Nimodipine but Not Nifedipine Promotes Expression of Fatty Acid 2-Hydroxylase in a Surgical Stress Model Based on Neuro2a Cells

Abstract

Nimodipine is well characterized for the management of aneurysmal subarachnoid hemorrhage and has been shown to promote a better outcome and less delayed ischemic neurological deficits. Animal and clinical trials show neuroprotective efficacy following nerve injuries. We showed a neuroprotective effect on Neuro2a cells. Subsequent microarray analysis revealed-among others-fatty acid 2-hydroxylase (FA2H) upregulated by nimodipine in vitro, which is a component of myelin synthesis. Differentiated Neuro2a cells were analyzed for nimodipine-mediated survival considering stress treatment in comparison to nifedipine-treatment. Cell survival was determined by measurement of LDH activity in the culture medium. Nimodipine decreased surgery-like stress-induced cell death of differentiated Neuro2a cells. Neuro2a cell culture was analyzed for changes in FA2H expression induced by nimodipine or nifedipine in surgery-like stress conditions. We analyzed expression levels of FA2H mRNA and protein by qPCR using fa2h specific primers or a FA2H-specific antibody in nimodipine or nifedipine non- and pre-treated Neuro2a cell culture, respectively. Nimodipine but not nifedipine increases FA2H protein levels and also significantly increases mRNA levels of FA2H in both undifferentiated and differentiated Neuro2a cells. Our findings indicate that higher expression of FA2H induced by nimodipine may cause higher survival of Neuro2a cells stressed with surgery-like stressors.

Keywords: FA2H; Neuro2a; myelin; neuroprotection; nifedipine; nimodipine; stress.

Figure 1

Lactate dehydrogenase (LDH) activity measurement after stress on differentiated, nimodipine pre-treated Neuro2a cells. Values are given as the mean ± SD (error bars) of one representative out of at least three biologically independent experiments. Nim = nimodipine (in µM); single asterisk = p < 0.05 compared to non-treated cells; double asterisks = p < 0.005 compared to non-treated cells. (A) Oxidative stress (EtOH, 2%); (B) osmotic stress (NaCl, 150 mM); (C) heat stress (4 or 6 h at 42 °C, respectively); (D) mechanical stress.

Figure 2

LDH activity measurement after stress on nifedipine pre-treated Neuro2a cells. (A) Undifferentiated Neuro2a; (B) differentiated Neuro2a. Values are given as the mean ± SD (error bars) of one representative out of at least three biologically independent experiments. Nif = nifedipine; mechanical = mechanical stress; NaCl = osmotic stress (150 mM); EtOH = oxidative stress (2%); 4 h = 4 h heat stress; 6 h = 6 h heat stress; single asterisk = p < 0.05 compared to non-treated cells.

Figure 3

Fatty acid 2 hydroxylase (FA2H) mRNA levels in undifferentiated Neuro2a cells. Values are given as the mean ± SD (error bars) of one representative out of at least three biologically independent experiments. (A) Oxidative stress; (B) osmotic stress; (C) heat stress; (D) mechanical stress. nt = no drug pre-treatment; Nim = nimodipine pre-treatment (20 µM); Nif = nifedipine pre-treatment (20 µM); nontreated = no stress application; mechanical = mechanical stress; NaCl = osmotic stress (150 mM); EtOH = oxidative stress (2%); 4 h = 4 h heat stress; 6 h = 4 h; single asterisk = p > 0.05 (NOT significant) compared to each other. All other samples show p < 0.05 compared to each other each.

Figure 4

FA2H mRNA levels in differentiated Neuro2a cells. Values are given as the mean ± SD (error bars) of one representative out of at least three biologically independent experiments. (A) Oxidative stress; (B) osmotic stress; (C) heat stress; (D) mechanical stress. nt = no drug pre-treatment; Nim = nimodipine pre-treatment (20 µM); Nif = nifedipine pre-treatment (20 µM); nontreated = no stress application; mechanical = mechanical stress; NaCl = osmotic stress (150 mM); EtOH = oxidative stress (2%); 4 h = 4 h heat stress; 6 h = 6 h heat stress; single asterisk = p > 0.05 (NOT significant) compared to each other. All other samples show p < 0.05 compared to each other each.

Figure 5

FA2H protein levels in undifferentiated Neuro2a cells. 20 µg total protein was loaded per sample on a 12% sodium dodecyl sulfate-polyacryl amide (SDS-PAA) gel. One representative out of at least three biologically independent experiments is displayed. Nim = nimodipine pre-treatment (20 µM); Nif = nifedipine pre-treatment (20 µM) nt = no drug pre-treatment; anti-FA2H = rabbit anti-FA2H antibody (1:1000); anti-GAPDH = mouse anti-GAPDH antibody (1:5000; loading control).

Figure 6

FA2H protein levels in undifferentiated Neuro2a cells. 20 µg total protein was on loaded per sample on a 12% SDS-PAA gel. One representative out of at least three biologically independent experiments is displayed. Nim = nimodipine pre-treatment (20 µM); Nif = nifedipine pre-treatment (20 µM) nt = no drug pre-treatment; anti-FA2H = rabbit anti-FA2H antibody (1:1000); anti-GAPDH = mouse anti-GAPDH antibody (1:5000; loading control).