based, medical management. Subsets of nonhuman primates were administered Neupogen (10 µg kg) starting on day 1, day 3, or day 5 until recovery (absolute neutrophil count ≥ 1,000 cells µL for three consecutive days). Mortality due to multiple organ injury at 180 d study duration: Mortality at 180 d post either 10.0 Gy or 11.0 Gy was the consequence of concurrent injury due to the acute radiation syndrome (gastrointestinal and hematological) and delayed radiation-induced lung injury. The 180-d all-cause mortality observed in the control cohorts at 10.0 Gy (53%) or 11.0 Gy (86%) did not vary from cohorts that received Neupogen at any administration schedule. Mortality ranged from 43-50% (10 Gy) to 75-100% (11.0 Gy) in the Neupogen-treated cohorts. The study, however, was not powered to detect statistical significant differences between mortality in the control and Neupogen-treated cohorts. Clinical and radiographic evidence of radiation-induced lung injury: The mean nonsedated respiratory rate in the control cohorts exposed to 10 or 11 Gy increased from a baseline value of 37 breaths min to >60 breaths min within 103 d and 94 d postexposure, and the incidence of nonsedated respiratory rate > 80 breaths min was 50% and 70%, respectively. The mean duration of latency to development of clinical pneumonitis and/or fibrosis (nonsedated respiratory rate > 80 breaths min) was not significantly different between the 10.0-Gy or 11.0 Gy-cohorts (range 100-107 d). Neupogen (granulocyte colony-stimulating factor) administration had no apparent effect of the latency, incidence, or severity of nonsedated respiratory rate within either radiation dose or administration schedule. Computed tomography scans were obtained and images were analyzed for evidence of lung injury, e.g., pneumonitis and/or fibrosis, pleural and pericardial effusion. A quantitative, semiautomated method was developed based on differences in radiodensity (Hounsfield units) and lung morphology to extract the volume of pneumonitis/fibrosis and pleural effusion as indexed against total lung at each time point obtained. At both irradiation doses, 100% of the nonhuman primates surviving acute radiation syndrome manifested radiographic evidence of radiation-induced lung injury as pneumonitis and/or fibrosis. There was no apparent effect of Neupogen administration on the latency, incidence, severity, or progression of pneumonitis/fibrosis:total lung volume or pleural effusion:total lung volume at either exposure. A comparative review of the data illustrated the concomitant time course of increased mortality, nonsedated respiratory rate, and pneumonitis/fibrosis:total lung volume and pleural effusion:total lung volume consequent to 10.0-Gy or 11.0-Gy partial-body irradiation with 5% bone marrow sparing. All key parameters proceeded from a latent period of approximately 60 d followed by an increase in all three indices of clinical and radiographic evidence of radiation-induced lung injury within the next 60 d to 120 d postexposure. The subsequent time course and longitudinal analysis was influenced by the persistent progression of radiation-induced lung injury, administration of dexamethasone, and loss of nonhuman primates due to lethality. Companion paper: Lung and Heart Injury in a Nonhuman Primate Model of Partial-body Irradiation With Minimal Bone Marrow Sparing: Histopathological Evidence of Lung and Heart Injury (Parker et al. 2019): Note that the computed tomography-based radiodensity data do not permit differentiation of pneumonitis and fibrosis. The companion paper employed Masson's trichrome, collagen 1, and selected staining to identify the key time and incidence parameters relative to excessive collagen deposition indicative of fibrosis and associated histopathology in the lung. This histological database provided valuable longitudinal analysis in support of the clinical and radiographic evidence associated with the time course of radiation-induced lung injury.