MRI indices of functional recovery following intracerebral implantation of a human neural stem cell line in a pre-clinical model of ischaemic stroke

Stem cell therapy for ischaemic stroke is an emerging field in light of an increasing number of patients surviving with permanent disability. Several allogenic and autologous cells types are now in clinical trials with preliminary evidence of safety. Some clinical studies have reported functional improvements in some patients. After initial safety evaluation in a Phase 1 study, the conditionally immortalised human neural stem cell line CTX0E03 is currently in a Phase 2 clinical trial (PISCES-II). Previous pre-clinical studies conducted by ReNeuron Ltd, showed evidence of functional recovery in the Bilateral Asymmetry test up to 6 weeks following transplantation into rodent brain, 4 weeks after middle cerebral artery occlusion. Resting-state fMRI is increasingly used to investigate brain function in health and disease, and may also act as a predictor of recovery due to known network changes in the post-stroke recovery period. Resting-state methods have also been applied to non-human primates and rodents which have been found to have analogous resting-state networks to humans. The sensorimotor resting-state network of rodents is impaired following experimental focal ischaemia of the middle cerebral artery territory. However, the effects of stem cell implantation on brain functional networks has not previously been investigated. Prior studies assessed sensorimotor function following sub-cortical implantation of CTX0E03 cells in the rodent post-stroke brain but with no MRI assessments of functional improvements. This thesis presents research on the effect of sub-cortical implantation of CTX0E03 cells on the resting- state sensorimotor network and sensorimotor deficits in the rat following experimental stroke, using protocols based on previous work with this cell line. The work in this thesis identified functional tests of appropriate sensitivity for long-term dysfunction suitable for this laboratory, and investigated non-invasive monitoring of physiological variables required to optimize BOLD signal stability within a high-field MRI scanner. Following experimental stroke, rats demonstrated expected sensorimotor dysfunction and changes in the resting-state sensorimotor network. CTX0E03 cells did not improve post-stroke functional outcome (compared to previous studies) and with no changes in resting-state sensorimotor network activity. However, in control animals, we observed changes in functional networks due to the stereotaxic procedure. This illustrates the sensitivity of resting-state fMRI to stereotaxic procedures. We hypothesise that the damage caused by cell or vehicle implantation may have prevented functional and network recovery which has not been previously identified due to the application of different functional tests. The findings in this thesis represent one of few pre-clinical studies in resting-state fMRI network changes post-stroke and the only to date applying this technique to evaluate functional outcomes following a clinically applicable human neural stem cell treatment for ischaemic stroke. It was found that injury caused by stereotaxic injection should be taken into account when assessing the effectiveness of treatment.

Tenecteplase and alteplase in acute ischaemic stroke thrombolysis: clinical and imaging study

Introduction: Intravenous thrombolysis in acute ischaemic stroke with alteplase improves clinical outcomes, but it has limited efficacy and is associated with increased risk of intracranial haemorrhage. An improved tissue plasminogen activator, tenecteplase, was evidenced to be at least equally effective with lower risk of haemorrhage in acute myocardial infarction thrombolysis. To date, two completed phase II randomised controlled studies comparing tenecteplase and alteplase in acute ischaemic strokes showed variable results. Methods: A literature review of thrombolytic agents used in myocardial infarction and acute ischaemic stroke was performed, followed by a retrospective investigation of the bolus-to- infusion delay of alteplase administration. The main focus of this thesis is the report of our single centre phase II randomised controlled trial that compared tenecteplase (0.25mg/kg, maximum 25mg) and alteplase (0.9mg/kg, maximum 90mg, 10% as the initial bolus, following by one hour infusion with the rest of the dose) in acute ischaemic stroke thrombolysis using advanced imaging as biomarkers. Imaging comprised baseline computed tomography (CT), CT perfusion (CTP) and CT angiography (CTA), and CT+CTA at 24-48 hours. The primary end-point was penumbral salvage (CTP-defined penumbra volume minus follow-up CT infarct volume). A sub-study of coagulation and fibrinolysis analysis of the two agents was performed by comparing a group of coagulation variables measured pre-treatment, 3-12 hours, and 24±3 hours post thrombolysis. An individual patient data (IPD) meta-analysis was carried out using all three completed tenecteplase/alteplase comparison studies in stroke thrombolysis. We compared clinical outcomes including modified Rankin scale at 3 months, early neurological improvement at 24 hours, intracerebral haemorrhage rate and mortality at 3 months between all three tenecteplase doses (0.1mg/kg, 0.25 mg/kg, and 0.4mg/kg) examined and standard alteplase. Imaging outcomes including penumbra salvage, recanalisation rates were also compared using the data from the two studies that had advance imaging carried out. Results: Delay between the initial bolus and the subsequent infusion in administration of alteplase is common. This may reduce the likelihood of achieving a good functional outcome. Among the 104 patients recruited in ATTEST trial, 71 contributed to the imaging primary outcome. No significant differences were observed for penumbral salvage [68 (SD 28) % tenecteplase vs 68 (SD 23) % alteplase], mean difference 1% (95% confidence interval -10%, 12%, p=0·81) or for any secondary end-point. The SICH incidence (1/52, 2% vs 2/51, 4%, by SITS-MOST definition, p=0·55; by ECASS-2 definition, 3/52, 6% tenecteplase vs 4/51, 8% alteplase, p=0.59) did not differed significantly. There was a trend towards lower ICH risk in the tenecteplase group (8/52 tenecteplase, 15% vs 14/51 alteplase, 29%, p=0·091). Compared to baseline, alteplase caused significant hypofibrinogenaemia (p=0.002), prolonged Prothrombin Time (PT) (p=0.011), hypoplasminogenaemia (p=0.001) and lower Factor V (p=0.002) at 3-12 hours after administration with persistent hypofibrinogenaemia at 24h (p=0.011), while only minor hypoplasminogenaemia (P=0.029) was seen in the tenecteplase group. Tenecteplase consumed less plasminogen (p<0.001) and fibrinogen (p=0.002) compared with alteplase. In a pooled analysis, tenecteplase 0.25mg/kg had the greatest odds to achieve early neurological improvement (OR [95%CI] 3.3 [1.5, 7.2], p=0.093), excellent functional outcome (mRS 0-1) at three months (OR [95%CI] 1.9 [0.8, 4.4], p= 0.28), with reduced odds of ICH (OR [95%CI] 0.6 [0.2, 1.8], P=0.43) compared with alteplase. Only 19 patients were treated with tenecteplase 0.4mg/kg, which showed increased odds of SICH compared with alteplase (OR [95% CI] 6.2 [0.7, 56.3]). In the two studies where advanced imaging was performed, the imaging outcomes did not differ in the IPD analysis. Conclusion: Tenecteplase 0.25 mg/kg has the potential to be a better alternative to alteplase. It can be given as a single bolus, does not cause disruption to systemic coagulation, and is possibly safer and more effective in clot lysis. Further phase III study to compare tenecteplase and alteplase in acute ischaemic stroke is warranted.