Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons

Abstract

Purpose: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD).

Methods: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 µM, 10 µM, and 100 µM) on OGD-induced neurotoxicity.

Results: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 µM and 100 µM of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 µM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05).

Conclusion: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

Keywords: Hypoxia-Ischemia; L-carnitine; Neurons; Neuroprotective effect; Oxygen-glucose deprivation.

Fig. 1

Assessment of cell number using the fluorescent dyes acridine orange (AO) and propidium iodide (PI). The figure shows the number of cells in the oxygen glucose deprivation (OGD) groups treated with various doses of L-carnitine (LC) compared with the untreated OGD group. The number of cells stained with AO was proportional to the dose of LC. LC did not reduce the number of cells that stained with PI. Data are expressed as mean±SEM. CON, control.

Fig. 2

Assessment of the propidium iodide/acridine orange (PI/AO) ratio using fluorescent dyes. The percentage of dying cells was evaluated by calculating the PI/AO ratio in the oxygen glucose deprivation (OGD) groups treated with L-carnitine (LC) versus the untreated OGD group. LC concentrations of 10 µM and 100 µM significantly reduced the number of dying cells (P<0.05). Data are expressed as mean±standard error of the mean. Different from control mean: ^*P<0.05. CON, control.

Fig. 3

Representative confocal microscope images of the effect of L-carnitine (LC) on the morphology of neuronal cells exposed to oxygen glucose deprivation (OGD). Higher LC doses had a greater neuroprotective effect than lower LC doses. CON, control; AO, acridine orange; PI, propidium iodide (magnification, ×400).

Fig. 4

Effect of L-carnitine (LC) on the oxygen glucose deprivation (OGD)-induced decrease in neuronal viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Exposure to OGD decreased cell viability whereas LC treatment (100 µM) significantly increased viability compared to the untreated OGD group. Data are expressed as mean±standard error of the mean. Different from control mean: ^*P<0.05. CON, control.

Fig. 5

Effect of L-carnitine (LC) on oxygen glucose deprivation (OGD)-induced increase in lactate dehydrogenase (LDH) release. Cells treated with 1 µM, 10 µM, or 100 µM of LC showed a significant decrease in LDH leakage compared with the untreated OGD group (P<0.05). Data are expressed as mean±standard error of the mean. Different from control mean: ^*P<0.05. CON, control.

Fig. 6

Representative microscopic images of reactive oxygen species (ROS) formation in cortical neurons after oxygen glucose deprivation (OGD) treatment. (A) Accumulation of ROS in neuronal cells exposed to OGD. Treatment with high doses of L-carnitine (LC) inhibited the accumulation of ROS in neuronal cells (magnification, ×400). (B) Significant decrease in ROS formation in OGD groups treated with 10 µM and 100 µM LC compared with the untreated OGD group. Data are expressed as mean±standard error of the mean. Different from control mean: ^*P< 0.05. CON, control; DCF, 2',7'-dichlorodihydrofluorescein diacetate.

Fig. 7

Representative microscopic image of the effect of L-carnitine (LC) on oxygen glucose deprivation (OGD)-induced apoptosis. TUNEL-positive cells are visible after OGD exposure. Note the dose-dependent decrease in the number of TUNEL-positive green cells in the OGD groups treated with L-carnitine compared with the untreated OGD group (magnification, ×400). CON, control; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labeling; DAPI, 4'-6-diamidino-2-phenylindole.

Fig. 8

Number of TUNEL-positive cells. L-carnitine (LC) treatment reduced the number of TUNEL-positive cells in the oxygen glucose deprivation (OGD) groups compared with the untreated OGD group in a dose-dependent manner, the difference between groups was not significant. Data are expressed as mean±standard error of the mean. CON, control; TUNEL, terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labeling.

Fig. 9

Ratio of TUNEL/PI positive cells. There was a significant decrease in the ratio of TUNEL/PI-positive cells in the oxygen glucose deprivation (OGD) groups treated with L-carnitine (LC) compared with the untreated OGD group. The OGD group treated with 100 µM LC had a significantly lower TUNEL/PI ratio than the untreated OGD group. Data are expressed as mean±standard error of the mean. Different from control mean: ^*P<0.05. CON, control; TUNEL, terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labeling; PI, propidium iodide.