MacGreen mice: A novel tool to investigate brain inflammation post stroke

Abstract

MacGreen transgenic mice carry a green fluorescent protein (GFP) reporter gene in activated monocytes and macrophage populations and provide a unique opportunity to directly visualize brain inflammation following experimental stroke. In order to evaluate MacGreen mice as a tool for experimental stroke studies, we characterised the response of MacGreen mice to transient middle cerebral artery occlusion (in comparison to standard C57bl/6j mice) and investigated the effect of a novel anti-inflammatory therapy (varenicline) in MacGreen mice following stroke. Both MacGreen mice and C57bl/6j mice had 100% survival rate up to 35 days post-stroke. Reproducible lesion damage was produced in both strains using monofilaments with sizes ranging between 150-180μm in diameter. The pattern of ischaemic damage for both strains was restricted to the ipsilateral striatum and cortex. C57 mice subjected to 60min MCAo had significantly larger lesions at Bregma +1.5mm compared to the MacGreen mice (P=0.003), although the overall volume of lesion damage were not different between the two strains (P=0.273). MacGreen mice showed increased GFP expression in the ipsilateral hemisphere after stroke. An exponential relationship was also found between the volume of tissue with increased GFP expression and the volume of lesion damage (P<0.001, R2=0.776). The increased in GFP signals in the ipsilateral hemisphere were attributed to both an increase cell density (P<0.001) and morphological activation (P<0.001) of GFP expressing cells. All of the GFP expressing cells in MacGreen mice were found to colabel with Iba1, a microglia/macrophage specific antigen. Seven day chronic administration of varenicline (2.5mg/kg/day and 5mg/kg/day) starting at 3 days appeared to reduce the intensity of ipsilateral GFP expression in a dose dependent manner in comparison to saline treated animals, however, significant difference in lesion volume and GFP expression volume were not found between the treatment groups. Varenicline treatment also increased body weight recovery (P<0.001) post stroke compared to saline treated controls. In contrast, varenicline treatment significantly delayed the rate of recovery of neurological function assessed by SHIRPA score and had no beneficial effect on sensorimotor and forelimb asymmetry. These results suggest that tMCAo in MacGreen mice is reproducible and reliable for future studies investigating therapeutic strategies. GFP expression in the MacGreen mouse brain is a useful marker to investigation general inflammation post stroke and preliminary evidence suggest that GFP expression can be modulated by varenicline through its putative activation of the cholinergic anti-inflammatory pathway. The reduction in GFP expression intensity within the ipsilateral hemisphere did not translate to improvements in sensorimotor function and forelimb asymmetry. However, the delayed improvement in neurological score in VAR treated animals in the present study suggest that a longer survival time, beyond 10 days may be required to see a beneficial effect in the functional performance of animals. Pharmacological activation of CAP in the sub-acute phase of stroke remains a viable therapeutic strategy and requires further investigation.