Neuroscience
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A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice
Chapters
Summary May 11th, 2022
The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.
Transcript
Our model is highly relevant to demyelination diseases, especially multiple sclerosis. And it can contribute to the related advancing clinically relevant research. It's a detailed protocol that could directly cause severe demyelination quickly and stably in mice by surgical procedure.
While trying this technique for the first time, be patient, and ensure that coordinates are accurate. Begin by connecting a 32 gauge needle to a 5 microliter syringe. Withdraw 5 microliters of LPC solution for the preparation of the injection.
Next, confirm the depth of anesthesia of the mouse by the lack of toe pinch reflex while the breathing is smooth. Position the mouse in the stereotaxic frame with the dorsal side up, and secure the head with a nose cone and tooth clamp. Fix the mouse to the stereotaxic apparatus with bilateral ear bars.
Ensure that the ear bars are level and the head is horizontal and stable. Disinfect the head skin by wiping with iodophor. Then use scalpel to cut a small incision to about 1.5 centimeters along the midline of the scalp to expose the skull.
Wipe the skill with a cotton swab dipped in 1%hydrogen peroxide until the bregma, lambda, and posterior fontanelle are exposed. Place the syringe on the stereotaxic apparatus. Ensure the horizontal positioning of the animal's head, and locate the corpus callosum.
Once the injection site is determined, record the coordinates, and make a mark with a marker on the skull. Gently drill the marked site with a skull drill. Take care to avoid any bleeding.
Slowly move the needle to the given coordinates. To induce corpus callosum demyelination, inject 2 microliters of LPC solution at each injection site at a rate of 0.4 microliters per minute. After injection, keep the needle in each site for an additional 10 minutes.
Finally, suture the skin with a 4-0 suture, and wait for the animal to wake up within 10 minutes. The representative image shows the detection of the white matter legions via Luxol fast blue staining. From the results, stable demyelination of the corpus callosum can be seen at 10 and 28 days post injection.
The quantification of the demyelination area at different time points is shown here. Through immunofluorescence staining degraded myelin basic protein, a remarkable increase of dMBP was observed in the LPC-injected group at 10 days post injection, which represents myelin loss in the corpus callosum. Western blot analysis of the myelin basic protein, or MBP, expression showed a significant loss of MBP after injection of LPC.
Beta-actin was used as the loading control to measure the baseline expression. Electron microscopy images confirm the myelinated ultrastructure in the corpus callosum of sham group and LPC-injected group. GST-pi is the marker of oligodendrocyte precursor cell, or OPC, differentiation maturation.
After 10 days of LPC injection, the GST-pi decreased compared with the sham group. At the same time, the proliferation ability of oligodendrocytes can be reflected by the ratio of oligodendrocytes proliferate and the total oligodendrocyte lineage cells. The higher ratio represents more proliferation of oligodendrocytes after 10 days post injection.
The representative images show the mice's swimming path in each group, in the platform's presence and after removing the platform. The results suggest that the spatial memory of demyelinated mice is significantly impaired in the two-point LPC injection model. While attempting this procedure, make sure that the injection rate is accurate, and keep the needle in each site for an additional 10 minutes.
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