Figure 1.
Some instructions for the methods.
(A) Experimental design. Rb fraction (Rb) was administrated intraperitoneally 40 min before intracerebroventricular (icv) KA injection. Rats were sacrificed at 48 h (acute period) or 3 weeks (chronic period) after KA for histological assay. The Morris water maze task was conducted from the 16th to 19th day for the training test and the 20th day for the probe trial after KA injection. (B) Division of the dorsal hippocampus. The CA2 subfield is small and difficult to separate from the CA1, and thus was included in the CA1 subfield. so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare; DGub, dentate gyrus upper blade; DGlb, dentate lower blade; gcl, granule cell layer; ml, molecular layer; hl, hilus.
Figure 2.
Seizure score and body weight loss after KA administration.
Seizure score and body weight loss at 24-induced acute hippocampal damage (A) and in the animals used to determine delayed neurodegeneration during chronic period (B) after KA injection. Data are expressed as means ± SEM and analyzed by a one-way analysis of variance followed by Dunnet's post-test; n = 6–8 per group.
Figure 3.
Rb fraction attenuates KA-induced acute degeneration in hippocampal pyramidal neurons.
Rats were pretreated with saline as KA controls, Rb fraction 30/kg (Rb30) or 40 mg/kg (Rb40) followed 40 min later by icv KA injection, and saline controls (Saline) received icv saline injection. After 48 h, brains were removed and stained for FJC and cresyl violet. (A) Representative images (200×) of the hippocampal subfields of CA1, CA3a–b, and CA3c stained with FJC or cresyl violet (Nissl staining). Images with higher magnification (400×) showed the details of pyramidal neurons (arrowhead) in each subfield. (B) Damage scores of the three subfields of hippocampus were measured in the sections stained with FJC in each experimental group. Data are expressed as means ± SEM; n = 6–8 per group; *p < 0.05, **p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 4.
Rb fraction prevents KA-induced acute loss of PV+ interneurons in the hippocampus.
Rats were pretreated with saline as KA controls, Rb fraction 30/kg (Rb30) or 40 mg/kg (Rb40) for 40 min followed by icv KA injection, and saline controls (Saline) received icv saline injection. After 48 h, brains were removed and PV+ interneurons were identified by immunohistochemical staining. (A) Numbers of PV+ interneurons in the hippocampal subfields of CA1, CA3a-b, hilus (including CA3c), and DG. (B) Representative photomicrographs (200×) of the CA1, CA3 a-b, and hilus subfields stained with anti-PV in the experimental groups. Data are expressed as means ± SEM; n = 6–8 per group; **p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 5.
Rb fraction prevents KA-induced acute loss of GAD67+ interneurons in the hippocampus.
Rats were pretreated with saline as KA controls, Rb fraction 30/kg (Rb30) or 40 mg/kg (Rb40) for 40 min followed by icv KA injection, and saline controls (Saline) received icv saline injection. After 48 h, brains were removed and GAD67+ interneurons were identified by immunohistochemical staining. (A) Quantification of GAD67+ interneurons in the hippocampal subfields of CA1, CA3a-b, hilus (including CA3c), and DG. (B) Representative photomicrographs (200×) of the CA1 and DG subfields stained with anti-GAD67 in each experimental group. Data are expressed as means ± SEM; n = 6-8 per group; *p < 0.05 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 6.
Rb fraction prevents KA-induced hippocampal-dependent spatial memory impairment.
Rats received a single injection of saline as KA controls, Rb (30 mg/kg) or Rb (40 mg/kg) 40 min before icv KA administration, and saline controls (Saline) received icv saline injection. Sixteen days later, the Morris water maze task was conducted. (A) Escape latency (the time to reach the platform) in the training test. (B) The numbers of times rats crossed the area where the platform was located in the probe trial. (C) Swimming speed of rats during the training test. (D) Swimming speed of rats in the probe trial. Data are expressed as means ± SEM; n = 6–8 per group; *p < 0.05, **p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test. Data of escape latency were analyzed by a two-way analysis of variance with repeated measures.
Figure 7.
Rb fraction decreases KA-induced hippocampal pyramidal neurodegeneration during chronic period after KA injection.
Rats received a single injection of saline as KA controls, Rb (30 mg/kg) or Rb (40 mg/kg) 40 min before icv KA administration, and saline controls (Saline) received icv saline injection. After the Morris water maze task (3 weeks after KA), brains were removed and stained for FJC or cresyl violet (Nissl staining). (A) Representative images (200×) of the hippocampal subfields of CA1, CA3a–b, and CA3c stained with FJC or cresyl violet. Images with higher magnification (400×) showed the details of pyramidal neurons (arrowhead) in each subfield. (B) Damage scores of the three subfields of hippocampus were measured in each experimental group. Data are expressed as means ± SEM; n = 6–8 per group; *p < 0.05, **p < 0.01 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 8.
Rb fraction decreases KA-induced loss of hippocampal GAD67+ interneurons during chronic period after KA injection.
Rats received a single injection of saline as KA controls, Rb (30 mg/kg) or Rb (40 mg/kg) 40 min before icv KA administration, and saline controls (Saline) received icv saline injection. After the Morris water maze task (3 weeks after KA), hippocampal GAD67+ interneurons were detected by immunohistochemical staining. (A) Numbers of GAD67+ interneurons in the hippocampal CA1, CA3a–b, hilus (including CA3c), and DG regions. (B) Representative photomicrographs (200×) of the CA1, CA3a–b, and hilus in the experimental groups. Data are expressed as means ± SEM; n = 6–8 per group; *p < 0.05, **p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 9.
Rb fraction decreases KA-induced loss of hippocampal PV+ interneurons during chronic period after KA injection.
Rats were pretreated with saline as KA controls, Rb fraction 30/kg (Rb30) or 40 mg/kg (Rb40) followed 40 min later by icv KA injection, and saline controls (Saline) received icv saline injection. After the Morris water maze task (3 weeks after KA), brains were removed and hippocampal PV+ interneurons were identified by immunohistochemical staining. (A) Numbers of PV+ interneurons in the hippocampal subfields of CA1, CA3a–b, hilus (including CA3c), and DG. (B) Representative photomicrographs (200×) of the CA1, CA3 a–b, and hilus in the experimental groups. Data are expressed as means ± SEM; n = 6–8 per group; *p < 0.05, **p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 10.
Rb fraction protects astrocytes during acute period and attenuates astrogliosis during chronic period after KA injection.
Rats were pretreated with saline as KA controls, Rb fraction 30/kg (Rb30) or 40 mg/kg (Rb40) followed 40 min later by icv KA injection, and saline controls (Saline) received icv saline injection. After 48 h or 3 weeks, brains were removed, and astrocytes in the hippocampus were detected by GFAP immunostaining. Images (40×) showed the distribution of astrocytes 48 h (A) or 3 weeks (C) after KA administration, the morphology of cells in the CA1 or CA3 stratum radiatum (arrowhead) were showed at high magnification (1000×). Numbers of astrocytes in the hippocampal subfields of CA1 stratum radiatum and CA3 stratum radiatum 48 h (B) or 3 weeks (D) after KA injection were determined and averaged across animals in each experimental group. Data are expressed as means ± SEM; n = 6–8 per group; **p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test.
Figure 11.
Rb fraction inhibits the activation of microglia after KA injection.
Rats were pretreated with saline as KA controls, Rb fraction 30/kg (Rb30) or 40 mg/kg (Rb40) followed 40 min later by icv KA injection, and saline controls (Saline) received icv saline injection. Forty-eight hours or 3 weeks later, brains were removed, and microglia were detected by Iba-1 immunostaining. Images (40×) showed the distribution of microglia in the hippocampus 48 h (A) or 3 weeks (C) after KA administration, the morphology of cells in CA1 or CA3 stratum radiatum (arrowhead) were showed at high magnification (1000×). Numbers of microglia in the hippocampal subfields of CA1 stratum radiatum and CA3 stratum radiatum 48 h (B) or 3 weeks (D) after KA injection were determined and averaged across animals in each experimental group. Data are expressed as means ± SEM; n = 6–8 per group; *p < 0.05,**p < 0.01, ***p < 0.001 by a one-way analysis of variance followed by Dunnet's post-test.