4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) Ameliorates Ischemia-Hypoxia-Induced White Matter Damage in Neonatal Rats through Inhibition of the Voltage-Gated Chloride Channel ClC-2

Abstract

Chronic cerebral hypoperfusion is believed to cause white matter lesions (WMLs), leading to cognitive impairment. Previous studies have shown that inflammation and apoptosis of oligodendrocytes (OLs) are involved in the pathogenesis of WMLs, but effective treatments have not been studied. In this study, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a chloride (Cl-) channel blocker, was injected into chronic cerebral ischemia-hypoxia rat models at different time points. Our results showed that DIDS significantly reduced the elevated mRNA levels and protein expression of chloride channel 2 (ClC-2) in neonatal rats induced by ischemia-hypoxia. Meanwhile, DIDS application significantly decreased the concentrations of reactive oxygen species (ROS); and the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha TNF-α in neonatal rats with hypoxic-ischemic damage. Myelin staining was weaker in neonatal rats with hypoxic-ischemic damage compared to normal controls in corpus callosum and other white matter, which was ameliorated by DIDS. Furthermore, the elevated number of caspase-3 and neural/glial antigen 2 (NG-2) double-labeled positive cells was attenuated by DIDS after ischemia anoxic injury. Administration of DIDS soon after injury alleviated damage to OLs much more effectively in white matter. In conclusion, our study suggests that early application of DIDS after ischemia-hypoxia injury may partially protect developing OLs.

Figure 1

(A) ClC-2 mRNA expression changes in cerebral white matter after hypoxic-ischemic injury; (B) Relative expression of corresponding to ClC-2 mRNA compared to sham-operation group. Well 1: sham-operation group, Wells 2–4: 1, 3, 7 days after injury, respectively; Values represent means ± S.E.M. (n = 5). ** p < 0.01.

Figure 2

(A) ClC-2 mRNA relative expression changes in cerebral white matter before and after hypoxic-ischemic injury observed three days after injury; (B) Relative expression of corresponding to ClC-2 mRNA compared to sham-operation group. Well 1: sham-operated group; Well 2: ischemic and hypoxia group; Well 3: administration of DIDS at 1 h after injury; Well 4: administration of DIDS at 6 h after injury; Well 5: administration of DIDS at 2h before injury. Values presented as means ± S.E.M. (n = 5), * p < 0.05; ** p < 0.01 vs. sham-operated group; # p < 0.05; ## p < 0.01 vs. ischemic and hypoxia group.

Figure 3

(A) ClC-2 protein relative expression changes, observed three days after injury, in cerebral white matter after hypoxic-ischemic injury; (B) Quantification of ClC-2 normalized to GAPDH expression; (C) Quantification of caspase-3 normalized to GAPDH expression. Well 1: sham-operated group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Values presented as means ± S.E.M. (n = 5), * p < 0.05; ** p < 0.01 vs. ischemic and hypoxia group.

Figure 4

The effect of DIDS treatment on reactive oxygen species (ROS) in cerebral white matter of each group at different time points after hypoxic-ischemic injury. Well 1: sham-operation group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Well 4: administration of DIDS at 6 h after injury. Well 5: administration of DIDS at 2 h before injury. Values presented as means ± S.E.M. (n = 5), * p < 0.05; ** p < 0.01 vs. sham-operated group; # p < 0.05, ## p < 0.01 vs. ischemic and hypoxia group.

Figure 5

(A) Inflammatory factors iNOS and TNF-α mRNA relative expression changes, observed three days after injury, in cerebral white matter after hypoxic-ischemic injury; (B) Relative expression of corresponding to iNOS and TNF-α mRNA compared to sham-operation group. Well 1: sham-operated group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Well 4: administration of DIDS at 6 h after injury. Well 5: administration of DIDS at 2 h before injury. Values presented as means ± S.E.M. (n = 5), * p < 0.05; ** p < 0.01 vs. sham-operated group.

Figure 6

The effect of DIDS treatment on cell division cycles by fluid cytology after hypoxic-ischemic injury. (A) sham-operated group; (B) ischemic and hypoxia group; (C) administration of DIDS at 1 h after injury; (D) Relative expression of corresponding to G0/G1 period of cell percentage compared to sham-operation group. Well 1: sham-operated group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Values presented as means ± S.E.M. (n = 5), * p < 0.05; ** p < 0.01 vs. sham-operated group.

Figure 7

The changes of myelin development by myelin specific LFB staining three days after hypoxic-ischemic injury. (A,B) sham-operated group; (C,D) ischemic and hypoxia group; (E,F) administration of DIDS at 1 h after injury; (G) Quantification of LFB staining by optical density. Well 1: sham-operated group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Values presented as means ± S.E.M. (n = 5), * p < 0.05; ** p < 0.01 vs. ischemic and hypoxia group.

Figure 8

The effect of DIDS treatment on the number of ClC-2 positive cells three days after hypoxic-ischemic injury. (A,B) sham-operated group; (C,D): ischemic and hypoxia group; (E,F): administration of DIDS at 1h after injury; (G) Quantification of ClC-2 positive cells. Well 1: sham-operated group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Values presented as means ± S.E.M, (n = 5), * p < 0.05; ** p < 0.01 vs. ischemic and hypoxia group.

Figure 9

The effect of DIDS treatment on the number of caspase-3 and NG-2 positive cells 3 days after hypoxic-ischemic injury. (A–C) sham-operated group; (D–F) ischemic and hypoxia group; (G–I) administration of DIDS at 1 h after injury; (J) Quantification of caspase-3 and NG-2 positive cells. Well 1: sham-operation group. Well 2: ischemic and hypoxia group. Well 3: administration of DIDS at 1 h after injury. Values presented as means ± S.E.M. (n = 5), ** p < 0.01 vs. ischemic and hypoxia group.