- Title
- Shear-activated nanoparticles with nitroglycerin to selectively enhance collateral-mediated penumbral perfusion during ischaemic stroke
- Creator
- Litman, Magdalena
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2025
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Background: Presence of good collateral circulation is a strong predictor of favourable outcome after stroke. There is increasing evidence that poor collaterals in hypertensive animals may be due to increased myogenic tone, indicating that collateral vasodilatory therapies may be particularly effective. To effectively develop such therapies, we need a greater understanding of the factors that regulate collateral blood flow in normotensive versus hypertensive patients. The most intuitive approach to pharmacologically open vasoconstricted vessels and improve flow to the penumbra in the presence of hypertension is use of vasodilators. However, treatment with vasodilators is associated with systemic vasodilation and hypotension, which can be harmful in compromised stroke patients. Nitroglycerin (glyceryl trinitrate; GTN) is a well-known vasodilator and the most studied nitric oxide (NO) donor in the treatment of ischaemic stroke. To date, clinical trials with systemic nitroglycerin have failed to improve patients’ functional outcome, with some indicators suggesting a possibility of harm. It is likely that reduced blood pressure in penumbra resulted from systemic hypotension counteracts benefits of collateral dilation. Therefore, a more targeted approach is needed. Recent work by our group suggests that leptomeningeal collateral fluid shear stress in normotensive Wistar rats during stroke is 3–7 times higher (100 dynes/cm2) than systemic vessels. This unique feature may provide a novel way to selectively deliver vasodilators to collaterals, using nanoparticle aggregates containing nitroglycerin (NG-NPAs), that only release the drug in areas with high levels of shear stress (≥ 100 dynes/cm2). Aims: The aims for this thesis were to: 1) quantify collateral blood flow and determine key regulators of collateral blood flow during stroke in spontaneously hypertensive rats (SHRs) (Chapter 4); 2) review the effect of nitroglycerin on infarct size and cerebral blood flow in experimental stroke (Chapter 5); 3) investigate whether shear stress in SHRs reaches prerequisite level for nanoparticle aggregate drug deployment (Chapter 6); 4) determine the efficacy of NG-NPAs in enhancing penumbral perfusion during stroke without causing systemic side effects (Chapter 6); 5) compare the effect of NG-NPAs versus free nitroglycerin on penumbral perfusion during cerebral ischaemia (Chapter 6). Methods: All experiments in this thesis used the model of transient middle cerebral artery occlusion (tMCAO) in male hypertensive rats. Aim 1: An open cranial window was made, and fluorescent imaging with fluorescent microspheres was performed to visualise leptomeningeal collaterals and measure collateral blood flow velocity and collateral diameter at baseline and during occlusion. Data obtained from this experiment was used to calculate total collateral blood flow. Aim 2: A systematic review and meta-analysis were conducted to determine the effect of nitroglycerin on infarct size in animal models of ischaemic stroke. Aim 3: The methodology is the same as in aim 2, but collateral blood flow characteristics data was used to calculate shear stress. Aim 4: Animals were randomised to receive intravenous infusion of NG-NPAs (containing 4μg/kg/min of nitroglycerin) or Blank-NPAs (control-NPAs) 25 minutes following tMCAO for 40 minutes. Laser speckle contrast imaging (LSCI) was performed through a cranial window to measure collateral supplied penumbral perfusion before and during drug/control infusion. Infarct volume was measured at 24 hours post-stroke. Aim 5: Animals received intravenous infusion of free nitroglycerin in a dose equivalent to this packaged in nanoparticles (4μg/kg/min), a 10-times-higher dose (40μg/kg/min) or saline (control), commencing 25 minutes after tMCAO. The experimental protocol is the same as in aim 4. Results: Aim 1: Average collateral blood flow post-stroke significantly increased relative to pre-stroke baseline (pre-MCAO: 16.8 ± 7.1nL/min vs. post-MCAO: 146.4 ± 37.7nL/min, p = 0.02). There was a strong positive correlation between changes in collateral blood flow and vessel diameter post-occlusion (r = 0.7–0.99, p = 0.3–0.002). Aim 2: Three publications met inclusion criteria (10 comparisons reporting infarct size). Overall, nitroglycerin did not significantly reduce infarct volume (normalised mean difference [NMD] point estimate = 20.2% [95% CI -1.52-52.7%, p = 0.068]). Aim 3: Shear stress significantly increased in collateral vessels following stroke in SHRs (baseline: 43.80 ± 11.60 dynes/cm2 vs. post-stroke: 252.2 ± 100.4 dynes/cm2, p = 0.03). Aim 4: NG-NPAs in SHRs significantly increased collateral-mediated penumbral perfusion relative to Blank-NPAs at 40 minutes post-infusion (NG-NPA: 44.9% ± 10.5% vs. Blank-NPAs: 11.9% ± 6.4% of pre-infusion baseline p = 0.03), without reducing blood pressure (40 minutes, NG-NPA: +5.6 ± 2.7% vs. Blank-NPA: -0.66 ± 3.5% of pre-infusion baseline p = 0.88). NG-NPA significantly reduced infarct volume at 24 hours (NG-NPA treated rats: 126 ± 15.6mm3 versus Blank-NPA treated: 172.1 ± 13.8mm3, p = 0.04). Aim 5: Free nitroglycerin at neither 4μg/kg/min nor 40μg/kg/min had a significant effect on penumbral perfusion versus saline (both p > 0.9), but significantly reduced blood pressure at 40 minutes after infusion onset (4μg/kg/min: p = 0.006; 40μg/kg/min: p < 0.0001). Conclusions: Aim 1: Collateral flow significantly increased at 30 minutes following stroke in hypertensive rats but was much less than collateral flow reported in Wistar rats from our previous work. Collateral blood flow was strongly correlated with collateral vessel diameter. Aim 2: The systematic review and meta-analysis indicated that, overall, systemically administered nitroglycerin was not effective in reducing infarct volume in experimental stroke. Aim 3: SHRs displayed a significant elevation in shear stress during stroke and reached well above the threshold of activation for NG-NPAs (> 100 dynes/cm2). Aim 4: Administration of shear-activated nanoparticles with nitroglycerin significantly enhanced penumbral perfusion and reduced infarct volume without reducing systemic blood pressure. Aim 5: Administration of free nitroglycerin (the same as and 10 times the dose of nitroglycerin in the NG-NPAs) did not significantly affect penumbral perfusion but caused significant drop in blood pressure. My results indicate that NG-NPAs may provide a novel and effective way to selectively enhance collateral flow, increase penumbral perfusion and reduce infarct volume in experimental stroke.
- Subject
- leptomeningeal collaterals; ischaemic stroke; nanoparticles; collateral therapeutics; shear stress
- Identifier
- http://hdl.handle.net/1959.13/1518376
- Identifier
- uon:57278
- Rights
- This thesis is under embargo and will be released 03.06.2025, Copyright 2025 Magdalena Litman
- Language
- eng
- Hits: 262
- Visitors: 251
- Downloads: 2
| Thumbnail | File | Description | Size | Format |
|---|