To establish functional and quantitative MRI biomarkers for monitoring of disease in a novel mouse model of β-thalassemia major to enable serial investigation in the development of new therapeutics.β-thalassemia major is a common blood disorder causing the synthesis of abnormal haemoglobin leading to severe anaemia. Current disease treatment consists of regular blood transfusions a consequence of which is iron overload. MRI evaluation of iron loading is key to the clinical management of patients with thalassemia¹. Research into therapy has been limited due to difficulties in producing a clinically relevant animal model of the disease. A novel humanised mouse model of thalassemia developed by Huo et al.² in which heterozygous animals are affected by anemia, splenomegaly and extramedullary hematopoiesis, provides a platform for the development of new treatments in a clinically relevant model. Here we show that in vivo MRI can be used to assess and quantify disease providing a method for serial long term assessment of the efficacy of therapies in rodents.γ_^HPFHδβ⁰/γβ^A knockin heterozygous thalassemia mice (n=6) received intraperitoneal injections of iron dextran solution (100mg/ml for 4 weeks (5 days/week)) to simulate repeated blood transfusions. Two control groups 1) wild type humanised controls γβ^A/γβ^A, n=7 and 2) γ^HPFHδβ⁰/γβ^A knockin heterozygous thalassemia mice, n=6 received injections of PBS. MR imaging was performed at 5 months of age using a 9.4T (Agilent technologies, USA) system equipped with 1000mT/m gradients and a 39mm volume resonator RF coil (Rapid biomedical, Germany). Spleen volume was measured using a gradient echo (GE) structural scan (156×156×500μm). Cardiac function was measured using a standard cine GE sequence segmented at systole and diastole to quantify ejection fraction (EF). T1 was measured using an inversion recovery look locker (LL) sequence (2.8 ≤ TI ≤ 30×RR interval (~110) [ms]) with regional means fitted to a LL corrected T1 relaxation curve. T2 was measured using an ECG+RESP gated spin-echo multislice sequence with 8 echo times (2.7 ≤ TE ≤ 20[ms]). T2* was measured with an ECG+RESP gated multi-GE sequence with 15 echo times (0.9 ≤ TE ≤ 14.9[ms]). T2 and T2* regional means were fitted to a standard T2 spin relaxation curve. Non-haem iron concentration was measured in excised heart, spleen and liver using the Bothwell iron assay.

Spleen volume to animal mass ratio was significantly higher in control thalassemia (9.5±1.2mm³/g) and iron overload thalassaemia mice (9.1±1.3mm³/g) relative to wild type mice (4.0±0.4mm³/g). Representative T2 weighted images in Figure 1 demonstrate the strong influence of high iron content on T2 relaxation in the liver (Lvr) and heart (Myo). Quantitative assessment showed that Iron overload reduced T1, T2 and T2* in all organs measured, while thalassemia reduced T2 in the liver and spleen, and T2* in the liver relative to wild type mice (Table 1). Quantification of non-haem tissue iron made using the Bothwell assay allows animal-specific correlations with relaxometry measurements, for example figure 2 shows evidence of correlation between liver iron with T2 relaxation.

Ejection fraction was found to be slightly higher in iron loaded thalassemia animals relative to unloaded but not significantly higher than controls (Figure 3). Cardiac volumes were not significantly different between groups suggesting that at 5 months the mice have not developed the high output state characteristic of chronic anaemia.

Hypersplenia observed in the thalassemia mice is a consequence of increased iron deposition in the organ due to poor processing of abnormal RBCs. This increased iron content shortened spleen and liver T2 and T2*, as confirmed by tissue iron assays. MR relaxation in the heart was unaffected by thalassemia, possibly due to the relatively early analysis time point, however T1, T2 and T2* were shortened in iron loaded animals making these measures useful for assessment of iron chelation therapies.MR imaging techniques can identify thalassemia mice through increased spleen volume and shortened T2 and T2* in the spleen and liver. Iron overload shortened T1, T2 and T2* in the heart, liver and spleen. These imaging methods provide a platform for assessing the severity of thalassaemia by the accumulation of iron in these organs in-vivo allowing for serial assessment and development of preclinical therapies such as iron chelation and gene therapy.