Innovative statistical approaches: the use of neural networks reduces the sample size in the splenectomy-MCAO mouse model.

AIM: To compare the effectiveness of artificial neural network (ANN) and traditional statistical analysis on identical data sets within the splenectomy-middle carotid artery occlusion (MCAO) mouse model. METHODS: Mice were divided into the splenectomized (SPLX) and sham-operated (SPLX-sham) group. A splenectomy was conducted 14 days before middle carotid artery occlusion (MCAO). Magnetic resonance imaging (MRI), bioluminescent imaging, neurological scoring (NS), and histological analysis, were conducted at two, four, seven, and 28 days after MCAO. Frequentist statistical analyses and ANN analysis employing a multi-layer perceptron architecture were performed to assess the probability of discriminating between SPLX and SPLX-sham mice. RESULTS: Repeated measures ANOVA showed no significant differences in body weight (F (5, 45)=0.696, P=0.629), NS (F (2.024, 18.218)=1.032, P=0.377) and brain infarct size on MRI between the SPLX and SPLX-sham groups post-MCAO (F (2, 24)=0.267, P=0.768). ANN analysis was employed to predict SPLX and SPL-sham classes. The highest accuracy in predicting SPLX class was observed when the model was trained on a data set containing all variables (0.7736±0.0234). For SPL-sham class, the highest accuracy was achieved when it was trained on a data set excluding the variable combination MR contralateral/animal mass/NS (0.9284±0.0366). CONCLUSION: This study validated the neuroprotective impact of splenectomy in an MCAO model using ANN for data analysis with a reduced animal sample size, demonstrating the potential for leveraging advanced statistical methods to minimize sample sizes in experimental biomedical research.

Reactivation of corticogenesis-related transcriptional factors BCL11B and SATB2 after ischemic lesion of the adult mouse brain.

The aim of this study was to characterize expression of corticogenesis-related transcription factors BCL11B and SATB2 after brain ischemic lesion in the adult mice, and to analyze their correlation to the subsequent brain recovery. Ischemic brain lesion was induced by transient middle cerebral artery occlusion followed by reperfusion, and the animals with ischemic lesion were compared to the sham controls. Progression of the brain damage and subsequent recovery was longitudinally monitored structurally, by magnetic resonance imaging, and functionally, by neurological deficit assessment. Seven days after the ischemic injury the brains were isolated and analyzed by immunohistochemistry. The results showed higher expression in the brain of both, BCL11B and SATB2 in the animals with ischemic lesion compared to the sham controls. The co-expression of both markers, BCL11B and SATB2, increased in the ischemic brains, as well as the co-expression of BCL11B with the beneficial transcriptional factor ATF3 but not its co-expression with detrimental HDAC2. BCL11B was mainly implicated in the ipsilateral and SATB2 in the contralateral brain hemisphere, and their level in these regions correlated with the functional recovery rate. The results indicate that the reactivation of corticogenesis-related transcription factors BCL11B and SATB2 is beneficial after brain ischemic lesion.

Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: Systematic review and isolation methods' evaluation.

The aim of our study was to systematically analyze the literature for published flow cytometry protocols for microglia isolation and compare their effectiveness in terms of microglial yield, including our own protocol using sucrose for myelin removal and accutase for enzymatic digestion. For systematic review, the PubMed was searched for the terms "flow cytometry," "microglia," "brain," and "mice." Three different myelin removal methods (Percoll, sucrose, and no removal) and five protocols for enzymatic digestion (accutase, dispase II, papain, trypsin, and no enzymatic digestion) were tested for the effectiveness of microglia (CD11b+CD45int cell population) isolation from the adult mouse brain using flow cytometry. Qualitative analysis of the 32 selected studies identified three most commonly used myelin removal protocols: Percoll, the use of myelin removal kit, and no removal. Nine enzymatic digestion protocols were identified, from which we selected dispase II, papain, trypsin, and no enzymatic digestion. A comparison of these myelin removal methods and digestion protocols showed the Percoll method to be preferable in removal of non-immune cells, and superior to the use of sucrose which was less effective in removal of non-immune cells, but resulted in a comparable microglial yield to Percoll myelin removal. Digestion with accutase resulted in one of the highest microglial yields, all while having the lowest variance among tested protocols. The proposed protocol for microglia isolation uses Percoll for myelin removal and accutase for enzymatic digestion. All tested protocols had different features, and the choice between them can depend on the individual focus of the research.