Diabetes and Experimental Stroke: Early Pathophysiology in the db/db mouse
Open Access
- Author:
- Patel, Shyama Dinesh
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 20, 2011
- Committee Members:
- Ian Alexander Simpson, Dissertation Advisor/Co-Advisor
Patricia Sue Grigson Kennedy, Committee Chair/Co-Chair
Susan Vannucci, Committee Member
Robert Harold Bonneau, Committee Member
Christopher Charles Norbury, Committee Member
Samuel Shaomin Zhang, Committee Member - Keywords:
- diabetes
stroke
neutrophils - Abstract:
- Diabetes is associated with an increased vulnerability to stroke and increased morbidity/mortality following a stroke. Although increased risk of stroke is attributable to increased rates of atherosclerosis, there is no well-established single mechanism explaining the poorer stroke recovery observed in diabetics. In animal models, diabetic db/db mice exhibit larger infarct volumes relative to their non-diabetic db/+ littermates following a cerebral hypoxic-ischemic (HI) insult. It was recently found that the enhanced morbidity in the diabetic db/db mice was associated with increased blood-brain barrier (BBB) permeability and increased levels of matrix metalloproteinase-9 (MMP-9) activity. In other experimental models of stroke, neutrophils are considered to be a source of MMP-9 and BBB breakdown. The overall objective of this thesis was to determine the role of neutrophil infiltration on stroke recovery in the brain of the db/db Type II diabetes mouse following cerebral HI. Specifically, the effects of diabetes on neutrophil infiltration, endothelial cell activation (ICAM-1 and P-Selectin), and BBB integrity was compared to the heterozygous control (db/+). The underlying hypothesis was that diabetic db/db mice, in comparison to the heterozygous control (db/+), will have more neutrophils adhering to the cerebral vessels and infiltrating the brain resulting in increased cell death following cerebral HI. 8-week old db/db mice, and their non-diabetic db/+ controls, were subjected to unilateral carotid ligation and hypoxia (HI) and their brains were analyzed at 4 and 24 hr post-HI. In another series of experiments, circulating neutrophils were depleted 24 hr prior to HI with the rat anti-mouse Ly6G 1A8 antibody and then 30 min prior to sacrifice bovine serum albumin was injected (i.v) to determine the level of BBB permeability. Brains were analyzed at 4 and 24 hr post-HI, as before. Blood and brain samples were collected for flow cytometry, immunohistochemistry (IHC), and Western blot analysis. Consistent with previous studies, HI resulted in a more rapid and extensive cell death in db/db mice, relative to the non-diabetic, db/+ mice. MMP-9 was variably associated with cell death in both the db/db and db/+ mice. Also, while there was more MMP-9 protein detected in the cortex and caudate-putamen of the db/db mice, there was also more tissue damage. P-selectin and ICAM-1 protein expression were not altered with diabetes. Additionally, while there was an effect of diabetes on circulating neutrophil levels, which significantly increased even in response to sham surgery in the db/db mice, brain neutrophil levels were not consistently associated with cell death in either the db/db or db/+ mice. Similar to MMP-9 protein, while there were more neutrophils in the cortex and caudate-putamen of the db/db mice, there was also more tissue damage. There was also early loss of BBB proteins (occludin, collagen IV, and laminin) in the db/db mice, but only in those brains where there was also more tissue damage. Finally, neutrophil depletion had no effect on infarct area in the db/+ mice and had a variable effect on infarct area in the db/db mice. Neutrophil depletion also had no effect on MMP-9 and BBB breakdown. Overall, future studies need to compare more comparably damaged db/db and db/+ mice in order to separate out the effect of diabetes versus extent of cell death.