Contrast-enhanced ultrasound with sub-micron sized contrast agents detects insulitis in mouse models of type1 diabetes

In type1 diabetes (T1D) autoreactive T-cells infiltrate the islets of Langerhans, depleting insulin-secreting β-cells (insulitis). Insulitis arises during an asymptomatic phase, prior to clinical diagnosis of T1D. Methods to diagnose insulitis and β-cell mass changes during this asymptomatic phase are limited, precluding early therapeutic intervention. During T1D the islet microvasculature increases permeability, allowing nanoparticles to access the microenvironment. Contrast enhanced ultrasound (CEUS) uses shell-stabilized gas bubbles to provide acoustic backscatter in vasculature. Here, we report that sub-micron sized ‘nanobubble’ ultrasound contrast agents can be used to measure increased islet microvasculature permeability and indicate asymptomatic T1D. Through CEUS and histological analysis, pre-clinical models of T1D show accumulation of nanobubbles specifically within pancreatic islets, correlating with insulitis. Importantly, accumulation is detected early in disease progression and decreases with successful therapeutic intervention. Thus, sub-micron sized nanobubble ultrasound contrast agents provide a predicative marker for disease progression and therapeutic reversal early in asymptomatic T1D.


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October 2018

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. No formal sample size calculation was performed. 3 cohorts of 4 NOD mice per cohort were first examined as a minimum number of animals required to examine disease-dependent differences, given reported inter and intra cohort variability in disease development. This determination was based on significant variability in NOD mice disease progression in general, and specifically in the blood flow measured in NOD mice in prior studies (St Clair et al, Nat Comm 2018). In this study a difference was observed between pancreas and kidney after this initial minimum required number of animals.
No data was excluded from analysis 2 different models of disease (NOD, adoptive transfer) were used, and 2 means to measure accumulation (ultrasound, histology) were performed. Each model and experimental approach reproduced initial fundings.
Experimental animals were randomly assigned into relevant treatment groups. where comparisons were made between different treatments (e.g. MB vs NB) measurements were made on the same animals, on separate days. (within 3 days). When caparison were made between animals, measurements were made on the same day. recordings were made in a randomized order Insulitis scoring was blinded. Contrast and fitting measurements were unbiased via automated matlab scripts (curve fitting or thresholding) and thus do not require blinding as analysis bias is negligible.
anti-mouse CD4 (BP0003-1; BioXCell). Note this was given for therapeutic purposes not for immunofluorescence or western blot analysis. Thus dilution is not applicable but treatment was a single 20 mg dose delivered IP Insulin and glucagon antibodies validated in WT mice by lacking exocrine staining and showing specific staining in core and periphery of adult mouse islets.