Rat optic nerve oligodendrocyte culture experiments

Summary

Rat optic nerve oligodendrocyte culture can (1) obtain rat optic nerve oligodendrocytes; (2) be used for optic nerve injury repair studies; (3) be used for oligodendrocyte-related topics.

Operation method

bovine serum culture method

Principle

Optic nerve, DMEM/F12 conditioned medium was used for culture, cell morphology and growth were observed, and immunohistochemical identification was performed.

Materials and Instruments

Wistar Rat
Sodium selenite, putrescine, transferrin, insulin, thyroxine, luteinizing hormone, glutamine, calf serum, rabbit anti-mouse GC antibody.
Ophthalmic Clipper Drip Tube Centrifuge Petri Dish CO2 Incubator

Move

I. Experimental Procedure

1. 10 newborn Wistar rats were taken at 2 days of age, and the optic nerves were removed bilaterally under aseptic conditions.2. Inoculate onto polylysine-treated coverslips in 24-well culture plates.3. Add DMEM/F12 medium containing ^30g-L-1 calf serum, and incubate continuously for 3 d at 37℃, 50mL/L _CO2, and 100% humidity.4. After 3 d, discard the waste solution and replace it with DMEM/F12 medium containing ^5g-L-1 calf serum.5. Change the solution twice a week. After obtaining a sufficient number of cells, the culture medium was changed to serum-free conditioned medium, and the medium was changed once every 2 d.
Morphological observation

The growth process and morphological changes of cultured cells were observed and photographed under an inverted microscope every day.

Immunocytochemical identification

1. Remove the cell-containing coverslips and rinse with 0.01 ^mol-L-1 PBS for 3 times x 5 min. 2.

2. Cold acetone fixation for 10 min. 3.

3. Rinse with PBS (3 times x 5 min). 4.

4. ^30g-L-1 hydrogen peroxide incubation at room temperature for 15 min. 5.

5. PBS rinse 3 times x 5 min. 6.

6. Normal goat serum was added dropwise and incubated at room temperature for 20 min. 7.

7. 1:100 GC antibody was added dropwise, negative control was incubated with PBS instead of primary antibody at 37℃ for 60 min. 8.

8. Rinse with PBS 3 times x 5 min. 9.

9. Dropwise addition of biotin-labeled sheep anti-rabbit IgG, 37℃ for 20 min. 10.

10. Wash with PBS 3 times x 5 min. 11.

11. Dropwise addition of horseradish enzyme labeled streptavidin working solution. 12.

12. DAB working solution develops the color and the reaction is terminated with tap water. 13.

13. Dewater, clear, DPX seal and store.
Results

1. Morphological observation
Tissue blocks began to adhere to the wall at 24 h, and a small number of cells could be seen emerging from the edge of the nerve tissue at 48-72 h, which were mainly round or spindle-shaped (Fig. 1). 8-9 days later, the cells basically filled up the culture wells. At this time, the culture medium was changed to serum-free conditioned medium for purification. Some of the cells died during the culture process, and the cytosol of the cells obtained at about 12 d was basically round or polygonal, with a diameter of about 6-10 μm, a large nucleus, and little cytoplasm (Fig. 2).
2. Immunohistochemical staining results
Immunohistochemical staining of cultured optic nerve oligodendrocytes was positive for GC protein and negative without primary antibody. The staining results showed that mature oligodendrocytes were rich in protrusions and formed a spider web with each other (Fig. 3). Hematoxylin restaining showed fewer heterogeneous cells and more than 90% were positive (Fig. 4).

Figure 1 Cells migrated from optic nerve tissue at the third day (10x10)Figure 1 Cells migrated from optic nerve tissue at the third day (10x10).


Figure 2 Purified oligodendrocytes of optic nerve at the fifteenth day (10x40)Figure 2. Purified oligodendrocytes of optic nerve at the fifteenth day of culture (10x40)


Figure 3 Positive expression of GC in oligodendrocytes at the seventh day (10x20)Figure 3 Positive expression of GC in oligodendrocytes at the seventh day of culture (10x20)



Figure 4 Positive expression of GC in purified oligodendrocytes at the fifteenth day (hematoxylin staining) (10x20)Figure 4 Positive expression of GC in purified oligodendrocytes at the fifteenth day (hematoxylin staining) (10x20)

Caveat

1. In vitro studies have found that thyroxine, insulin, transferrin, putrescine, luteinizing hormone, and the trace element selenium, among others, can cause O2A progenitor cells to differentiate toward oligodendrocytes.

2. and serum, etc. can induce O2A progenitor cells to differentiate into type II astrocytes. Gradually reduce the serum concentration in the conditioned medium to promote the differentiation of O2A progenitor cells toward oligodendrocytes.

Common Problems

The results of immunostaining showed that the purity was higher than 90%. This method is simple, reliable, and easy to popularize, which is convenient for the research of oligodendrocyte-related topics.

I. DISCUSSION

The discovery of Nogo, a gene that inhibits nerve regeneration, has brought hope for the repair of optic nerve injury and the protection of visual function. Optic nerve glial cells include astrocytes and oligodendrocytes, which are differentiated and have different functions. Mature oligodendrocytes no longer have the ability to divide and proliferate, and are terminal cells ^{[1], which} are difficult to culture.

In 1980, McCarthy ^{[2] and others} established a model for separating and purifying astrocytes and oligodendrocytes from gray matter tissue of the brain. At present, this method is mostly used at home and abroad ^[3]. The separation and culture of oligodendrocytes and astrocytes were carried out using the vibration separation method by utilizing the difference in wall adherence ability of oligodendrocytes and astrocytes. This method is mainly used to obtain astrocytes, and fewer oligodendrocytes are obtained. There are shortcomings such as more operation steps are easy to be contaminated, easy to cause mechanical damage due to oscillation centrifugation during the separation process leading to cell death.

In this experiment, we used the optic nerve block culture method to conditioned primary culture of optic nerve glial cells from neonatal rats, and used conditioned medium to purify oligodendrocytes and astrocytes by taking advantage of the different growth conditions of the two types of cells.

Oligodendrocytes and type II astrocytes develop from a common bipotential oligodendrocyte-type 2 astrocyte progenitor ^[1].

Oligodendrocytes mature approximately 1 week after birth in rats.

Therefore, O2A progenitor cells can be obtained from neonatal rats.

In vitro studies have shown that thyroxine, insulin, transferrin, putrescine, luteinizing hormone, and the micronutrient selenium can direct the differentiation of O2A progenitor cells toward oligodendrocytes.

And serum and others can induce the differentiation of O2A progenitor cells into type II astrocytes ^{[1, 4].}

We took advantage of this feature and gradually reduced the serum concentration in the conditioned medium to promote the differentiation of O2A progenitor cells toward oligodendrocytes.

Eventually, serum-free conditioned medium was used to inhibit the proliferation of astrocytes and other heterogeneous cells, whereas oligodendrocytes were largely unaffected under this condition.

GC is a major lipid expressed in oligodendrocyte membranes as well as myelin sheaths, and it is a universally applied specific chemical marker for recognizing oligodendrocytes ^{[1, 3].}

Immunostaining results showed a high purity of more than 90%. This method is simple, reliable, and easy to generalize, which is convenient for the research of oligodendrocyte-related topics.

Literature

1. He P, Shen XY. Progress of oligodendrocyte proliferation and differentiation[J],Journal of Neuroanatomy,2000,16(2):183-188.

2. McCarthy K D, DeVellis J. Preparation of separate astroglial and oligodendroglia cell cultures from rat cerebral tissue [J], J Cell Biol 1980,85: 890- 902.

3. WU Yamin, MA Haihan, LIAO Weihong. Purification and characterization of rat astrocytes and oligodendrocytes [J], Trauma Surgery 2000,4: 207-209.

4. Raff MC, Miller RH, Noble M. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium [J], Nature 1983,303(591616) Nature 1983,303(5916): 390-6.

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