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Updated: Jul 14, 2026
Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry
Published on: June 24, 2025
Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical
J Blackbeard1, K P O'Dea, V C J Wallace
1Department of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital Campus, 369 Fulham Road, London SW10 9NH, UK. j.blackbeard@imperial.ac.uk
Abstract:
Microgliosis is implicated in the pathophysiology of several neurological disorders, including neuropathic pain. Consequently, perturbation of microgliosis is a mechanistic and drug development target in neuropathic pain, which highlights the requirement for specific, sensitive and reproducible methods of microgliosis measurement. In this study, we used the spinal microgliosis associated with L5 spinal nerve transection and minocycline-induced attenuation thereof to: (1) evaluate novel software based semi-quantitative image analysis paradigms for the assessment of immunohistochemical images. Microgliosis was revealed by immunoreactivity to OX42. Several image analysis paradigms were assessed and compared to a previously validated subjective categorical rating scale. This comparison revealed that grey scale measurement of the proportion of a defined area of spinal cord occupied by OX42 immunoreactive cells is a robust image analysis paradigm. (2) Develop and validate a flow cytometric approach for quantification of spinal microgliosis. The flow cytometric technique reliably quantified microgliosis in spinal cord cell suspensions, using OX42 and ED9 immunoreactivity to identify microglia. The results suggest that image analysis of immunohistochemical revelation of microgliosis reliably detects the spinal microgliosis in response to peripheral nerve injury and pharmacological attenuation thereof. In addition, flow cytometry provides an alternative approach for quantitative analysis of spinal microgliosis elicited by nerve injury.
Insights
New methods for measuring microgliosis, a key factor in neuropathic pain, were developed. Image analysis and flow cytometry offer robust quantification of microglial activation in spinal nerve injury models.
Area of Science:
- Neuroscience
- Immunology
- Pain Research
Background:
- Microgliosis, the activation of microglia, is central to neuropathic pain pathophysiology.
- Targeting microgliosis is crucial for developing new pain therapeutics.
- Accurate and reproducible methods for measuring microgliosis are essential.
Purpose of the Study:
- To evaluate novel software-based image analysis for assessing spinal microgliosis.
- To develop and validate a flow cytometry approach for quantifying spinal microgliosis.
- To compare these methods against established techniques using a neuropathic pain model.
Main Methods:
- Immunohistochemistry using OX42 to detect microgliosis in spinal nerve transection models.
- Semi-quantitative image analysis including grayscale measurement of OX42 immunoreactivity.
- Flow cytometry utilizing OX42 and ED9 to quantify microglia in spinal cord cell suspensions.
Main Results:
- Grayscale measurement of OX42 immunoreactivity proved a robust image analysis paradigm for microgliosis.
- Flow cytometry reliably quantified spinal microgliosis in cell suspensions.
- Both methods successfully detected microgliosis changes in response to nerve injury and drug treatment.
Conclusions:
- Software-based image analysis of immunohistochemistry is a reliable method for assessing spinal microgliosis.
- Flow cytometry offers a validated alternative for quantitative analysis of nerve injury-induced microgliosis.
- These advancements support mechanistic studies and drug development for neuropathic pain.
