Semi-Automated Cell and Tissue Analyses Reveal Regionally Specific Morphological Alterations of Immune and Neural Cells in a Porcine Middle Cerebral Artery Occlusion Model of Stroke.

Samantha E Spellicy,Steven L Stice,Brian J Jurgielewicz,Franklin D West,Emily W Baker,Holly A Kinder,Kelly M Scheulin,Janet A Grimes,Elizabeth S Waters

doi:10.3389/fncel.2020.600441

Abstract

Histopathological analysis of cellular changes in the stroked brain provides critical information pertaining to inflammation, cell death, glial scarring, and other dynamic injury and recovery responses. However, commonly used manual approaches are hindered by limitations in speed, accuracy, bias, and the breadth of morphological information that can be obtained. Here, a semi-automated high-content imaging (HCI) and CellProfiler histological analysis method was developed and used in a Yucatan miniature pig permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke to overcome these limitations. Evaluation of 19 morphological parameters in IBA1+ microglia/macrophages, GFAP+ astrocytes, NeuN+ neuronal, FactorVIII+ vascular endothelial, and DCX+ neuroblast cell areas was conducted on porcine brain tissue 4 weeks post pMCAO. Out of 19 morphological parameters assessed in the stroke perilesional and ipsilateral hemisphere regions (38 parameters), a significant change in measured IBA1+ parameters, GFAP+ parameters, NeuN+ parameters, FactorVIII+ parameters, and DCX+ parameters were observed in stroked vs. non-stroked animals. Principal component analysis (PCA) and correlation analyses demonstrated that stroke-induced significant and predictable morphological changes that demonstrated strong relationships between IBA1+, GFAP+, and NeuN+ areas. Ultimately, this unbiased, semi-automated HCI and CellProfiler histopathological analysis approach revealed regional and cell specific morphological signatures of immune and neural cells after stroke in a highly translational porcine model. These identified features can provide information of disease pathogenesis and evolution with high resolution, as well as be used in therapeutic screening applications.

Highlights

Stroke remains a leading cause of death and long-term disability worldwide despite numerous preclinical and clinical trials to develop novel treatments (Virani Salim et al, 2020)
Positive area outlines generated through CellProfiler analysis revealed a visual difference in the size of IBA1+ areas throughout the ipsilateral sections and perilesional regions in S animals compared to NS animals (Figures 2A,B)
There was a significant decrease in ipsilateral hemispheric section IBA1+ area eccentricity (Figure 2H; Median NS = 0.8382px, S = 0.8122px, p < 0.0001) in S animals compared to NS animals

Summary

Introduction

Stroke remains a leading cause of death and long-term disability worldwide despite numerous preclinical and clinical trials to develop novel treatments (Virani Salim et al, 2020). Modern histological approaches have led to significant breakthroughs in understanding thrombus etiology (Sporns Peter et al, 2017; Heo et al, 2020), patterns of immune cell activation (Savchenko et al, 2016; Rayasam et al, 2018), and other important pathological and recovery cellular changes. These standard approaches are limited by the number of cellular features that can be assessed, accompanied by decreased spatial information, slow data processing speeds, and subjectivity. Assessment of micro-scale cellular morphological changes, garnered through HCI analysis of stroked tissues, is likely to result in more predictive studies of neural injury and recovery responses- a critical need in the stroke field (Kaiser and West, 2020)

Methods

Results

Conclusion