"Introduction. Despite being the youngest branch of regenerative medicine, neural tissue engineering has rapidly developed, with numerous advances close to clinical translation. One of the most researched areas are tissue engineering and regenerative medicine approaches for brain repair after ischaemic stroke, with more than 70 preclinical studies testing therapeutic combinations of biomaterials, stem cells, and/or therapeutic proteins. Many of these studies have administered the regenerative therapies at the target site, in the stroke cavity resulted following the clearance of dead tissue in the chronic phase of stroke. This localised administration favours tissue repair by overcoming the blood brain barrier, which drastically limits drug accessibility into the brain. Most studies assess behavioural and histological outcomes. Here, we propose a more specific approach to assess the success of a combination therapy based on the use of an injectable synthetic peptide hydrogel and two multifunctional therapeutic proteins for inducing recovery following ischaemic stroke.
Methodology. We propose outcome evaluation at three main levels: behaviour functional (through sickness, wellbeing, and memory behavioural tests), functional vascular, and histological level. The behaviour functional level assesses brain function, representing the ultimate goal of regenerative therapies. In this regard, sickness, wellbeing, sensory-motor, and memory tests are performed. The functional vascular level assesses the cerebral blood flow in potentially regenerated areas through magnetic resonance imaging (MRI) arterial spin labelling (ASL). The histological level assesses markers of interest for the main repair processes which should be targeted by novel therapies: reduction of inflammation, immune-modulation, angiogenesis, and neurogenesis.
Results. Here, we have assessed the effect on functional recovery of a regenerative construct using wellbeing and sickness behaviour tests like the burrowing and nest building tests, and the sensorimotor neuroscore test. Functional vascular assessment through MRI ASL can discern cerebral blood flow differences after stroke, differences which minimise at 28 days post stroke. Histological assessment of brain inflammation was done through scoring of brain immune cells, Iba1+ (ionising calcium binding adaptor molecule 1) microglia, into discrete activation states. Relevant for tissue repair, measurement of glial scar thickness was made by GFAP+ (glial fibrillary acidic protein) integrated density quantification. Assessment of angiogenesis, the most targeted repair process in stroke, was done by measuring the ratio between endoglin, marker of new endothelial cells, and PECAM-1, an endothelial cell adhesion molecule. Finally, neurogenesis is assessed by integrated density of doublecortin positive cells infiltrated into the infarct and ratio of ipsilateral to contralateral doublecortin integrated density in the neurogenic niche-containing brain ventricles.
Conclusions. Our approach proposes the stroke outcome assessment in pre-clinical models at three different levels, behaviour functional, vascular functional, and histological functional. These are instrumental in evaluating the success of regenerative therapies for brain recovery after stroke, before further progression into clinical practice."