Adverse effects of bone marrow stromal cell treatment of stroke in diabetic rats

Abstract

Background and purpose: Cell therapy with bone marrow stromal cells (BMSCs) improves functional recovery after stroke in nondiabetic rats. However, its effect on diabetics with stroke is unknown. This study investigated the effect of BMSCs on stroke outcome in Type 1 diabetic (T1DM) rats.

Methods: T1DM was induced in adult male Wistar rats by injecting streptozotocin. Nondiabetic and T1DM rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO), treated with or without BMSCs (3×10(6)) at 24 hours after MCAO, and monitored for 14 days.

Results: Functional benefit was not detected in T1DM-MCAO treated with BMSC rats compared with corresponding T1DM-MCAO controls. BMSC treatment in T1DM-MCAO rats had increased mortality, blood-brain barrier leakage, brain hemorrhage, and angiogenesis. Internal carotid artery neointimal formation and cerebral arteriole narrowing/occlusion were also observed in T1DM-MCAO+BMSCs rats compared with T1DM-MCAO controls (P<0.05), but not in nondiabetic stroke rats. We further studied the underlying mechanisms responsible for BMSC-induced blood-brain barrier leakage and accelerated vascular damage in T1DM-MCAO rats. We found that the expression of angiogenin (an angiogenic factor) and ED1 (a marker for macrophages) was significantly increased in the T1DM-MCAO+BMSC rats in the ischemic brain and internal carotid artery compared with nontreated T1DM-MCAO rats, but not in nondiabetic stroke rats.

Conclusions: BMSC therapy in T1DM-MCAO rats does not improve functional outcome. On the contrary, it increases blood-brain barrier leakage and cerebral artery neointimal formation, and arteriosclerosis, which possibly is due to increased expression of angiogenin. Thus, BMSC treatment starting 24 hours after MCAO may not be beneficial for diabetic subjects with stroke.

Figure 1

BMSC treatment of stroke in T1DM rats does not improve functional outcome and lesion volume. A Adhesive removal test. B, mNSS test. C, Foot-fault test. D, Lesion volume measurement. BMSC indicates bone marrow stromal cell; T1DM, Type 1 diabetes mellitus; mNSS, modified neurological severity score.

Figure 2

BMSC treatment in T1DM-MCAO rats significantly increases BBB leakage, brain hemorrhage volume, and vascular density in the ischemic brain. A, Evans blue dye assay for BBB leakage (n=5/group). B–C, Brain hemorrhage (B) and vascular density (C) measurement. Scale bar in B and C=0.1 mm. BMSC indicates bone marrow stromal cell; T1DM, Type 1 diabetes mellitus; MCAO, middle cerebral artery occlusion; BBB, blood–brain barrier.

Figure 3

BMSC treatment in T1DM-MCAO rats significantly increases cerebral artery wall thickness and occlusion artery number. A, α-SMA staining. B–D, Quantitative data for artery density (B), cerebral artery wall thickness (C), and cerebral artery occlusion (D) in the ischemic brain. Scale bar in A=0.1 mm. BMSC indicates bone marrow stromal cell; T1DM, Type 1 diabetes mellitus; MCAO, middle cerebral artery occlusion; α-SMA, α-smooth muscle actin.

Figure 4

BMSC treatment in T1DM-MCAO rats significantly accelerates arteriosclerosis in the ICA. A, Trichrome immunostaining; (B) quantitative data for ICA artery intima thickness, artery media thickness, and artery diameter in ipsilateral ICA. C–D, Collagen staining and measurement in ICA. Scale bar in A and C=0.1 mm. BMSC indicates bone marrow stromal cell; T1DM, Type 1 diabetes mellitus; MCAO, middle cerebral artery occlusion; ICA, internal carotid artery.

Figure 5

T1DM-MCAO rats increases angiogenin expression and BMSC treatment in T1DM-MCAO rats significantly enhances angiogenin and ED1 expression. A–B, Angiogenin immunostaining and quantitative data in IBZ (A) and in the ICA (B). C, ED1 immunostaining and quantitative data in ICA. Scale bar in A–C=0.1 mm. T1DM indicates Type 1 diabetes mellitus; MCAO, middle cerebral artery occlusion; BMSC, bone marrow stromal cell; IBZ, ischemic border zone; ICA, internal carotid artery.

Figure 6

Angiogenin expression is colocalized with ED1. Angiogenin expression significantly correlated with ED1 expression and cerebral artery thickness in the ischemic brain. A, Angiogenin and ED1 double immunostaining in the ischemic brain. B, Correlation analysis of angiogenin and ED1. C, Correlation analysis of angiogenin and cerebral artery thickness.