rat cerebral cortical neurons  (Dawley Inc)

Journal: International Journal of Molecular Sciences

Article Title: Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons

doi: 10.3390/ijms24098328

Figure Lengend Snippet: Pue alleviated Cd-induced neuronal injury in vitro. Primary rat cerebral cortical neurons were pretreated with Pue (100 μM) for 1 h and then incubated with or without Cd (10 μM) for 12 h. The nuclei and mitochondria in primary rat cerebral cortical neurons were observed using TEM. The blue arrow indicates the damaged nucleus and the yellow arrow indicates the damaged mitochondria. Scale bar in nuclei: 5 μm; scale bar in mitochondria: 1 μm.

Article Snippet: As previously described [ ], the primary rat cerebral cortical neurons were isolated from Sprague Dawley (SD) rats at 18–19 d of gestation by trypsinization and culturing for 6 d for the subsequent experiments.

Techniques: In Vitro, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons

doi: 10.3390/ijms24098328

Figure Lengend Snippet: Pue activated autophagy and alleviated Cd-induced autophagic blockade in primary rat cerebral cortical neurons in vitro. The primary rat cerebral cortical neurons were treated with ( A ) 10 μM Cd for different lengths of time (0, 3, 6, 12, and 24 h) or ( B ) with 0–20 μM Cd for 12 h or ( C ) with 10 μM Cd and/or 25 μM Baf A1 for 12 h or ( D ) pretreated with 100 μM Pue for 1 h, followed by 10 μM Cd exposure 12 h. Western blot analysis was then performed to measure LC3II and P62 protein expression. Each value is presented as the mean ± SD ( n = 3). Completely different letters mean significant differences ( p < 0.05); the same letters mean no significant differences ( p > 0.05). ( E ) The representative images of autophagosomes (blue arrows) and autophagolysosome (yellow arrows) in primary cerebral cortical neurons were observed using TEM. The selected areas were magnified. Scale bars: 1 μm.

Article Snippet: As previously described [ ], the primary rat cerebral cortical neurons were isolated from Sprague Dawley (SD) rats at 18–19 d of gestation by trypsinization and culturing for 6 d for the subsequent experiments.

Techniques: In Vitro, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons

doi: 10.3390/ijms24098328

Figure Lengend Snippet: Pue attenuated Cd-induced inhibition of LC3 colocalization with LAMP2 in vitro and in vivo. ( A ) The primary rat cerebral cortical neurons were treated with 10 μM Cd and/or 100 nM Rap (positive control) and 25 nM Baf A1 (negative control) for 12 h. Scale bar: 10 μm. ( B ) The primary rat cerebral cortical neurons grown on coverslips were treated with 10 µM Cd and/or 100 µM Pue for 12 h and then successively stained with LC3 (green), LAMP2 (red), and DAPI (blue). Colocalization of LC3 and LAMP2 was assessed using confocal microscopy. Scale bar: 10 μm. ( C ) The rat cerebral cortices were analyzed for LC3 (red) and LAMP2 (green) colocalization signals using immunofluorescence. Representative confocal images showing colocalization of LC3 with LAMP2. Scale bar: 20 μm. Percent of colocalization of LC3 with LAMP2. The data are represented as the mean ± SD of three independent experiments with 50 cells per condition in each experiment. Completely different letters mean significant differences ( p < 0.05); the same letters mean no significant differences ( p > 0.05).

Article Snippet: As previously described [ ], the primary rat cerebral cortical neurons were isolated from Sprague Dawley (SD) rats at 18–19 d of gestation by trypsinization and culturing for 6 d for the subsequent experiments.

Techniques: Inhibition, In Vitro, In Vivo, Positive Control, Negative Control, Staining, Confocal Microscopy, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons

doi: 10.3390/ijms24098328

Figure Lengend Snippet: Pue restored the expression of key proteins of autophagosome–lysosome fusion down-regulated by Cd in vitro and in vivo. ( A ) The primary rat cerebral cortical neurons were pretreated with 100 µM Pue for 1 h, followed by 10 μM Cd exposure for 12 h. Then, the expression levels of Rab7, SNAP29, and VPS41 were examined using Western blot analysis. ( B ) The protein samples were prepared from the cerebral cortex homogenate of the rats in each group, and the expression levels of Rab7, SNAP29, and VPS41 were detected using Western blot analysis. Each value is presented as the mean ± SD ( n = 3). Completely different letters mean significant differences ( p < 0.05); the same letters mean no significant differences ( p ˃ 0.05).

Article Snippet: As previously described [ ], the primary rat cerebral cortical neurons were isolated from Sprague Dawley (SD) rats at 18–19 d of gestation by trypsinization and culturing for 6 d for the subsequent experiments.

Techniques: Expressing, In Vitro, In Vivo, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons

doi: 10.3390/ijms24098328

Figure Lengend Snippet: Pue alleviates Cd-induced lysosomal degradation dysfunction in vitro and in vivo. ( A ) The primary rat cerebral cortical neurons were treated with 100 μM Pue and/or 10 μM Cd or 25 nM Baf A1 (negative control) or 100 nM Rap (positive control) for 12 h and then stained with 75 nM LTR and incubated at 37 °C for 30 min to determine the pH of the lysosomes. Scale bar: 100 μm. ( B ) The primary rat cerebral cortical neurons were pre-incubated with 10 μg/mL DQ-BSA Red for 2 h and then treated with 100 μM Pue and/or 10 µM Cd or 25 nM Baf A1 (negative control) or 100 nM Rap (positive control) for 12 h. Hoechest 33,342 staining labeled nucleus (blue). The images were obtained by confocal microscopy. Scale bar: 10 μm. The mean fluorescence intensity of LTR and DQ-BSA Red were quantified in a relative way to the control group, whose value of fluorescence is set at “1”. The data in the (( A , B ) left panel) represent the mean ± SD of three separate experiments and each one was performed in duplicate ( n = 3). ( C ) The primary rat cerebral cortical neurons were treated with 100 μM Pue and/or 10 μM Cd for 12 h and then the expression levels of LAMP2, CTSB, and CTSD were examined using Western blot analysis. ( D ) The protein samples were prepared from the cerebral cortex homogenate of the rats in each group, and the expression levels of LAMP2, CTSB, and CTSD were detected using Western blot analysis. Each value is presented as the mean ± SD ( n = 3). Completely different letters mean significant differences ( p < 0.05); the same letters mean no significant differences ( p ˃ 0.05).

Article Snippet: As previously described [ ], the primary rat cerebral cortical neurons were isolated from Sprague Dawley (SD) rats at 18–19 d of gestation by trypsinization and culturing for 6 d for the subsequent experiments.

Techniques: In Vitro, In Vivo, Negative Control, Positive Control, Staining, Incubation, Labeling, Confocal Microscopy, Fluorescence, Control, Expressing, Western Blot