ADC of Rat Lungs

The secondary in vivo use of the Sectoral pulse sequence was to validate 129Xe diffusion- weighting with histology to explore whether the sequence can be accepted as a reliable technique to measure lung tissue density. The work presented here shows for the first

time 129Xe ADC maps of healthy and irradiated rat lungs obtained at low field. Anatomical projection images showing ventilation (Figure 3-13) or ADC (Figure 3-14) maps, in general, were not very demonstrative of RILI in this pilot study. The present images were generally very similar between the two cohorts although some minor ventilation non-uniformities can be seen in the irradiated rat lung ADC map (Figure 3-14, right).

The data presented here provide the first opportunity to directly compare Sectoral 129Xe global ADC with histological measurements in the same lungs. The measured ADC values in the lungs (Table 3-7) overall are significantly reduced compared to the self- diffusion coefficient (0.061 cm2/s) and phantom ADC (Table 3-3) values. This is expected because the diffusion of the 129Xe gas atoms is restricted by the walls of the distal air spaces (i.e. alveoli). Therefore, the ADC measured with 129Xe is a quantitative measure of microstructure, and is potentially useful for detecting changes associated with alveolar wall thickening during inflammation, post-irradiation. It was previously hypothesized that 2-weeks post-irradiation is the initial stage of lung damage where 129Xe diffusion MRI techniques might be most valuable (32). Both the healthy and the irradiated rat cohorts appeared to have heterogeneous distributions of ADC values, with a slightly more heterogeneous distribution in the irradiated rat (Figure 3-14). A decrease in ADC values in the irradiated rats (Figure 3-14, right) was expected based on alveolar wall thickening confirmed by histology. However, the heterogeneous distribution of ADC in the healthy rat cohorts (Figure 3-14, left) is surprising because much greater homogeneity was expected with the larger alveolar dimension in the normal healthy lung, and therefore subjecting 129Xe gas to less restriction to diffusion. Furthermore, some rats in the healthy cohort demonstrated reduced ADC and correspondingly showed inflammation by histology. The rats were housed in open-barred cages, exposing them to potential cross-contamination between rats with infections and accidental exposure to infectious material brought in from exposure to personnel. The use of enclosed cage systems and independent high-efficiency particulate air (HEPA) filtration to minimize infection will be an easy implementation for future cohort studies.

The mean ADC values for the healthy and irradiated rats were initially compared but showed no significant difference (p > 0.05) between the two cohorts. This may be due to the heterogeneous contributions of radiation damage to the mean ADC and/or the possibility that some irradiated rats did not demonstrate RILI (discussed further below). The calculations were then repeated by extracting the FWHM ADC, which was calculated from the Gaussian distribution of each lung ADC histogram (as described in

section 2.12). Figure 3-15 shows good distinction between the healthy and irradiated rats for the global FWHM ADC Sectoral results. A clear separation with a strong correlation (p < 0.05) between healthy rats and for irradiated rats was demonstrated using the FWHM ADC of 129Xe with Sectoral. FWHM ADC increases may be attributable to heterogeneous distribution of air space sizes following irradiation, presumably by RILI. Therefore, ADC was directly compared to mean linear intercept, Lm, measured by

histology.

Table 3-7 and Figure 3-16 summarize the correlation between 129Xe diffusion measurements (i.e. mean ADC) and Lm following removal of one outlier (as described

below). The mean ADC values from Sectoral ADC maps did show a strong correlation when compared with Lm, as measured by lung histology. The Lm is the mean distance

from tissue interfaces, between the air spaces, seen on histological slide samples obtained from fixed lungs. The reason why some (3) irradiated rats did not show evidence of RILI using the mean ADC is unclear, but possibly due to the difference in immunological response of different animals or the difference in the time course of RILI for those specific animals.

One healthy outlier was excluded in the statistical analysis because the low mean ADC value did changed the distribution skewness to just above significance (p > 0.05). The rat with the low mean ADC of the healthy outlier was housed in an open-barred cage, exposing it to accidental infection from cross-contamination between rats, which lead to inflammation. To address this concern in future in vivo studies, HEPA filters will be used to prevent infection. Sometimes the affected areas of the diseased lung may be minor, heterogeneous, or spread out. This introduces a challenge for quantification strategies of disease severity such as Lmbecause it tends to underestimate the influence of variable air

space size and outliers (67). Therefore, future work should investigate other histological analysis that may better assess the ability to detect very early stages of airway constriction such as tissue thickness and percent area.

The ability of Sectoral to detect lung injury supports the hypothesis that the Sectoral

diffusion-weighted sequence, with 129Xe, possesses the ability and potential to assess RILI and correlate it with histology. Despite the limitation of a relatively low number of rats used in this study, even with the current techniques (i.e. low polarization and high gradient-associated noise), both a strong correlation is seen between Lm and ADC,

allowing separation between healthy and irradiated cohorts using FWHM ADC. A limitation of Sectoral diffusion-weighting is that ADC maps are only acquired from measured areas in ventilated regions of the lung. In this study, all the lungs showed good whole lung ventilation, indicating that imaging was not constrained by unventilated areas. However, extending the Sectoral diffusion-weighted sequence to lung cancer or human patients with COPD, where regions of low ventilation are commonly seen due to airway obstruction, would be a limitation. Future extensions of this type of study with Sectoral

will include time courses following the progression of ADC in this and other disease models (i.e. emphysema). A larger number of rats could potentially permit the detection of a separation between healthy and irradiated cohorts using mean ADC instead of FWHM ADC.

Even though Figure 3-16 shows good correlation between morphometric and ADC measurements, the ADC values in healthy lungs can exhibit broad variability. This is because the ADC not only depends on the lung microstructure, but also on pulse sequence parameters (i.e. diffusion time, b-value). Since Sectoral is a new pulse sequence and very few in vivo129Xe ADC studies have been performed to study RILI, the choice of optimal parameters is limited as there is little guidance. Therefore, the sensitivity of

Sectoral to detect structural changes of RILI with 129Xe may have been limited due to the choice of imaging parameters. The optimization of Sectoral129Xe imaging for measuring changes associated with RILI will be an area for future research.