Abstract
Each year, millions of people are affected by stroke resulting in death or long-term disabilities. Due to improvements in stroke treatment resulting in improved survival following having a stroke, more stroke survivors are living with long-lasting disabilities in sensorimotor and cognitive functions. Due to this, it is critical to find new therapeutic interventions to improve mechanisms of recovery and improve functional outcomes in the field of stroke research. An interesting class of biological molecules for stroke recovery are glycosaminoglycans (GAGs), as they can be expressed in hundreds of different configurations, playing many critical roles in biological processes, whist acting on a large variety of cell types. Due to recent advances in the field, glycomimetics can be efficiently synthesised. The overall aim of this thesis was to explore the potential therapeutic effects of glycomimetic compounds on recovery following stroke. Specifically, we aimed to assess the effects of glycomimetic compounds on inflammation and immune cell activation, functional recovery and axonal sprouting patterns and networks following stroke.
A panel of glycomimetic compounds of different sizes and sulfation patterns were first screened in vitro to investigate whether they altered inflammation of immune cell maturation under conditions of LPS-stimulation. All compounds successfully reduced the inflammatory response following LPS-stimulation, whilst many also modulate the maturation of dendritic cells (DCs) by altering the expression of co-stimulatory molecules CD86 and MCHII. The therapeutic potential of two glycomimetic compounds showing similar success in vitro, were then tested in vivo to assess changes in immune cell signalling and populations, axonal sprouting and behavioural recovery following photothrombotic stroke to the motor cortex (MC) in mice. Glycomimetic compounds were given at a delay of 3-days to promote recovery during the sub-acute phase of stroke. Interestingly, we saw a decrease in the stroke-induced infiltration of B-lymphocytes into the brain following treatment with compound A, but not G. In addition, both glycomimetic compounds, A and G improved gross motor impairments on the grid-walking task and changed the distribution of axonal sprouting by increasing connections to the premotor and prefrontal cortex, whilst dramatically decreasing the sprouting response into the direction of somatosensory cortex. Consistent with this loss in sprouting to the somatosensory cortex, we also observed impaired recovery in the cylinder task as a measure of forepaw asymmetry, which corroborates prior publications assessing some glycomimetic compounds for stroke recovery.
As rehabilitation clinically overlaps with our intervention window, we thought that it would be imperative to train a cohort of mice on the pasta matrix reaching task (PMRT) in order to try and maximise the extent of recovery. A second rational for this is that rehabilitation may also interfere with potential pharmacological treatments. Assessment of the rehabilitation intervention alone, using the pasta matrix reaching task (PMRT), was able to abolish gross motor impairments and forepaw asymmetries observed at 4-weeks post-stroke in all stroke groups. In addition, a combinational approach of rehabilitation and treatment with glycomimetic compounds changed the distribution of axonal sprouting to support functional recovery by the rescue of axonal sprouting to the somatosensory cortex. There is also potential to limit any aberrant axonal sprouting using a combination approach, as we observed a reduction in the sum of axons coordinates compared to rehabilitation alone. Together, this provides evidence that glycomimetic compounds have the potential to improve recovery following stroke, however, a combinational approach with rehabilitation was more successful. Additionally, these studies highlight that a component of rehabilitation should be undertaken in preclinical experiments to support the accuracy and translatability of mechanisms involved in therapeutic agents.
Normal cognitive processing is the result of dynamic connections between multiple brain regions. Previously, the Clarkson group has observed delayed impairments in spatial working memory following photothrombotic stroke to the prefrontal cortex (PFC) in mice. The mechanisms underpinning this impairment remain poorly unknown, but may involve loss of connections to other brain regions. Therefore, we assessed neural projections by injecting retrograde tracers into two regions associated with cognition, the prelimbic cortex (PLC) and ventral hippocampus (vHPC). A global loss of projections into the PLC was observed, in particular from the midline thalamic nuclei mediodorsal (MD and nucleus reuniens (Re). To confirm the involvement of these thalamic nuclei in spatial working memory function, a “silent stroke” induced by L-NIO injection targeting either midline thalamic nuclei was used and which resulted in the same delayed spatial memory impairment. These data support our hypothesis that a loss in connections and in particular, a loss of connections between the PLC and MD and Re, likely underpins the development of delayed memory impairments we have observed following stroke to the PFC and which is similar to reports also observed in humans. In addition to the changes in PLC connections, we also observed altered projections into the vHPC that may be a compensatory mechanism due to global loss of inputs into the PLC. The current thesis provides evidence that functional recovery is, in part, reliant on intact and correct connections being maintained or produced between multiple brain regions, which are likely compromised by the chronic changes in inflammatory signalling pathways. In addition, this thesis offers further support for the use of glycomimetics as a therapeutic option. However, much needed work is still required, especially around understanding differences in sulfation as this can greatly affect the different areas of functional recovery, inflammation, sprouting, angiogensisis, and growth factor support to name just a few.