Korean J Physiol Pharmacol.  2022 Jan;26(1):47-57. 10.4196/kjpp.2022.26.1.47.

Therapeutic effects of stiripentol against ischemia-reperfusion injury in gerbils focusing on cognitive deficit, neuronal death, astrocyte damage and blood brain barrier leakage in the hippocampus

• Shin MC
• Lee TK
• Lee JC
• Kim HI
• Park CW
• Cho JH
• Kim DW
• Ahn JH
• Won MH
• Lee CH

• KMID: 2524530
• DOI: http://doi.org/10.4196/kjpp.2022.26.1.47

Abstract

Stiripentol is an anti-epileptic drug for the treating of refractory status epilepticus. It has been reported that stiripentol can attenuate seizure severity and reduce seizure-induced neuronal damage in animal models of epilepsy. The objective of the present study was to investigate effects of post-treatment with stiripentol on cognitive deficit and neuronal damage in the cornu ammonis 1 (CA1) region of the hippocampus proper following transient ischemia in the forebrain of gerbils. To evaluate ischemia-induced cognitive impairments, passive avoidance test and 8-arm radial maze test were performed. It was found that post-treatment with stiripentol at 20 mg/kg, but not 10 or 15 mg/kg, reduced ischemia-induced memory impairment. Transient ischemia-induced neuronal death in the CA1 region was also significantly attenuated only by 20 mg/kg stiripentol treatment after transient ischemia. In addition, 20 mg/kg stiripentol treatment significantly decreased ischemia-induced astrocyte damage and immunoglobulin G leakage. In brief, stiripentol treatment after transient ischemia ameliorated transient ischemia-induced cognitive impairment in gerbils, showing that pyramidal neurons were protected and astrocyte damage and blood brain barrier leakage were significantly attenuated in the hippocampus. Results of this study suggest stiripentol can be developed as a candidate of therapeutic drug for ischemic stroke.

Keyword

Blood-brain barrier; Brain ischemia; Hippocampus; Neuroprotection; Stiripentol

Figure

• 

  Fig. 1 Experimental timeline. Vehicle (saline) and STP (10, 15, and 20 mg/kg) are treated 30 min after TFI. 8-ARMT is conducted at 3, 2, and 1 day before TFI, and 1, 2, 3, 4, and 5 days after TFI. PAT is carried out at 1 day before, and 5 days after TFI. The animals are sacrificed at 6 h, 2 and 5 days after TFI respectively, and histologically analyzed. STP, stiripentol; TFI, transient forebrain ischemia; 8-ARMT, 8-arm radial maze test; PAT, passive avoidance test.

• 

  Fig. 2 Latency time in PAT (A) and mean numbers of errors in 8-ARMT (B) in the Sham-vehicle, Sham-STP (10, 15, and 20 mg/kg), TFI-vehicle and TFI-STP (10, 15, and 20 mg/kg) groups before and after TFI. The substantial trials for PAT are conducted one day before and five days after TFI and gerbils are undergone pre-training trial at 20 min before each substantial trial. In the TFI-20 mg/kg STP group, a significantly delayed latency time in PAT is recorded compared with that in the TFI-vehicle, TFI-10 mg/kg STP and TFI-15 mg/kg STP groups. 8-ARMT is daily performed at three to one day before TFI, and one to five days after TFI. In the TFI-20 mg/kg STP group, the numbers of the errors in 8-ARMT are significantly decreased from two days after TFI. The bars indicate the means ± SEM (n = 7, respectively; *p < 0.05 vs. Sham-vehicle or Sham-STP group; #p < 0.05 vs. TFI-vehicle group). 8-ARMT, 8-arm radial maze test; PAT, passive avoidance test; STP, stiripentol; TFI, transient forebrain ischemia.

• 

  Fig. 3 (A–H and a–h) NeuN immunohistochemistry (A–H) and F-J B histofluorescence (a–h) in the CA1 region of the Sham-vehicle (A and a), TFI-vehicle (B and b), Sham-STP (10, 15, and 20 mg/kg) (C, c, E, e, G, and g) and TFI-STP (10, 15, and 20 mg/kg) (D, d, F, f, H, and h) groups five days after TFI. In the TFI-vehicle group, NeuN+ cells are rarely found, and F-J B+ cells are abundant in the stratum pyramidale (SP). However, in the TFI-20 mg/kg STP group, NeuN+ cells are abundant and F-J B+ cells are very few (arrows) in the SP. Scale bars = 100 μm. (I and i) The mean numbers of NeuN+ cells (I) and F-J B+ cells (i). The bars indicate the means ± SEM (n = 5 or 7, respectively; *p < 0.05 vs. Sham-vehicle or Sham-STP group; #p < 0.05 vs. TFI-vehicle group). NeuN, neuronal nuclei; F-J B, Fluoro-Jade B; CA1, cornu ammonis 1; TFI, transient forebrain ischemia; STP, stiripentol; SO, stratum oriens; SR, stratum radiatum.

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  Fig. 4 (A) GFAP immunohistochemistry in the CA1 region of the Sham-vehicle (a), TFI-vehicle (b–d), Sham-20 mg/kg STP (e) and TFI-20 mg/kg STP (f–h) groups six hours (b and f), two days (c and g) and five days (d and h) after TFI. In the TFI-vehicle group, GFAP+ astrocytes become gradually hypertrophied after TFI, showing that the ends of the astrocytes are blunt (arrow) five days after TFI. In contrast, the hypertrophy of GFAP+ astrocytes is apparently attenuated in the TFI-20 mg/kg STP group. Scale bars = 100 μm. (B) ROD of GFAP immunoreactive structures. The bars indicate the means ± SEM (n = 5 or 7, respectively; *p < 0.05 vs. Sham-vehicle or Sham-STP group; #p < 0.05 vs. TFI-vehicle group). GFAP, glial fibrillary acidic protein; CA1, cornu ammonis 1; TFI, transient forebrain ischemia; STP, stiripentol; ROD, relative optical density; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

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  Fig. 5 Double immunohistofluorescence for GFAP (green) and GLUT-1 (red) in the CA1 region of the Sham-vehicle (A), TFI-vehicle (B–D), Sham-20 mg/kg STP (E) and TFI-20 mg/kg STP (F–H) groups six hours (B and F), two days (C and G) and five days (D and H) after TFI. GFAP+ AEF (arrows in A) are contact with GLUT-1+ endothelial cells in the Sham-vehicle group; however, in the TFI-vehicle group, AEF disappear gradually (arrows in B–D). In contrast, GFAP+ AEF (arrows in F–H) are well co-localized with GLUT-1+ endothelial cells in the TFI-20 mg/kg STP group. Scale bars = 50 μm. GFAP, glial fibrillary acidic protein; GLUT-1, glucose transporter 1; CA1, cornu ammonis 1; TFI, transient forebrain ischemia; STP, stiripentol; AEF, astrocyte endfeet; SP, stratum pyramidale; SR, stratum radiatum.

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  Fig. 6 (A) IgG immunohistochemistry in the CA1 region of the Sham-vehicle (a), TFI-vehicle (b–d), Sham-20 mg/kg STP (e) and TFI-20 mg/kg STP (f–h) groups six hours (b and f), two days (c and g) and five days (d and h) after TFI. In the Sham-vehicle-group, IgG immunoreactivity is shown only within blood vessels (arrow). In the TFI-vehicle-group, IgG immunoreactivity is increased in cellular components (arrowheads) and parenchyma from six hours after TFI. In contrast, IgG immunoreactivity in the TFI-20 mg/kg STP group is apparently decreased. Scale bars = 100 μm. (B) ROD of IgG immunoreactive structures. The bars indicate the means ± SEM (n = 5 or 7, respectively; *p < 0.05 vs. Sham-vehicle or Sham-STP group; #p < 0.05 vs. TFI-vehicle group). IgG, immunoglobulin G; CA1, cornu ammonis 1; TFI, transient forebrain ischemia; STP, stiripentol; ROD, relative optical density; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

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