2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature

Abstract

Purpose: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2^nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney.

Procedures: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3^rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2^nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection.

Results: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31⁺ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction.

Conclusions: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

Keywords: Consomic rat model; Delta-like ligand 4 (Dll4); Indocyanine green (ICG); NIR-1 window; NIR-2 window; Near-infrared (NIR) fluorescence imaging; Vascular permeability.

Fig. 1

SS.BN3 rats have reduced Dll4 expression and reduced susceptibility to late radiation-induced morbidity. a Consomic map illustrating the SS.BN3 rat is generated by the substitution of chromosome 3 from the Brown Norway rat into the Dahl-SS genetic background. b mRNA expression of Dll4 from CD31⁺ endothelial cells in non-irradiated SS (Dll4-high) and consomic SS.BN3 (Dll4-low) rats (n = 4 (SS) or 5 (SS.BN3) rats per group). c Survival following 13 Gy partial body irradiation (PBI) in adult SS (n = 13) and SS.BN3 rats (n = 19). P value for log-rank analysis: P = 0.0002 for SS vs SS.BN3 following 13 Gy PBI. d Change in body weight relative to pre-irradiation body weight for the rats represented in the survival curve (**P = 0.0066, ****P < 0.0001, two-way ANOVA Sidak’s multiple testing correction). Note the increased drop in body weight at 90 and 120 days in SS as compared to SS.BN3 rats, that occurs during pneumonitis and nephropathy, respectively

Fig. 2

Imaging setup and image processing technique. a NIR Fluorescence imaging setup using Indocyanine Green optical imaging for the lung and kidney 42 and 90 days after PBI. b Determination of region of interest (ROI) for the lung and kidney using principal component analysis (PCA) for 3 different camera setups. Reference lung and kidney locations have been illustrated by using a cross-sectional CT image of a representative SS rat via the SmART 225 kV ortho-voltage x-ray system (Precision X-Ray, North Branford, Connecticut) at 40 kV and 5 mA with 0.2 mm voxels. Time-dependent fluorescence intensity data at each pixel was decomposed into principal components along the time-dimension. The principal components identified to represent lungs and kidneys are illustrated for ICG fluorescence emission windows of 830 nm bandpass, 950 nm long pass, and 1100 nm long pass

Fig. 3

SS.BN3 (Dll4-low) rats are protected from radiation-induced increases in lung vascular permeability. a Kinetics of ICG uptake and clearance in the right lung at 42 days (a total of 32 SS and 35 SS.BN3 rats, i.e., SS 0 Gy (n = 17), SS 13 Gy (n = 15), SS.BN3 0 Gy (n = 13), and SS.BN3 13 Gy (n = 22)) (solid lines are the average intensity for each group and ribbons represent standard error). b ICG fluorescence kinetics of right lung at 90 days following PBI (a total of 22 SS and 29 SS.BN3 rats, i.e., SS 0 Gy (n = 9), SS 13 Gy (n = 13), SS.BN3 0 Gy (n = 12), and SS.BN3 13 Gy (n = 17)) (solid lines are the average intensity for each group and ribbons represent standard error). c The mixed-effects covariance model for 8192 and 300 (cropped) timesteps with appropriate time-varying covariates, used to analyze the average fluorescence intensity of ICG in the right lung where subject numbers serve as the repeated measure indicator and the rat strain serving as covariate. d The diagonal projection of covariance on a time axis. e Physiology-based pharmacokinetic modeling (PBPK) of NIR fluorescence-derived ICG uptake and clearance (ref. 18). The model involves two regions (vascular and tissue regions) and describes the ICG exchange and retention and is used to estimate parameters that significantly affect the ICG uptake by lung, including the permeability of the pulmonary vascular endothelium. f The PS (permeability-surface area) product in the right lung at 42 days and 90 days (representing in the a and b) post PBI (solid and dashed lines represent median and mean respectively)

Fig. 4

SS.BN3 (Dll4-low) rats have reduced radiation-induced loss of CD31⁺ endothelial cells. a Left, representative FACS contour plots showing the percentage of CD45⁻CD31⁺ lung ECs at day 70 following 13 Gy partial body irradiation in SS (Dll4-high) and SS.BN3 (Dll4-low) rats. Right, quantification of percent CD45⁻CD31⁺ cells (n = 9 rats per group). b Left, representative FACS contour plots showing the percentage of CD45⁻CD31⁺ kidney ECs at day 70 following 13 Gy partial body irradiation in SS (Dll4-high, n = 4) and SS.BN3 (Dll4-low, n = 5) rats. Right, quantification of percent CD45⁻CD31⁺ cells

Fig. 5

SS.BN3 (Dll4-low) are less sensitive to radiation-induced renal vascular damage. a ICG fluorescence kinetics in the right kidney at 90 after PBI (a total of 16 SS and 19 SS.BN3 rats, i.e., SS 0 Gy (n = 11), SS 13 Gy (n = 15), SS.BN3 0 Gy (n = 7), and SS.BN3 13 Gy (n = 12)) (**P < 0.01, two-way ANOVA Sidak’s multiple testing correction; solid lines are the average intensity for each group and ribbons represent standard error). b Blood urea nitrogen (BUN) levels at days 90 and 120 following 13 Gy PBI in SS (Dll4-high; n = 30 for 90 days and n = 8 for 120 days) and SS.BN3 (Dll4-low; n = 37 for 90 days and n = 24 for 120 days) rats. The dashed line depicts mean BUN measurement for all non-irradiated rats (n = 58). c RECA-1 staining (brown) of renal cortex region, showing the glomeruli at day 70 following 13 Gy PBI in SS and SS.BN3 rats (n = 4 for each group). One glomerulus outlined in white from each group is shown in higher magnification (40x) below. d Quantification of RECA-1 staining in each group

Fig. 6

Radiation dynamically regulates pulmonary endothelial Dll4 expression. a In Vitro transcriptional analysis of Notch target genes hes1 and hey2 following ex vivo irradiation of rat pulmonary endothelial cells with 8 Gy (n = 3/group). b Ex vivo transcriptional regulation of Dll4 in pulmonary ECs from SS (Dll4-high) and SS.BN3 (Dll4-low) rats at day 70 following 13 Gy PBI (n = 4 for SS 0 Gy, n = 5 for SS 13 Gy, n = 4 for SS.BN3 0 Gy, and n = 5 for SS.BN3 13 Gy). c Ex vivo transcriptional regulation of Dll4 and Notch target genes hes1 and hey2 in pulmonary ECs from WagRij/Cmcr at days 28, 42, and 70 following 13.5 Gy PBI (n = 6/group each time point). d FACS expression of Dll4 within pulmonary ECs from SS (Dll4-high) and SS.BN3 (Dll4-low) rats at day 70 following 13 Gy (n = 8 for SS 0 Gy, n = 9 for SS 13 Gy, n = 8 for SS.BN3 0 Gy, and n = 9 for SS.BN3 13 Gy). e FACS expression of Dll4 within pulmonary ECs from WagRij/Cmcr at days 28, 42, and 70 following 13.5 Gy (n = 6/group each time point)