Characterization of Perineuronal Nets in the Paraventricular Nucleus of the Hypothalamus and their Alteration in Neurogenic Hypertension

Figures

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  Fig. 1. Developmental regulation of PNNs in the PVN. a WFA staining (magenta) shows no visible PNNs at P6, with progressive appearance at P14 and maturation by 1Mo. b-c Quantification of WFA+ area (Time, p < 0.0001; Sex, p = 0.1140; Interaction, p = 0.6546) and PNN intensity (Time, p < 0.0001; Sex, p = 0.0874; Interaction, p = 0.7332) across developmental timepoints (each data point = average of 3 mice, 7–12 sections per mouse). Two-way ANOVA with Tukey’s multiple comparisons. *p > 0.05 vs. P6 for each sex. PVN-F denotes females, and PVN-M denotes males. d Representative images of PNN-enwrapped neurons at P14, 3Mo, and 25Mo. e-f Quantification of WFA+ area (%) and fluorescence intensity in 3- vs. 25-month-old naïve male mice (3Mo n = 4; 25Mo n = 5). Data are shown as mean ± SEM; circles represent individual mice (average of 7–12 bilateral PVN sections/mouse). DAPI (blue) indicates nuclei and HuC/D (green) indicates neurons

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  Fig. 2. PNNs enwrap multiple neuronal cell types in the PVN. a Representative images of PNN-enwrapped neurons within the PVN. Arrows point to double-labeled neuron. Asterisk indicates WFA+ blood vessel. b Quantification of the number and percent of PNN-enwrapped neurons per cell type (n = 3/cell type). Parvalbumin (PV)-neurons were not detected within the PVN and are therefore not shown. c Distribution of absolute PNN number across all mice analyzed for neuronal cell type quantification in B (females vs. males: n = 18; 87.1 ± 4.9 vs. 73.6 ± 5.7, p = 0.0798). d PNN number normalized to PVN area in an independent cohort of 3-month-old mice (females vs. males: n = 3; 43.3 ± 1.7 vs. 46.5 ± 3.5, p = 0.4506, unpaired). Data are mean ± SEM; circles represent independent mice (average of 7–12 bilateral PVN sections/mouse). *p = 0.0169, #p = 0.0077

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  Fig. 3. DOCA-salt increases PVN PNNs in male but not female mice. a Change in systolic blood pressure (SBP) after 21 days of DOCA-salt treatment (males: SHAM n = 11, DOCA n = 9, ∆SBP − 5.0 ± 4.1 vs. 20.6 ± 4.3, p = 0.0004, unpaired t-test; females: SHAM n = 12, DOCA n = 13, ∆SBP − 6.7 ± 4.7 vs. 21.0 ± 5.2, p = 0.0024, Mann-Whitney). b Schematic showing PVN sections included in quantification. c-e Quantification in males: c WFA area (%) and normalized WFA area (SHAM n = 10, DOCA n = 9; 4.2 ± 0.3 vs. 5.7 ± 0.5, p = 0.0255; normalized 1.0 ± 0.1 vs. 1.4 ± 0.1, p = 0.0013), d PNN number (35.6 ± 2.0 vs. 44.8 ± 3.9, p = 0.0447), and e WFA intensity (1.0 ± 0.1 vs. 1.1 ± 0.0, p = 0.2677). f Ratio of glutamate (Glu) to GABA (GABA) neurotransmitters after DOCA-salt. Pooled PVN tissue-punches from the PVN of both DOCA and SHAM male mice (n = 3; 2 mice/sample) (1.0 ± 0.0 vs. 1.2 ± 0.1, p = 0.0190). g-i Quantification in females: g WFA area (%) and normalized WFA area (SHAM n = 7, DOCA n = 8; 6.7 ± 0.7 vs. 6.3 ± 0.5, p = 0.5532; normalized 1.0 ± 0.1 vs. 1.0 ± 0.1, p > 0.9999), h PNN number (71.5 ± 4.4 vs. 64.9 ± 6.0, p = 0.4011), and i WFA intensity (1.0 ± 0.1 vs. 1.0 ± 0.1, p > 0.9999). Data are mean…