Investigating Treatments for Hemorrhagic Stroke Using Rodent Models

Intracerebral hemorrhage (ICH), when a blood vessel ruptures within the brain, affects approximately 15% of stroke victims in Canada. There are no proven neuroprotective treatments ICH but two therapies, therapeutic hypothermia (TH) and iron-chelators, have gained preclinical and clinical interest. Therapeutic hypothermia involves cooling the whole-body or brain to typically 32-35°C and has robust neuroprotective effects for neonatal hypoxic-ischemic encephalopathy and cardiac arrest. However, the literature on treating ICH is inconclusive. Cooling lessens inflammation, edema and blood brain barrier damage after ICH; yet this has not consistently led to neuroprotection or functional benefit. To better understand these conflicting reports, this thesis investigated whether TH 1) does not mitigate key mechanisms of injury following ICH and/or 2) causes side effects that negate protective effects. Also, since iron-induced injury is a popular target in preclinical studies and causes greater neuronal death in ICH compared to other types of brain injury that TH protects against, the iron-chelator bipyridine was also evaluated. Our aim was to better understand the effects of TH on specific mechanisms of injury and evaluate a potential therapy that could then be studied in the future as a combination therapy with TH (e.g., an iron-chelator). The first two sets of experiments used simplistic models of injury to directly study the impact of TH on thrombin- and iron-induced injury. Thrombin is an important clotting factor that prevents on-going bleeding, but is also neurotoxic causing cell death, inflammation and edema. In the first study, thrombin was infused into the rats’ striatum followed by TH or maintained normothermia. TH treated rats had a greater number of degenerating neurons two weeks post-infusion but there was no effect on overall tissue loss, edema and functional impairment. This suggested that TH delays but does not prevent ongoing thrombin-induced neuronal damage. In the second study, FeCl2 was infused into the striatum to evaluate the influence of TH on iron-induced injury. This model of injury causes tissue loss and neurodegeneration, edema, functional impairment and a small amount of intracerebral bleeding. Therapeutic hypothermia significantly reduced bleeding after FeCl2 infusion but had no beneficial effect on any other measure. These results suggest that two key mechanisms of damage after ICH are not treated by TH. The third study focused on inflammation and hematoma resolution using a rodent model of ICH. It is well known that TH is a potent anti-inflammatory which can be protective, however, we hypothesized that this influence on inflammation would hamper the brain’s natural defenses for containing and resolving the hematoma. Interestingly, despite a considerable reduction in inflammatory cells, TH did not lead to a greater spread of iron into the perihematoma zone nor did it impact the rise in non-heme iron, an indirect measurement of hematoma resolution. Most importantly, we found that 40% of treated animals had significantly greater amounts of blood volume a week following stroke (i.e., 3 days following end of rewarming). At time points when treated animals were still being cooled, there was no increase in blood volume in treated groups compared to controls. These data suggest that TH does not impair endogenous mechanisms of hematoma containment and resolution but likely causes rebleeding following either treatment or rewarming in some animals. This substantial increase in blood volume, even in only a few animals, would likely counteract our ability to detect protective effects of TH in group averages. The intermittent nature of the rebleeding could explain the inconsistent neuroprotection seen in the literature and has important implications for clinical use. In the last set of experiments, bipyridine was investigated using two rodent models of ICH, collagenase and whole-blood, and the simplistic FeCl2 model. The collagenase model causes on-going bleeding by breaking down blood vessel walls. Bipyridine did not impact non-heme iron levels, edema or functional impairments in this model. In the whole-blood model, a bolus injection of autologous blood, bipyridine was not protective. As well, bipyridine did not impact FeCl2-induced tissue loss, neurodegeneration or behavioural impairment. Bipyridine did cause significant, transient hypothermia. Despite thorough evaluation of bipyridine, we found no beneficial effects, which suggests to us that bipyridine would not be a suitable therapy to combine with TH. In conclusion, TH did not effectively treat thrombin- or iron-induced injury and may cause re-bleeding complications.

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