Semester

Summer

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Jason D Huber

Abstract

Many studies point to vascular dysfunction as an underlying cause for the increased incidence of cognitive dysfunction and risk for development of Alzheimer's disease during diabetes. Vascular dysfunction is not an uncommon occurrence in patients with diabetes and microvascular dysfunction commonly leads to clinical complications such as blindness, peripheral neuropathy, and kidney failure. Microangiopathies of the retina, kidney, and peripheral nerves have been well-characterized; however, the effects of diabetes on blood-brain barrier (BBB) function have been understudied.;Pathophysiological changes defining microvascular dysfunction include basement membrane thickening, cytoskeleton rearrangement, and increased paracellular leakage. Increased paracellular leakage of the BBB suggests a functional break down of the tight junction. To investigate changes in functional integrity, we used three different sized vascular space markers [sucrose (342 Da), inulin (5000 Da), and evans blue (68,000 Da)] to measure time-dependant paracellular permeability changes. Our findings revealed that the smallest vascular space marker (sucrose) showed subtle region-specific permeability changes that may represent an altered neuronal microenvironment. Previously published clinical data coincides with these region-specific changes observed in the hippocampus, cortex and midbrain. Patients with diabetes have a higher incidence of midbrain-related lacunar infarcts and cognitive deficiencies can be correlated to areas like the hippocampus and cortex.;Sesamol, a natural antioxidant, has been shown to improve cognitive function in STZ-induced diabetic rats. Furthermore, microangiopathy studies show that oxidative stress plays a major role in microvascular dysfunction; therefore, we investigated if oxidative-stress contributed to BBB permeability. Rats were randomly divided into four treatment groups (CON- control; STZ- STZ-induced diabetes; CON+S- control+sesamol; STZ+S-STZ-induced diabetes+sesamol). Functional and structural BBB changes were measured by in situ brain perfusion with sucrose and tight junction expression was assessed by real time RT-PCR and western blot analyses. Oxidative stress markers were visualized by fluorescent confocal microscopy and assayed by spectrophotometric analyses. Results demonstrated that STZ+S rats showed increased tight junction protein expression and decreased permeability as compared to STZ treated rats. Furthermore, STZ+S treated rats show increased antioxidant enzyme activity and decreased markers of oxidative stress in the brain. In conclusion, this study showed that sesamol treatment enhanced antioxidant capacity of the diabetic brain and led to decreased perturbation of oxidative stress-induced changes in BBB structure and function.;Next, we investigated the antioxidant mechanism for sesamol and oxidative mechanisms that may contribute to enhanced BBB permeability. The chemical properties of sesamol permit passage through the BBB and suggest that Fenton-induced lipid peroxidation can be inhibited. The brain, possessing iron stores and high levels of polyunstaturated fatty acids, may be vulnerable to Fenton-induced lipid peroxidation under pro-oxidant conditions during diabetes. Spectrophotometric assays were used to assess ferrous iron levels, hydrogen peroxide production, and lipid peroxidation in the brain. Furthermore, oxidative stress influences vascular remodeling and aberrant neovascularization of blood-retinal barrier (BRB) during diabetes. Because the BRB and BBB possess a similar structure and function, we examined whether similar pathophysiological changes occurred in the brain and if sesamol treatment influenced pathological changes. Gel zymography and real time RT-PCR were used to assess these parameters. Sesamol treatment reduced lipid peroxidation and enhanced mitochondrial superoxide dismutase (SOD) activity. Sesamol-related lignans can upregulate lipolytic enzymes, thus, sesamol may have exert similar effects. Elevated PDGF transcription in the STZ group was attenuated in the STZ+S group. PDGF plays a role in tight junction rearrangement and neovascularization in diabetic retinopathy, thus demonstrating neovascularizing factors may influence BBB integrity. This study suggests that sesamol may be beneficial as an adjuvant therapy for minimizing lipid peroxidative damage during diabetes.;The present results suggest that oxidative stress is a key factor promoting BBB dysfunction during STZ-induced diabetes and that sesamol or sesamol-related compounds might be beneficial adjuvant therapies for minimizing oxidative damage to the cerebral endothelium. Understanding the oxidative mechanisms contributing to BBB permeability may elucidate novel pharmacological targets for maintaining BBB function and promoting neuron survival. To accomplish this, more studies are needed to understand the signaling pathways connecting BBB integrity and supporting cells (e.g. astrocytes, microglia, pericytes).

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