Culturing Microglia from the Neonatal and Adult Central Nervous System

Summary

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Abstract

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

Introduction

Microglia are the resident immune cells of the CNS, most closely resembling peripheral macrophages in structure and function ¹. It has recently been demonstrated that postnatal microglial cells derive from primitive myeloid progenitors and are generated before the eighth day of embryogenesis, upending the previous notion that postnatal hematopoietic progenitors are the source of microglia in the adult brain ². They play key roles in several neurological diseases and can quickly respond to infection or injury by releasing pro-inflammatory or anti-inflammatory cytokines ³. Thus microglia encompass a standalone unit in the CNS that can be....

Protocol

1. Dissection (Day 0)

1. Induce anesthesia to p0-2 mouse pups with hypothermia. Wipe the heads of the pups with a Kimwipe soaked in 70% ethanol to disinfect the tissue.
2. Remove the heads with a pair of scissors, using 4 pups per 10 cm culture plate. Place the heads in a petri dish containing ice-cold Hank's buffer.
3. Anchor the heads using curved forceps through the eye sockets and carefully remove the skin covering the skull with straight microforceps.
4. Remove the cranial bones with straight microforceps, using caution to not puncture or damage the cortices. The most effective way is to begin the removal starting near the cerebellum....

Results

An example of resting and activated microglial cells is shown in Figure 1. The microglia were visualized 24 hr after plating (Figure 1a) and exhibit ramified (resting) morphology. Exposure to the priming reagent, bacterial lipopolysaccharide (LPS) results in changes in microglial morphology as the cells become activated (Figure 1b).

An example of counting the cells for plating is shown in Figure 2. Due to the presence of cell .......

Discussion

Microglia modulate CNS normal functioning as well as inflammatory responses to various pathologies. Functional synaptic remodeling by microglia has been implicated in the maintenance of normal brain homeostasis ¹⁵. During the neurogenic cascade they participate in the clearance of neural progenitor cells from the dentate gyrus of the hippocampus ^{4, 16}. Therefore, it is necessary to develop a culture system in which to study neonatal and adult microglia, which will cover the vast swath of deve.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EDTA, 5mM	Invitrogen	15567-028
Lidocaine, 60mM	Sigma	L-5647
Trypan Blue	Sigma	T8154
Trypsin/EDTA	Cellgro	25-052-CI
DMEM, 1X	Cellgro	10-017
Sodium Pyruvate	Cellgro	25-000-Cl
Gentamycin Sulfate	Biowittaker	17-518Z
35 x 10mm tissue culture dish	Falcon	353001
Poly-D-Lysine, 100 μg/ml			Dilute 1:20
HBSS, 1X	Cellgro	20-023-CV