Chapter 3. Delayed Varenicline Administration Reduces Inflammation and Improves Forelimb Use Following Experimental Stroke
Delayed hospital admission, which limits the application of recanalization treatments, is a major hurdle in the translational of experimental stroke research to the clinic. Novel treatment strategies that display extended therapeutic windows (efficacy beyond 4.5h post stroke) are therefore an attractive drug development approach.
The current study investigates the potential of pharmacological interventions targeting pathophysiological mechanisms, such as post-stroke inflammation, in the sub-acute (days to weeks after) and chronic phases (weeks to months after) of stroke to enhance post-stroke recovery. The current study proposed that a delayed pharmacological intervention starting at 3 days post-stroke would be sufficient in producing significant improvements in the functional recovery post-stroke.
The previous chapter optimized the experimental protocols for the established MCAo model of stroke in mice to account for the cumulative changes in mice colonies over time. These studies use the optimized mouse model of stroke outlined in the previous chapter as a platform to investigate whether delayed treatment with a novel anti-inflammatory therapy improves short-term recovery of function.
One manuscript has been generated from the data of this chapter and published in the journal of stroke and cerebrovascular diseases (135).
Brain injuries, such as stroke, are associated with profound activation of inflammatory pathways. Whilst inflammation is important in resolving injury, prolonged inflammation hinders tissue repair and ultimately, patient prognosis (400-402).
Immune responses and inflammation are regulated in part by neural mechanisms (202). The parasympathetic nervous system can inhibit cytokine release and prevent tissue injury via an efferent neural signalling pathway termed the CAP (202, 403). Stimulation of the vagus nerve attenuates production of TNF-a and other pro-inflammatory cytokines from macrophages in the spleen through a mechanism dependent on α7 nAChRs (202, 249, 404, 405). Activation of the a7 nAChR suppresses activation of leukocytes such as the M/M thereby limiting the extent of inflammatory responses within the CNS (249, 406).
It remains unclear why this homeostatic mechanism fails to limit the extent of brain inflammation that occurs after stroke. Previous studies have identified that dysfunction of the ANS is closely associated with and affected by ischemic stroke (203, 407). While a complex reciprocal relationship exists between the onset of ischemic stroke and ANS dysfunction, where one may impact the other and vice versa, previous studies have established that activation of ANS prior to ischemic stroke appeared to have a neuroprotective effect via vasodilation and increase of cerebral blood flow (408). Clinically, low heart-rate variability and chronic inflammation correlate with poor neurological outcomes in stroke patients (409-413). Due to its intimate relationship with the immune system, suppressed ANS activity and reduced HRV have been proposed to be partially responsible for the enhanced activity of sympathetic nervous system (SNS) after stroke (407). This autonomic imbalance leads to suboptimal splenic activation which excessively recruits macrophages, monocytes, and neutrophil into the ischemic core shortly following stroke (109, 169, 187). As indicated previously, suppression of excessive inflammatory responses via splenectomy was neuroprotective thereby indicating that autonomic regulation may be an effective therapeutic strategy in managing post-stroke inflammation (414).
Autonomic regulation via stimulation of the vagus nerve has been shown to be a viable strategy for targeting diseases with an inflammatory component such as rheumatoid arthritis and hemorrhagic shock (231, 415). Vagal nerve stimulation within 30 mins of injury has also been shown to reduce brain damage in experimental models of stroke (233, 240, 416). More recently direct activation of α7nAChRs has been investigated as a less invasive and more applicable therapeutic strategy to reduce brain inflammation and ultimately promote functional recovery after stroke. Both a7 nAChR agonists and allosteric modulators display neuroprotective and anti-inflammatory effects in rodent models of cerebral ischemia (242, 243) and traumatic brain injury (417). To date, these studies have initiated administration within 6 hours of injury and none have demonstrated functional improvement beyond 7 days post stroke (243). It is not known whether activation of nAChRs in the sub-acute phase (>24h) after stroke can also modulate brain inflammation and promote recovery of function.
Pharmacological activation of α7 nAChR in experimental studies often utilize selective full agonists such as CNI-1493 (213, 418), GTS-21 (419-421), and PHA568487 (243, 244) to pin point the underlying mechanisms responsible for their therapeutic benefits. Given a wide range of background research provided in previous literature, the current study proposes that the use of a currently clinically approved pharmacological agent would hasten the process of translational studies when therapeutic benefits are proven in proof of concept and experimental studies. Varenicline is currently used as a first line nicotine replacement for smoking cessation therapies (422). It is a full agonist for the α7 nAChR agonist, partial agonist for the α4β2 and α3 nAChR (423-425). While previous studies investigated the therapeutic potential of α4β2 nAChR activation in smoking cessation, little is known regarding the therapeutic potential of α7 nAChR agonist property of varenicline for suppression of inflammation in neurological disorders such as stroke. Here I hypothesize that delayed administration the a7 nAChR agonist varenicline starting at 3 days post stroke would enhance spontaneous motor function via suppression of inflammation.
The MacGreen mouse has been shown previously to provide a model system in which to investigate brain inflammation following experimental stroke (190). The specific aims of this study were:
- To investigate whether delayed administration of the a7 nAChR agonist varenicline would reduce brain inflammation following stroke in CSF-1R-EGFP mice.
- To investigate whether delayed administration of the a7 nAChR agonist varenicline would improve forepaw use in spontaneous exploratory movement following stroke.Material and methodsExperimental design considerations
Male MacGreen mice (25-30g) were subjected to 45 min occlusion of the right MCA as described in section 22.214.171.124. Mini-osmotic pumps (Alzet® 1007D, Canada) containing 0.9% saline (n=10) or varenicline tartrate at 2.5mg/kg/day (n=10) were implanted subcutaneous between the scapulae on day 3 post-stroke (as described in section 3.2.4). The spontaneous exploratory movement of all animals was assessed at baseline one day prior to stroke and on days 2, 4, 6, 8 and 10 post-stroke using the cylinder test (297). Functional testing was chosen to be performed every two days to minimize the effect of rehabilitative training on functional performances and maximize the frequency of longitudinal monitoring on spontaneous motor function over the study period. Animals were killed at 10 days post-stroke and brain tissues were harvested for Nissl histology, immunofluorescence analyses for EGFP and GAP43 (as described in section 3.2.9). The study was chosen to be terminated at 10 days for as early activation of molecular precursors for neuroplasticity changes and early improvements in functional performances were expected to be observable at this time (368, 426). A relatively short longitudinal study would therefore be ideal for preliminary proof of concept studies. See Figure 3-1 for a summary of experimental procedures.
Chapter 1. General introduction
1.1. Epidemiology of stroke
1.2. Pathophysiology of stroke
1.3. Long-term pathologies
1.4. Experimental modelling of stroke
1.5. Thesis outline
Chapter 2. Characterization of Monofilament Middle Cerebral Artery Occlusion Model of
Stroke in MacGreen mice
2.2. Material and methods:
Chapter 3. Delayed Varenicline Administration Reduces Inflammation and Improves
Forelimb Use Following Experimental Stroke
3.2. Material and methods
Chapter 4. Delayed Administration of Citalopram is Associated with Long-Lasting
Improvements in Skilled Motor Function after Stroke
4.2. Material and methods
Chapter 5. Delayed Administration of Varenicline Impaired Sensorimotor Function,
Increased Inflammatory Cytokine Expression, and Disrupted Myelinated Fiber Tracts in
the Ipsilateral Hemisphere
5.2. Material and methods
Chapter 6. General discussion
6.1. Summary of key research findings
6.2. Pre-clinical models of stroke
6.3. Delayed pharmacological intervention: when is the right time?
6.4. The effect of delayed pharmacological intervention on neuropathology
6.5. The cholinergic anti-inflammatory pathway as a target for stroke
6.6. Recommendation for management of post-stroke complications
6.8. Future directions
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Enhancement of Post-Stroke Recovery via Delayed Pharmacological Intervention Using a Mouse Model of Stroke