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Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons.

Xu SY, Hu FY, Ren LJ, Chen L, Zhou ZQ, Zhang XJ, Li WP - Neural Regen Res (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Postdoctoral Workstation, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province, China ; Department of Brain Center, Shenzhen Key Laboratory of Neurosurgery, Shenzhen Second People's Hospital, Shenzhen University First Affiliated Hospital, Shenzhen, Guangdong Province, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: The animal experimental protocol was approved by the Animal Ethics Committee of Sun Yat-sen University, China. Pathogen-free and pregnant Sprague-Dawley rats were purchased from Guangdong Experimental Animal Center, Guangzhou, Guangdong Province, China (license No. SCXK (Yue) 2011-0015), and they were sacrificed by cervical dislocation. Fetuses at gestational day 18 were micro-dissected, and primary cortical neurons were cultured, according to our previous study (Xu et al., 2012). In brief, the apical cortex was removed and digested using papain and DNase I (Sigma-Aldrich, St. Louis, MO, USA). The sifted cell suspension was seeded onto 0.1 mg/mL poly-L-lysine-coated culture plate at a density of 50,000 cells per cm2. A 4-hour incubation in high-glucose Dulbecco's Modified Eagle's Medium (HyClone, Logan, UT, USA) allowed the neurons to adhere, and the media was then changed to Neurobasal Medium (Gibco Invitrogen, Carlsbad, CA, USA). Neurons were identified (via immunofluorescence) by Tuj1, a neuron-specific class III β-tubulin III/Tuj1 immunofluorescence (Figure 1). Briefly, cells were incubated with rabbit anti-β-tubulin III (Tuj1) polyclonal antibody (1:1,000; Abcam, Cambridge, UK) for 1 hour at 37°C. Neurons were then co-incubated with the secondary antibody, Alexa Fluor® 488 goat anti-rabbit IgG (1:200; Abcam), for an additional 1 hour at 37°C. The nuclei were labeled with 4,6-diamidino-2-phenylindole. The images were acquired with an inverted fluorescence microscope (Olympus, Tokyo, Japan).


Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons.

Xu SY, Hu FY, Ren LJ, Chen L, Zhou ZQ, Zhang XJ, Li WP - Neural Regen Res (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590241&req=5

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.
Mentions: The animal experimental protocol was approved by the Animal Ethics Committee of Sun Yat-sen University, China. Pathogen-free and pregnant Sprague-Dawley rats were purchased from Guangdong Experimental Animal Center, Guangzhou, Guangdong Province, China (license No. SCXK (Yue) 2011-0015), and they were sacrificed by cervical dislocation. Fetuses at gestational day 18 were micro-dissected, and primary cortical neurons were cultured, according to our previous study (Xu et al., 2012). In brief, the apical cortex was removed and digested using papain and DNase I (Sigma-Aldrich, St. Louis, MO, USA). The sifted cell suspension was seeded onto 0.1 mg/mL poly-L-lysine-coated culture plate at a density of 50,000 cells per cm2. A 4-hour incubation in high-glucose Dulbecco's Modified Eagle's Medium (HyClone, Logan, UT, USA) allowed the neurons to adhere, and the media was then changed to Neurobasal Medium (Gibco Invitrogen, Carlsbad, CA, USA). Neurons were identified (via immunofluorescence) by Tuj1, a neuron-specific class III β-tubulin III/Tuj1 immunofluorescence (Figure 1). Briefly, cells were incubated with rabbit anti-β-tubulin III (Tuj1) polyclonal antibody (1:1,000; Abcam, Cambridge, UK) for 1 hour at 37°C. Neurons were then co-incubated with the secondary antibody, Alexa Fluor® 488 goat anti-rabbit IgG (1:200; Abcam), for an additional 1 hour at 37°C. The nuclei were labeled with 4,6-diamidino-2-phenylindole. The images were acquired with an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Postdoctoral Workstation, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province, China ; Department of Brain Center, Shenzhen Key Laboratory of Neurosurgery, Shenzhen Second People's Hospital, Shenzhen University First Affiliated Hospital, Shenzhen, Guangdong Province, China.

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.

No MeSH data available.


Related in: MedlinePlus