GFAP expression in the BRAIN during human postnatal development.

Glial fibrillary acidic protein (GFAP) immunohistochemistry was investigated in the developing human brain using two measures; the number of GFAP-positive cells (density, GFAP+/mm2), and a reactivity score (R-score), which we recently introduced to indicate astrogliosis, with scores ≥120 indicative of pathological processes. The primary aim was to report on GFAP expression and cell soma size in 26 microscopically defined regions of the amygdala, basal ganglia, cerebellum, hippocampus and medulla, and to determine whether they are affected by postconceptional age (PCA) from 40 to 83 weeks. The secondary aim was to determine if GFAP expression differs according to the classification of sudden infant death syndrome (SIDS) as opposed to infant deaths of known causes, or for the presence of major SIDS risk factors of male sex, cigarette smoke exposure, upper respiratory tract infection (URTI), bed-sharing and prone sleeping. The cerebellar molecular layer was void of GFAP+ cells, while the internal granular layer (IGL) had the highest density, with >60% of infants having an R-Score >120. GFAP expression decreased with increasing PCA in the entorhinal and temporal cortex, subiculum and regions of the cerebellum and medulla. GFAP cell soma size corresponded with astrogliosis score and no effect of PCA was evident. Various region-dependent GFAP expressional differences were seen according to SIDS classification and the risk factors studied. The findings indicate that the density of GFAP decreases in specific regions of the brain within the first year of postnatal development, and that reactive astrocytes are common, particularly within the early postnatal months.

Quantifying GFAP immunohistochemistry in the brain - Introduction of the Reactivity score (R-score) and how it compares to other methodologies.

BACKGROUND: Immunohistochemical upregulation of glial fibrillary acidic protein (GFAP) is commonly used to detect astrogliosis in tissue sections and includes measurement of intensity and/or distribution of staining. There remains a lack of standard objective measures when diagnosing astrogliosis and its severity. NEW METHOD: Aim was to test a novel semi-quantitative assessment of GFAP which we term reactivity (R)-score, on its reproducibility and sensitivity to measure astrogliosis. The R-score, which is based on the proportion of astrocytes seen at each level of reactivity, was compared to 3 other commonly employed quantification methods in research: (1) thresholding, (2) point-counting, and (3) qualitative grading. Sub-regions of the hippocampus, medulla, and cerebellum were studied in piglet, and 4 human cases with clinically reported astrogliosis. Intra-assay coefficient of variation (CV) and percentage agreement cut-offs of ≤ 20% and ≥ 75% were used respectively to compare amongst the methods, with outcome measures being reproducibility across serial and non-serial sections, resilience to changes in experimental conditions, and inter- and intra-rater concordance. RESULTS: Averaged across 3 brain regions, the intra-assay coefficient of variation (CV) was 5% for R-score, with inter and intra-rater kappa scores being 0.99 and 0.95 respectively. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: Based on CV values, the R-score was superior to thresholding (CV of 51%) and point-counting (CV of 16%), with the qualitative grade being found to be on par (percentage agreement 95%). Given the ease, reproducibility and selectivity of the R-score, we propose its validity in future research purposes and clinical application.

Immunostaining for NeuN Does Not Show all Mature and Healthy Neurons in the Human and Pig Brain: Focus on the Hippocampus.

Neuronal nuclei (NeuN) is a neuron-specific nuclear protein, reported to be stably expressed in most postmitotic neurons of the vertebrate nervous system. Reduced staining has been interpreted by some to indicate loss of cell viability in human studies, while others suggest this may be because of changes in the antigenicity of the target epitope. Preliminary studies in our laboratory found low immunostaining for the NeuN antibody on formalin fixed and paraffin embedded (FFPE) human brain tissue. We report on the techniques and results used to enhance the staining for NeuN in that tissue. In parallel, we stained NeuN in piglet brain tissue, sourced from an experimental model where methodological parameters, including those for tissue fixation and storage, were tightly controlled. In human FFPE brain tissue, we were unable to enhance NeuN immunostaining to a degree sufficient for cell counting. In contrast, we found consistently high levels of staining in the piglet brain tissue. We conclude that processes used for fixation and storage of human FFPE brain tissue are responsible for the reduced staining. These results emphasize that a cautionary approach should be taken when interpreting NeuN staining outcomes in human FFPE brain tissue.