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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria <t>floribunda</t> <t>agglutinin</t> (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.
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Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria floribunda agglutinin (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.

Journal: Advanced Science

Article Title: Neonatal Sevoflurane Exposure Induces Long‐Term Cognitive Impairment via Epigenetically Mediated MMP9 Activation and Perineuronal Net Disruption in the Hippocampal CA2

doi: 10.1002/advs.78006

Figure Lengend Snippet: Neonatal sevoflurane exposure induces PNNs damage in the hippocampus. (A) Schematic of the experimental protocol for hippocampal extracellular matrix (ECM) analyses. (B) Representative scanning electron micrographs (SEM) of hippocampal ECM ultrastructure in Con and Sev ‑treated groups. Scale bars = 5 µm (5K×), 1.2 µm (20K×), and 500 nm (50K×). (C) Quantification of mean gray value (left) and fiber diameter (right) of hippocampal ECM in Con and Sev groups. (D, E) Wisteria floribunda agglutinin (WFA) staining and quantitative analysis of PNN intensity in the CA2 region at 7, 14, 30, and 60 days post‐exposure (dpe). Scale bar = 50 µm. (F, G) Western blotting and quantification of hapln1 and aggrecan protein levels in the hippocampus of Con and Sev groups. (H, I) Immunofluorescence staining and quantitative analysis of aggrecan in the hippocampus of Con and Sev groups at 60 dpe. Green: aggrecan. Blue: DAPI. Scale bar = 50 µm. (J–M) PNNs in the Con group exhibit bright, well‐organized mesh‐like architecture, whereas PNNs in the Sev group display reduced WFA intensity and a disrupted staining pattern. (J) Representative WFA staining. Purple: WFA. Scale bar = 10 µm. (K) Binary images of the regions in (J). (L) Quantification of black/white pixel ratio. (M) Representative surface intensity profiles of WFA‐positive PNNs. (N) Immunoelectron microscopy of PNNs in the Con and Sev groups. High‐magnification images (right panels) display immunogold labeling within WFA (red arrows) in both groups. Scale bars = 1 µm (left), 500 nm (right). Data represent the mean ± SEM. n = 3 mice per group for SEM (B, C, J–N); n = 6 mice per group for all other experiments. Statistical comparisons were conducted using unpaired Student's t‐test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: Serial coronal brain sections were blocked in PBS containing 3% BSA and 0.3% Triton X‐100, then incubated overnight at room temperature with the following primary antibodies: biotinylated Wisteria floribunda agglutinin (WFA; Vector Laboratories, 1:300), rabbit anti‐Aggrecan (Merck‐Millipore, MABT83, 1:200), rabbit anti‐NeuN (Merck Millipore, ABN78, 1:600), rabbit anti‐GFAP (GeneTex, GTX108711, 1:200), mouse anti‐CC1 (Sigma–Aldrich, OP80, 1:100), rabbit anti‐Iba1 (WAKO, Lek0542, 1:200), rabbit anti‐Camk2 (GeneTex, GTX135117, 1:200), mouse anti‐CaMKIIα (Abcam, ab22609, 1:200), goat anti‐BDNF (Vector Laboratories, EB08117, 1:500), rabbit anti‐VGlut2 (Abcam, ab216463, 1:200), chicken anti‐MAP2 (Abcam, ab5392, 1:500), and rabbit anti‐MMP9 (Abclonal, A25299, 1:200).After PBS washes, sections were incubated for 3 h at room temperature in the dark with corresponding Alexa Fluor 488‐ or 594‐conjugated secondary antibodies (donkey anti‐rabbit, donkey anti‐mouse, donkey anti‐chicken, and donkey anti‐goat; Jackson ImmunoResearch, 1:500) or Alexa Fluor 488‐conjugated streptavidin (Cell Signaling Technology, 89067S, 1:500).

Techniques: Staining, Analysis, Western Blot, Immunofluorescence, Immuno-Electron Microscopy, Labeling