Anatomy and Neuroscience - Theses
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ItemMechanisms that increase vascular reactivity following spinal cord injury( 2013)People with spinal cord injury (SCI) can experience episodes of dangerously high blood pressure, termed autonomic dysreflexia, in response to a range of sensory stimuli. While SCI severs bulbospinal inputs to sympathetic preganglionic neurons, the spinal reflex pathways below the lesion remain intact and are unopposed by inhibitory inputs from the brainstem. As a result, somatosympathetic reflexes can produce pronounced constriction of arterial vessels. Studies in man indicate that SCI not only modifies spinal reflexes but also increases neurovascular transmission in arterial vessels. The objective of this thesis was to gain insight into the mechanisms underlying the augmentation of neurovascular transmission that occurs following SCI. In Chapter 2, I investigated the mechanisms that underlie SCI-induced enhancement of neurovascular transmission in the rat tail artery. Isometric contractions of arterial segments from T11 spinal cord-transected and sham-operated rats were compared 6 weeks postoperatively. SCI more than doubled the amplitudes of contractions evoked by nerve stimulation. In arteries from SCI rats, but not those from sham-operated rats, the L-type Ca2+ channel blocker nifedipine reduced nerve-evoked contractions. Furthermore, while the sensitivity to the agonists phenylephrine (α1-adrenoceptor selective) and clonidine (α2-adrenoceptor selective) was unaffected by SCI, nifedipine had a greater inhibitory effect on contractions to both agents in arteries from SCI rats. In arteries from unoperated rats, the L-type Ca2+ channel agonist Bay K8644 mimicked the effects of SCI. These findings demonstrate that the SCI-induced enhancement of neurovascular transmission in rat tail artery can largely be accounted for by an increased contribution of L-type Ca2+ channels to activation of the vascular muscle. In Chapter 3, the mechanisms underlying the enhancement of neurovascular transmission produced SCI and Bay K8644 were further investigated in rat tail artery. In situ electrochemical detection of noradrenaline and electrophysiological monitoring of purinergic transmission were used to assess if Bay K8644 changed neurotransmitter release. In addition, isometric contractions of arterial segments were used to assess if SCI and Bay K8644 similarly changed the contribution of α1-adrenoceptors to nerve-evoked contractions and if interfering with sarcoplamic reticulum (SR) Ca2+ uptake modified the contribution of L-type Ca2+ channels to activation of tail arteries. Bay K8644 did not change noradrenaline-induced oxidation currents or purinergic excitatory junction potentials. Both SCI and Bay K8644 reduced blockade of nerve-evoked contractions by BMY7378 (α1D-adenoceptor antagonist), but did not change that by RS100329 (α1A-adrenoceptor antagonist). Disruption of the SR Ca2+ stores with ryanodine increased both nerve-evoked contractions and blockade of these responses by nifedipine. The findings demonstrate that SCI and Bay K8644 increase the α1A-adrenoceptor-mediated component of nerve-evoked contractions. The findings also suggest that Ca2+ entering smooth muscle via L-type channels is rapidly sequestered by the SR limiting its access to the contractile mechanism. Studies in individuals with SCI suggest the vasculature is hyperreactive to angiotensin II (Ang II). In Chapter 4, the effects of SCI on the reactivity of rat tail and mesenteric arteries to Ang II were investigated. SCI increased contractions of both vessels evoked by Ang II. In tail arteries, the facilitatory effect of Ang II on neurovascular transmission was greatly increased. In contrast, SCI did not change the facilitatory action of Ang II on neurovascular transmission in mesenteric arteries. These findings provide the first direct evidence that SCI increases the reactivity of arterial vessels to Ang II. In addition, in tail artery, the findings indicate that Ang II may contribute to amplifying spinal reflex activation of this vessel.
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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.