Neamine induces neuroprotection after acute ischemic stroke in type one diabetic rats

Abstract

Introduction: Angiogenin is a member of the ribonuclease superfamily and promotes degradation of the basement membrane and the extracellular matrix. After stroke in type one diabetes (T1DM) rats, Angiogenin is significantly increased and the Angiogenin is inversely correlated with functional outcome. Neamine, an aminoglycoside antibiotic, blocks nuclear translocation of Angiogenin, thereby abolishing the biological activity of Angiogenin. In this study, we therefore investigated the effect and underlying protective mechanisms of Neamine treatment of stroke in T1DM.

Methods: T1DM was induced in male Wistar rats by streptozotocin (60mg/kg, ip), and T1DM rats were subjected to embolic middle cerebral artery occlusion (MCAo). Neamine (10mg/kg ip) was administered at 2, 24 and 48h after the induction of embolic MCAo. A battery of functional outcome tests was performed. Blood-brain barrier (BBB) leakage, and lesion volume were evaluated and immunostaining, and Western blot were performed.

Results: Neamine treatment of stroke in T1DM rats significantly decreased BBB leakage and lesion volume as well as improved functional outcome compared to T1DM-control. Neamine also significantly decreased apoptosis and cleaved caspase-3 in the ischemic brain. Using immunostaining, we found that Neamine treatment significantly decreased nuclear Angiogenin, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activity, advanced glycation endproducts receptor (RAGE) number, the positive area of toll-like receptor 4 (TLR4) and increased Angeopoietin-1 expression compared to T1DM-MCAo control rats. Western blot results are consistent with the immunostaining.

Conclusion: Neamine treatment of stroke is neuroprotective in T1DM rats. Inhibition of neuroinflammatory factor expression and decrease of BBB leakage may contribute to Neamine-induced neuroprotective effects after stroke in T1DM rats.

Keywords: Ang1; Angiogenin; BBB; BP; BT; DM; ECA; ECM; IBZ; ICA; MCAo; NFκB; Neamine; PBS; RAGE; T1DM; TLR4; TUNEL; WT; advanced glycation end products receptor; angiopoietin-1; blood pressure; blood–brain barrier; body temperature; diabetes mellitus; external carotid artery; extracellular matrix; internal carotid artery; ischemic border area; mNSS; middle cerebral artery occlusion; modified neurological severity score; neuroprotection; nuclear factor kappa-light-chain-enhancer of activated B cells; phosphate-buffered saline; stroke; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; toll-like receptor 4; type one diabetes; wild type.

Figure 1. Functional tests and lesion volume measurements

Neamine treatment of stroke in T1DM rats significantly improved functional outcome and reduces lesion volume compared to PBS treated T1DM-MCAo rats. A: mNSS test. B: Adhesive removal test. C: Foot-fault test. D: Lesion volume measurement.

Figure 2. BBB leakage and apoptosis changes in the ischemic brain

Neamine treatment of stroke in T1DM rats significantly decreased BBB leakage and apoptotic cell numbers in the ischemic brain compared to PBS treated T1DM-MCAo rats. A: Evans blue dye assay for BBB leakage. B-E: TUNEL and cleaved caspase-3 immunostaining and quantitative data in the IBZ.

Figure 3. Angiogenin, Angiopoietin-1, TLR4, NFkB and RAGE expression in the ischemic brain

Neamine treatment of stroke in T1DM rats significantly increased angiopoietin-1 and decreased TLR4, RAGE, nuclear Angiogenin and NFkB expression in the ischemic brain compared to PBS treated T1DM-MCAo rats. A-E: Angiogenin, TLR4, NFkB, RAGE and Angiopoietin-1, immunostaining and quantitative data in IBZ. Scale bar in A-E=0.1 mm.

Figure 4. Western blot assay

Neamine treatment of stroke significantly increased brain tissue Ang-1, but decreased Angiogenin, NFkB, RAGE and TLR4 levels in T1DM rats compared to non-treatment T1DM-MCAo rats (P<0.05). A: Western blot assay; B-E: Ang1 (B), Angiogenin (C), RAGE (D) and TLR4 (E) quantitative data from Western blot assay; F-G: Nuclear NFkB activity measured by Western blot assay (F) and quantitative data (G).