Stereotaxic Injection of Lysophosphatidylcholine Into Mouse Corpus Callosum for Establishment of a Focal Demyelination Model

Background

Demyelinating diseases such as multiple sclerosis (MS) involve damage to the myelin sheath surrounding axons in the central nervous system. Focal demyelination models allow investigators to study the cellular and molecular mechanisms of demyelination and remyelination in a spatiotemporally controlled manner. Current animal models for studying demyelination and remyelination fall mainly into three categories: the experimental autoimmune encephalomyelitis (EAE) model, the cuprizone model, and focal toxin-injection models [such as lysophosphatidylcholine (LPC) and ethidium bromide (EB) injection]. Because no single animal model recapitulates all features of human demyelinating disease, the choice of model should depend on the biological question being addressed. Experimental autoimmune encephalomyelitis (EAE) is suitable for studying autoimmune inflammation, but its lesions are temporally asynchronous, and spatial control is limited. Cuprizone intoxication produces pronounced corpus callosum demyelination after several weeks of systemic feeding, but the lesions are diffuse rather than experimentally targeted. Ethidium bromide can produce focal lesions but is broadly cytotoxic, damaging astrocytes and oligodendrocyte lineage cells in addition to myelin. By contrast, LPC is a membrane-disrupting lysophospholipid that produces spatially confined lesions, relatively spares axons, and exhibits a predictable demyelination–spontaneous remyelination sequence [1–9]. The LPC injection model is widely used because it induces focal demyelination without damaging axons, making it an ideal system for studying remyelination. Stereotaxic injection of LPC into the corpus callosum targets an anatomically well-defined and functionally important white matter tract, which is highly suitable for studying the recruitment and differentiation of oligodendrocyte progenitor cells. Compared with systemic demyelination models (such as the cuprizone model), this protocol allows precise control of lesion location and size, enables paired comparisons with the contralateral hemisphere, and facilitates the correlation of lesions with specific behavioral deficits [10–12]. Based on these advantages, this protocol describes in detail a standardized procedure for establishing a focal demyelination model by stereotaxic injection of LPC into the mouse corpus callosum and provides a complete histological validation method, with the aim of offering researchers in this field—especially beginners—a reproducible and easy-to-perform experimental reference (Table 1).

Table 1. Comparison of commonly used demyelination models

The LPC paradigm is not restricted to the corpus callosum. Focal LPC lesions have been established in spinal cord white matter, the caudal cerebellar peduncle, the internal capsule, the optic nerve, and the optic chiasm [5,13–19]. Therefore, this method is broadly applicable to region-specific questions of myelin injury and repair. However, coordinates, injection volumes, needle gauges, and tissue collection endpoints must be re-optimized for each target structure and species.

In the corpus callosum, demyelination can be detected within the first few days after LPC injection, typically peaking at around 3–7 dpi, followed by recruitment and differentiation of oligodendrocyte progenitor cells and remyelination at approximately 14–21 dpi. The exact time course depends on species, strain, age, injection volume, concentration, and target site [4,12,14,19,20]. Published volumetric analyses using defined mouse corpus callosum protocols report demyelination volumes of approximately 0.2–0.3 mm³ at 4 dpi and 0.05–0.15 mm³ at 14 dpi; these values are protocol-specific and should not be regarded as universal constants [16].

Adult C57BL/6 mice aged 8–10 weeks are commonly used because of their robust baseline remyelination capacity and well-defined time course. Aged mice may also be used when the experimental question concerns age-dependent repair; however, remyelination in aged animals is slower and less efficient, so age-matched controls should be included and tissue collection times adjusted to account for the slower remyelination in aged animals [15,21–23].

The target coordinates used in this protocol—AP +1.10 mm, ML +0.95 mm, DV -2.15 mm from the skull surface—are designed to place the injectate within the corpus callosum at the level of the fornix, dorsal to the dorsal hippocampus, while avoiding the superior sagittal sinus. If bilateral lesions are required, the procedure is repeated at ML -0.95 mm. Coordinates should be verified against the Paxinos and Franklin mouse brain atlas and adjusted according to atlas edition, animal age, body weight, skull geometry, and local instrument calibration [11,12,16,20,32]. The coordinates in this protocol were determined with reference to The Mouse Brain in Stereotaxic Coordinates by Paxinos and Franklin [32] and cross-validated using the Allen Mouse Brain Common Coordinate Framework 3D digital atlas. The AP +1.10 mm plane lies at the level of the frontal/motor cortex, where the corpus callosum has developed into a broad, thick, horizontally oriented, well-demarcated dense white matter tract with a large target cross-sectional area and good tolerance to targeting error; moreover, this plane is rostral to the hippocampus, so vertical needle insertion passes only through the motor/somatosensory cortex without traversing deep nuclei such as the hippocampus or striatum, thereby minimizing needle track damage. ML +0.95 mm corresponds to the lateral segment of the corpus callosum body (approximately dorsal to the lateral ventricle), the region where the white matter tract extends most widely laterally, ensuring adequate diffusion of the injectate within the callosal fibers. DV -2.15 mm (from the skull surface) is set according to the atlas, which shows the corpus callosum at this plane lying approximately 1.3–2.1 mm below the skull surface; placing the needle tip slightly deeper than its ventral border, combined with slow, low-flow-rate injection, allows the injectate bolus to spread within the corpus callosum and reflux mildly along the needle track, covering the full thickness of the corpus callosum while avoiding excessively deep penetration into the lateral ventricle that would cause injectate loss. The reasons for not injecting at the midline are as follows: first, the superior sagittal sinus runs along the sagittal suture directly beneath the midline, and drilling or needle insertion at the midline readily injures this venous sinus, causing difficult-to-control hemorrhage and markedly increasing surgical failure and animal mortality; a lateral offset of approximately 1 mm completely avoids the sinus body and its major bridging veins. Second, the corpus callosum at the midline is a narrow structure connecting the two cerebral hemispheres, and midline needle insertion would simultaneously damage the bilateral cingulate cortex and the corpus callosum on both sides of the midline, resulting in an uncontrollable extent of injury [11,20].