Magnetic resonance spectroscopy to detect functional improvement by adipose tissue-derived stromal cell transplantation in the ischemic hind limb in diabetic rats

Abstract

Adipose tissue-derived stromal cells (ADSCs) are a prominent cellular source in regenerative medicine. We tested whether transplantation of ADSCs into ischemic muscular tissue of diabetic animals would attenuate the impairment in cell metabolism and microcirculatory with a combination of techniques including magnetic resonance imaging and spectroscopy (1H-MRS). Methods: We induced unilateral hind limb ischemia in male streptozotocin-treated rats and non-diabetic controls. One day after femoral artery ligation, 6 rats per group were randomly injected intramuscularly allogeneic ADSCs (C1: 106, C2: 107, C3: 108 cells/mL); or conditioned media from ADSC cultures (CM); or saline (control). Rats underwent magnetic resonance angiography (MRA); short time inversion recovery (STIR) edema-weighed imaging; 1H-MRS; immunoblotting and immunofluorescence on both hind limbs for 4 weeks. Results: T1-weighted and STIR images showed the presence of tissue swelling and signal hyperintensity respectively in tissue affected by occlusion. Mean total ratio of tissue creatine/water (tCr/water) for the occluded limb was significantly lower than for the non-occluded limbs in both non-diabetic and diabetic rats. At 4 weeks, ADSC and CM groups had greater recovery of tCr/water in ischemic limbs compared with controls in both diabetic- and non-diabetic rats (**p<0.01; *p<0.05) (Figure 1, panel A), with less tissue swelling (panel B), increased expression of α-sarcomeric actinin, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) (Table 1), as well as increased vessel density (panel C) and suppression of the up-regulation of water channel aquaporin (AQP)-1 and pro-inflammatory early response gene such as cycloxygenase (COX)-2 (panel D). Figure 1 Figure 1 Table 1. VEGF and HGF expression in diabetic rat ischemic tissues after femoral artery ligation and treatments with ADSCs conditioned medium and controls Values (pg/mL) are mean ± SD from 3 independent experiments of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in frozen tissues from limbs with femoral artery ligation treated with multiple injections of PBS (1 mL), FB (107 cells/mL), ADSCs (at concentration C1 = 106 cells/mL, C2 = 107 cells/mL, C3 = 108 cells/mL) or supernatants obtained from primary ADSCs (conditioned medium. CM, 1 mL) or non-conditioned medium (1 mL). N=6 rats per each treatment group. **P<0.01 vs non-diabetic non-occluded PBS-treated; °°P<0.01 vs diabetic non-occluded PBS-treated; #P<0.05 vs diabetic occluded PBS treated, ##P<0.01 vs diabetic occluded PBS-treated. Legend: PBS, phosphate buffer saline; ADSCs, adipose tissue-derived stromal cells; CM, conditioned medium; NCM, non-conditioned medium; FB, fibroblasts; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor. Conclusions: ADSCs improve ischemic muscle metabolism and increase neovasculogenesis in diabetic rats with hind limb ischemia. 1H-MRS is a useful tool to monitor attempts at salvaging the ischemic tissues with cell-derived novel therapies.