Moreover, high glucose concentration was reported to induce apoptosis of hippocampal neurons via inhibiting the phosphorylation of Akt (p-Akt). It also regulated cellular survival 30, proliferation 31, 32, differentiation 32 and apoptosis 33. Previous studies have demonstrated that PI3K-Akt signal pathway played a crucial role in central nervous system (CNS) myelination 26 - 29. In our study, we made a research on whether abnormal ClC-2 activation was associated with GDM in the early stage of white matter development and whether OPCs/Pre-OLs injury or white matter damage involved GDM. Moreover, serum and glucocorticoid inducible kinases (SGK1), which is up-regulated by high glucose concentration 22, 23, can enhance ClC-2 expression in plasma membrane in vitro 24, 25. Study on white matter of neonatal rats also showed that ischemia-hypoxia elevated ClC-2 activation which initiated apoptosis of OLs 10. Previous studies have demonstrated that ischemia and hypoxia induced the excessive opening of Cl - channels, which was involved in the apoptosis-induced volume decrease of the myocardial cells 20, 21. It is also connected with the regulation of cellular volume, proliferation and migration 18, 19. Type 2 voltage-gated chloride channels (ClC-2) is a member of the Cl - channel family. However, in the early development stage for the white matter, whether high glucose can induce OPCs/Pre-OLs injury and affected myelination development still remains unclear. Besides, Yu, J et al 17 demonstrated the involvement of oxidative stress in NTDs under maternal diabetic in vivo or high glucose in vitro. In diabetic neuropathy, the apoptosis of rat primary dorsal root ganglion neurons is associated with generation of a variety of reactive oxygen species (ROS) 16. In addition, high blood sugar concentration can induce cell apoptosis by increasing oxidative stress response 14 and glycosylation level 15. which led to OPCs/Pre-OLs injury and myelination disturbance 13. Numerous studies have shown that during the period when OPCs differentiate into early OLs, immature OLs were vulnerable to injury factors, such as hypoxia-ischemia 10, toxicity 11, oxidative stress 12 etc. This process usually occurs in the third trimester of pregnancy and even last after birth 9. In early stage of myelination, oligodendrocyte progenitor cells(OPCs) differentiate into OLs and form myelin sheath 8. Most OLs are located in white matter regions 7. In addition, study in neuropathological showed that hyperglycemia is connected with oligodendrocytes (OLs) injury 6. Clinical studies have provided evidence that high glucose level could affect the development of fetus' nervous system and result in a high possibility of neural tube defects (NTDs) 4, 5. Patients develop high blood sugar concentration which can do harm to the intrauterine fetus.
Gestational diabetes mellitus (GDM) refers to any degree of glucose intolerance with onset or first recognition during pregnancy 1 - 3. Application of ClC-2 inhibitor DIDS showed protective effects on cerebral white matter damage stimulated by high glucose concentration. The effect was caused by repressing PI3K-Akt signaling pathway.
In conclusion, ClC-2 and caspase-3 were induced by GDM, which resulted in apoptosis and myelination inhibition. While DIDS (a chloride channel blocker) can reverse these changes. Caspase-3 was also increased in those white matter regions in GDM group, but p-Akt expression was inhibited. However, PDGFα positive cell number at P0 stage and CNPase expression at P3 stage were significantly decreased.
In GDM group, ClC-2 expression in the corpus callosum (CC) and cingulate gyrus (CG) regains, and TUNEL positive cell number were increased at P0 and P3 stage. Furthermore, the expression level of myelin transcription factor Olig2 at P0 stage and CNPase at P3 stage were strikingly lower than that of the control group. Meanwhile, In GDM group, reactive oxygen species (ROS) levels of the white matter at E18, P0, and P3 stage were significantly higher than control group. Our results showed that ClC-2 mRNA and protein expressions in GDM group were significantly increased in white matter of fetal rats after E18 stage, and elevated the level of TNF-α and iNOS in white matter at P0 and P3 stage of newborn rats. The alteration of ClC-2 expression in various developmental stages of cerebral white matter with/without being exposed to high glucose was analyzed using RT-PCR, active oxygen detection, TUNEL staining, Western Blot as well as immuno-histochemical staining. In this study, GDM model was induced in late pregnant rat model. This study aims to investigate the effect and mechanism of type 2 voltage-gated chloride channel (ClC-2) on myelin development of newborn rats' cerebral white matter with gestational diabetes mellitus (GDM).
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