Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons

Abstract

Therapeutic hypothermia is the most promising non-pharmacological neuroprotective strategy against ischemic injury. However, shivering is the most common adverse reaction. Many studies have shown that dantrolene is neuroprotective in in vitro and in vivo ischemic injury models. In addition to its neuroprotective effect, dantrolene neutralizes the adverse reaction of hypothermia. Dantrolene may be an effective adjunctive therapy to enhance the neuroprotection of hypothermia in treating ischemic stroke. Cortical neurons isolated from rat fetuses were exposed to 90 minutes of oxygen-glucose deprivation followed by reoxygenation. Neurons were treated with 40 μM dantrolene, hypothermia (at 33°C), or the combination of both for 12 hours. Results revealed that the combination of dantrolene and hypothermia increased neuronal survival and the mitochondrial membrane potential, and reduced intracellular active oxygen cytoplasmic histone-associated DNA fragmentation, and apoptosis. Furthermore, improvements in cell morphology were observed. The combined treatment enhanced these responses compared with either treatment alone. These findings indicate that dantrolene may be used as an effective adjunctive therapy to enhance the neuroprotective effects of hypothermia in ischemic stroke.

Keywords: apoptosis; calcium overload; flow cytometry; fluorescent probe; ischemic stroke; nerve regeneration; neural regeneration; neurons; oxygen-glucose deprivation; reactive oxygen.

Figure 1

Immunofluorescence identification of primary cortical neurons. (A) DAPI nuclear staining (blue); (B) skeleton protein staining of neuron-specific β-tubulin III (Tuj1; green); (C) more than 98% of the cells are stained by both Tuj1 and DAPI (merge). Scale bar: 100 μm. DAPI: 4′,6-Diamidino-2-phenylindole.

Figure 2

JC-1 fluorescence probe labeling and flow cytometry. The change in JC-1 fluorescence reflects mitochondrial membrane function and is used as an early indicator of apoptosis. (A) J-aggregates show red fluorescence at a high ΔΨm; (B) JC-1 monomer shows green fluorescence at a low ΔΨm. Scale bars: 50 μm. (C) The mean fluorescence intensity of J-aggregates (upper panel) and the JC-1 monomer (lower panel). ΔΨm: Mitochondrial membrane potential.

Figure 3

Annexin V/PI double staining of the neuronal cell suspension. (A) Annexin V-positive cells (green fluorescence) indicate early apoptosis. (B) PI-positive cells (red fluorescence) indicate necrosis. (C) Both annexin V/PI-positive cells (merge) indicate late apoptosis. Scale bars: 50 μm. PI: Propidium iodide.

Figure 4

Effects of hypothermia plus dantrolene on the morphology of OGD/R cells. Phase-contrast microscopy of (A) normal group; (B) OGD/R group in which OGD/R induces injury, observed as neuronal loss, axonal rupture, and network connection fracture; (C) hypothermia group; (D) dantrolene group in which hypothermia or dantrolene treatment ameliorates the morphological abnormality; (E) hypothermia + dantrolene group in which the combined treatment enhances neuroprotection compared to either treatment alone. Scale bars: 50 μm. OGD/R: Oxygen-glucose deprivation/reoxygenation.

Figure 5

Neuroprotection against OGD-mediated injury occurs with hypothermia, dantrolene, or their combined administration. DNA fragmentation and annexinV/PI labeling was performed 48 hours after OGD treatment to assess cell death, while other parameters were measured after 12 hours of hypothermia (or dantrolene) administration after OGD. (A) CCK-8: Cell viability. (B) DCFH-DA: ROS. (C) Fluo-3 AM: Free calcium ions. (D) JC-1: Monomers/J-aggregates. (E) Cell death detection: Cytoplasmic histone-associated DNA fragments. (F) Q1 + Q2: Late apoptosis and necrosis cells. Experiments were performed six times. *P < 0.05, vs. normal group; #P < 0.05, vs. OGD group; †P < 0.05, vs. H + D group. I: Normal group; II: OGD/R group; III: Hypothermia group; IV: Dantrolene group; IV: H + D. CCK-8: Cell counting kit-8; DCFH-DA: dichlorofluorescin diacetate; Fluo-3 AM: fluo-3-acetoxymethyl ester; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reoxygenation; H + D: hypothermia and dantrolene; ROS: reactive oxygen species.

Figure 6

Detection of annexin V/PI double-labeled neuronal cells via flow cytometry. (A) Normal group; (B) OGD/R group; (C) hypothermia group; (D) dantrolene group; (E) hypothermia and dantrolene group. Q1, Q2, Q3, and Q4 quadrants represent necrosis, late apoptosis, viable cells, and early apoptotic cells, respectively. PI: Propidium iodide; OGD/R: oxygen-glucose deprivation/reoxygenation.