Principal component analysis, a useful tool to study cyclin-dependent kinase-inhibitor’s effect on cerebral ischaemia

Stroke is a leading cause of acute death related in part to brain oedema, blood–brain barrier disruption and glial inflammation. A cyclin-dependant kinase inhibitor, (S)-roscovitine, was administered 90 min after onset on a model of rat focal cerebral ischaemia. Brain swelling and Evans Blue tissue extravasation were quantified after Evans Blue injection. Combined tissue Evans Blue fluorescence and immunofluorescence of endothelial cells (RECA1), microglia (isolectin-IB4) and astrocytes (glial fibrillary acidic protein) were analysed. Using a Student’s t-test or Mann–Whitney test, (S)-roscovitine improved recovery by more than 50% compared to vehicle (Mann–Whitney, P < 0.001), decreased significantly brain swelling by 50% (t-test, P = 0.0128) mostly in the rostral part of the brain. Main analysis was therefore performed on rostral cut for immunofluorescence to maximize biological observations (cut B). Evans Blue fluorescence decreased in (S)-roscovitine group compared to vehicle (60%, t-test, P = 0.049) and was further supported by spectrophotometer analysis (Mann–Whitney, P = 0.0002) and Evans Blue macroscopic photonic analysis (t-test, P = 0.07). An increase of RECA-1 intensity was observed in the ischaemic hemisphere compared to non-ischaemic hemisphere. Further study showed, in the ischaemic hemisphere that (S)-roscovitine treated group compared to vehicle, showed a decrease of: (i) endothelial RECA-1 intensity of about 20% globally, mainly located in the cortex (−28.5%, t-test, P = 0.03); (ii) Microglia’s number by 55% (t-test, P = 0.006) and modulated reactive astrocytes through a trend toward less astrocytes number (15%, t-test, P = 0.05) and astrogliosis (21%, t-test, P = 0.076). To decipher the complex relationship of these components, we analysed the six biological quantitative variables of our study by principal component analysis from immunofluorescence studies of the same animals. Principal component analysis differentiated treated from non-treated animals on dimension 1 with negative values in the treated animals, and positive values in the non-treated animals. Interestingly, stroke recovery presented a negative correlation with this dimension, while all other biological variables showed a positive correlation. Dimensions 1 and 2 allowed the identification of two groups of co-varying variables: endothelial cells, microglia number and Evans Blue with positive values on both dimensions, and astrocyte number, astrogliosis and brain swelling with negative values on dimension 2. This partition suggests different mechanisms. Correlation matrix analysis was concordant with principal component analysis results. Because of its pleiotropic complex action on different elements of the NeuroVascular Unit response, (S)-roscovitine may represent an effective treatment against oedema in stroke.