Cortical primary neurons culture

Cortical primary neurons were isolated from neonatal or one-day old wild type or pups lacking AT_1a receptors (AT₁R) specifically on neurons (AT1N).(Xu et al. 2017) Brains were dissected immediately and the cortex was collected in ice-cold Hank’s balanced salt solution (HBSS) (Gibco 14175-079). Following brief dissociation, the cortices were washed and dispersed in HBSS containing 1% trypsin (Sigma-Aldrich T1426) and 1.5 KU/mL DNaseI (Sigma-Aldrich D5025), then digested for 10 min at 37 °C. The digested samples were then dissociated by gentle pipetting in HBSS containing 1.5 KU/mL DNase I. After brief centrifugation, the cell pellets were re-suspended in complete Neurobasal culture medium, supplemented with 2% B27 and 0.5 mM GlutaMax (Gibco), and plated at a density of 10⁶ cells/ml onto poly-L-lysine coated plates. Cytosine arabinofuranoside (Ara-C, 2 μM, Sigma-Aldrich C1768) was added to the cultures 48 h after plating to arrest the growth of non-neuronal cells. Cultured cortical neurons were validated via immunofluorescence labeling with MAP2 (microtubule associated protein 2), as shown in Figure 1A. Cortical primary neurons were cultured for at least 10 days and then treated with specific agonist or blockers. All blockers, except for MitoTEMPOL and 1400W, were given 1 hour before L-glutamic acid treatment (100 μM): NBQX (AMPA, α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, receptor antagonist, 100 μM), D-AP5 (NMDA receptor, 50 μM), and DL-AP4 (non-selective ionotropic receptors antagonist, 50 μM), SB203580 (p38 MAPK blocker, 10 μM). MitoTEMPOL (mitochondria-targeted antioxidant, 10 μM) and 1400W (iNOS inhibitor, 5 μM) were given 30 min before L-glutamic acid treatment.

(A) Immunofluorescence labeling of primary cultured cortical neurons for visualization of MAP2 (red). The scale bar represents 20 μm. (B) ACE2 activity assay was performed in primary cultured cortical neurons isolated from wild type neonates. The cultured neurons were treated with L-glutamic acid (glutamate, 100 μM) or lipopolysaccharide (LPS, 100 ng/ml) for 18 h (n=3 independent cultures/group). (C) ACE2 activity assay was performed in cultured cortical neurons isolated from neonates with neuronal AT_1aR deficiency. The cultured neurons were treated with glutamate (100 μM) or Angiotensin II (Ang-II, 300 nM) for 18 h (n=4 independent cultures/group). Statistical significance: One-way ANOVA: *P<0.05 vs. vehicle. (D-E) Glutamate significantly upregulates the gene expression of pro-inflammatory cytokines in cultured cortical neurons: qPCR was performed to assess the mRNA levels of IL-6, (D) and Cox-2 (E) in neurons treated with glutamate (100 μM) or vehicle. Statistical significance: student t-test: *P<0.05 vs. vehicle.