3-dimensional morphological analysis of microglia in the sheep brain

Activated microglia change their morphology from small cell bodies with highly ramified branches to become more amoeboid with shorter branches. Accurate morphometric assessment is key to detecting subtle changes in microglia state. Whilst there are many existing methods of analysis, 3-dimensional analysis using Imaris® software is widely used. Despite its widespread use, there is a lack of reporting on what is required from users to ensure accurate and reproducible tracing - including little to no rational for the parameters chosen, and no comparisons between microscope types and magnifications.

We first investigated the possibilities and pitfalls of using Imaris, exploring the effect of microscope type, magnification and different investigators on analysis of microglia from naïve Merino ewes.

Tracings of widefield images were highly variable, whereas tracings of confocal images provided more accurate results. The greatest consistency between analysts was achieved using confocal images taken with a 20x objective; however, the greatest tracing accuracy was achieved using confocal images taken with a 40x objective.

Having established this, changes in microglia morphology were assessed in two groups of sheep; naïve and a group that underwent cardiopulmonary bypass, a procedure known to induce microglial activation. Three-dimensional microglial analysis allowed detection of more markers of activation than previous 2-dimensional analysis.

This paper describes step-by-step instructions, as well as notes of caution at critical points in the analysis workflow, with the aim of helping those new to microglial analysis avoid pitfalls and improve analysis consistency and accuracy.