Anatomy and Neuroscience - Theses
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ItemThe effects of diabetes on sympathetic neurovascular transmission( 2013)Impaired neural control of arteries is implicated in the etiology of diabetic foot, a major complication of diabetes. The loss of sympathetic nerve-mediated control of blood flow to plantar skin may be an early change that contributes to the later development of microvascular disease in foot skin. The mechanisms that modify sympathetic regulation of arterial vessels are not understood, but are suggested to be due to diabetes-induced neuropathy. Therefore the primary aim of this thesis was to investigate the effects of diabetes on the sympathetic innervation and activation of plantar metatarsal arteries (PMAs) that supply blood to plantar skin of the hind paw digits in rats. The streptozotocin (STZ) rat model of type I diabetes was chosen as it has been widely used to investigate mechanisms that lead to diabetic complications. Eight-week-old male Wistar rats were treated with STZ (60 mg/kg i.p.) or vehicle (citrate-buffer i.p.; controls). STZ-treated rats received no insulin (STZ-NI) or were treated with a low (~1 unit/day; STZ-LI) or a high (~4 units/day; STZ-HI) dose of insulin. The STZ-NI and STZ-LI rats were hyperglycemic (blood glucose >20 mM), whereas STZ-HI rats were normoglycemic (blood glucose <15 mM). Rats were maintained for 12 weeks when arteries were isolated for in vitro studies. In the first study, wire myography was used to assess vascular function. In comparison with PMAs from control rats, those from STZ-NI rats had reduced nerve-evoked contractions. PMAs from STZ-NI rats also had a decreased density of perivascular nerve fibers revealed by immunolabeling for the pan-neuronal marker β-tubulin III. No changes in vascular function and innervation density were observed in PMAs from STZ-LI and STZ-HI rats. However, in PMAs from both STZ-NI and STZ-LI rats, the β-tubulin III immunoreactive (IR) nerve fibers were thickened. The majority of perivascular nerve fibers were tyrosine hydroxylase (TH)-IR (i.e. originated from sympathetic neurons) and the labeling intensity for this protein increased in PMAs from both STZ-NI and STZ-LI rats. The effects of diabetes on mesenteric arteries (MAs) from STZ-NI rats were also determined. Compared to control MAs, nerve-evoked contractions were not changed in MAs from STZ-NI rats. The density of nerve fibers in the perivascular nerve plexus of MAs was reduced but this change could be explained by an increase in vascular dimensions. There was no change in the width or TH immunolabeling of the nerve fibers. These findings suggest PMAs are particularly sensitive to the effects of diabetes. Thickening of the sympathetic nerve fibers in the perivascular nerve plexus of PMAs suggests diabetes may induce axon remodeling. Peripherin and β-tubulin III are structural proteins that are reported to increase in regenerating axons. The second study investigated whether diabetes changed expression of these neuron-specific proteins in PMAs. Western blotting revealed an increase in peripherin protein content of PMAs from STZ-LI rats compared to those from STZ-HI and control rats. The number of fibers in the perivascular nerve plexus that were peripherin-IR also increased in PMAs from STZ-LI rats. Co-labeling with antibodies to peripherin and neuropeptide Y (a marker for sympathetic axons) revealed that peripherin expression increased in sympathetic axons. No changes in β-tubulin III protein content were detected. These findings are consistent with diabetes stimulating remodeling of the sympathetic nerve terminals. No changes in peripherin protein expression were detected in the tail artery, again suggesting that PMAs are selectively affected by diabetes. The third study investigated whether changes in the structure of the perivascular nerve plexus were accompanied by changes in mRNA expression levels (assessed by quantitative RT-PCR) of genes involved in neurotransmission, axon structure, plasticity, neurotrophin signaling and stress. No diabetes-induced changes in mRNA expression were detected in neuronal cell bodies within the L1-L4 sympathetic chain ganglia. In all experiments, changes observed in PMAs from STZ-NI and/or STZ-LI rats were not observed in those from STZ-HI, suggesting they are due to hyperglycemia. The possibility the changes are explained by loss of a direct influence of insulin on the sympathetic neurons/PMAs, however, cannot be excluded. PMAs appear to be particularly vulnerable to the effects of diabetes. This may be explained by these vessels, which are located close to the plantar surface of the hind paw, also being subjected to biomechanical stress from weight-bearing and locomotion. Together the findings indicate that PMAs provide a suitable model for the assessment of treatments for the prevention of diabetes-induced neurovascular dysfunction seen in diabetic humans.