Ann Rehabil Med.  2016 Oct;40(5):757-768. 10.5535/arm.2016.40.5.757.

A Model of Glial Scarring Analogous to the Environment of a Traumatically Injured Spinal Cord Using Kainate

• Yoo JY
• Hwang CH
• Hong HN

• KMID: 2382907
• DOI: http://doi.org/10.5535/arm.2016.40.5.757

Abstract

OBJECTIVE
To develop an in vitro model analogous to the environment of traumatic spinal cord injury (SCI), the authors evaluated change of astrogliosis following treatments with kainate and/or scratch, and degree of neurite outgrowth after treatment with a kainate inhibitor.
METHODS
Astrocytes were obtained from the rat spinal cord. Then, 99% of the cells were confirmed to be GFAP-positive astrocytes. For chemical injury, the cells were treated with kainate at different concentrations (10, 50 or 100 µM). For mechanical injury, two kinds of uniform scratches were made using a plastic pipette tip by removing strips of cells. For combined injury (S/K), scratch and kainate were provided. Cord neurons from rat embryos were plated onto culture plates immediately after the three kinds of injuries and some cultures were treated with a kainate inhibitor.
RESULTS
Astro-gliosis (glial fibrillary acidic protein [GFAP], vimentin, chondroitin sulfate proteoglycan [CSPG], rho-associated protein kinase [ROCK], and ephrin type-A receptor 4 [EphA4]) was most prominent after treatment with 50 µM kainate and extensive scratch injury in terms of single arm (p<0.001) and in the S/K-induced injury model in view of single or combination (p<0.001). Neurite outgrowth in the seeded spinal cord (β-III tubulin) was the least in the S/K-induced injury model (p<0.001) and this inhibition was reversed by the kainate inhibitor (p<0.001).
CONCLUSION
The current in vitro model combining scratch and kainate induced glial scarring and inhibitory molecules and restricted neurite outgrowth very strongly than either the mechanically or chemically-induced injury model; hence, it may be a useful tool for research on SCI.

Keyword

Spinal cord injuries; In vitro techniques; Neuroglia; Kainic acid

MeSH Terms

Animals
Arm
Astrocytes
Chondroitin Sulfate Proteoglycans
Cicatrix*
Embryonic Structures
In Vitro Techniques
Kainic Acid*
Neurites
Neuroglia
Neurons
Plastics
Protein Kinases
Rats
Spinal Cord Injuries
Spinal Cord*
Vimentin
Chondroitin Sulfate Proteoglycans
Kainic Acid
Plastics
Protein Kinases
Vimentin

Figure

• 

  Fig. 1 Confirmation of depletion of microglia. Immunocytochemical staining of the culture system representing a pure astrocyte. GFAP, glial fibrillary acidic protein.

• 

  Fig. 2 Effects of injury intensity on scratch- and kainate-induced astrogliosis. (A) The optical immune-densities representing astrogliosis were the strongest at 50 µM kainate at 48 post-treatment hours. (B) The optical immune-densities representing astrogliosis were more prominent in the extensive (Ex) scratch injury model than in the moderate (M) scratch injury model. Values are presented as mean±standard deviation. GFAP, glial fibrillary acidic protein; CSPG, chondroitin sulfate proteoglycan; ROCK, rho-associated protein kinase; EphA4, ephrin type-A receptor 4; CON, control. *p<0.001 vs. CON; n=3–6.

• 

  Fig. 3 Effects of single or combined injury on astrogliosis. The optical densities representing glia scarring were the strongest in the combined injury model among the control (CON), chemical (KA), mechanical (S), or combined injury (S/K) models and glial scarring in the S/K-induced injury model was the most intense at post-treatment 48 hours. Values are presented as mean±standard deviation. GFAP, glial fibrillary acidic protein; CSPG, chondroitin sulfate proteoglycan; ROCK, rho-associated protein kinase; KA, kainate; S, scratch; S/K, scratch/kainate. *p<0.001 vs. CON; n=3–6.

• 

  Fig. 4 Effects of kainate on expression of phosphacan (scale bar, 75 µm). Western blot was performed (n=3–6 for immunoblot analysis at each time point in each group). Stained GFAP and phosphacan representing astrogliosis were the largest in the scratch/kainate (S/K)-induced injury model among the control (CON), scratch (S), or S/K-induced injury models. Merged images (Merged) showed double-immunolabeling for GFAP and phosphacan. GFAP, glial fibrillary acidic protein; CNQX, 6-cyano-nitroquinoxaline-2,3-dione.

• 

  Fig. 5 Effects of the kainate inhibitor on neurite outgrowth. Neurite stained with anti-Tuj1 in the scratch/kainate (S/K)-induced injury model was the smallest among the control (CON), scratch (S), or S/K-induced injury models (*p<0.001 vs. S) and this effect was reversed by treatment with a kainate inhibitor (**p<0.001 vs. S/K).

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