Neuroprotective effects of pre-treatment with l-carnitine and acetyl-L-carnitine on ischemic injury in vivo and in vitro

Abstract

The therapeutic effect of stroke is hampered by the lack of neuroprotective drugs against ischemic insults beyond the acute phase. Carnitine plays important roles in mitochondrial metabolism and in modulating the ratio of coenzyme A (CoA)/acyl-CoA. Here, we investigate the neuroprotective effects of l-carnitine (LC) and Acetyl-l-carnitine (ALC) pre-treatment on ischemic insults under the same experimental conditions. We used a transient middle cerebral artery occlusion (MCAO) model to evaluate the protective roles of LC and ALC in acute focal cerebral ischemia in vivo and to understand the possible mechanisms using model of PC12 cell cultures in vitro. Results showed that ALC, but not LC, decreased infarction size in SD rats after MCAO in vivo. However, both LC and ALC pretreatment reduced oxygen-glucose deprivation (OGD)-induced cell injury and decreased OGD-induced cell apoptosis and death in vitro; at the same time, both of them increased the activities of super oxide dismutase (SOD) and ATPase, and decreased the concentration of malondialdehyde (MDA) in vitro. Thus, our findings suggested that LC and ALC pre-treatment are highly effective in the prevention of neuronal cell against ischemic injury in vitro, however, only ALC has the protective effect on neuronal cell injury after ischemia in vivo.

Keywords: Acetyl-l-carnitine; ischemia; l-carnitine; neuroprotection; oxygen-glucose deprivation.

Figure 1

Chemical structure of l-carnitine (LC) (a) and Acetyl-l-carnitine (ALC) (b).

Figure 2

Effects of LC and ALC on infarct size following middle cerebral artery occlusion (MCAO) in rats. (a) 2,3,5-triphenyltetrazolium chloride (TTC) staining of cerebral infarct size in brain sections from sham groups, MCAO groups, LC (100 mg/kg) and ALC (100 mg/kg) pretreated groups at 24 h before MCAO; (b) Quantification of infarct size by TTC staining 6 h after MCAO. Values are mean ± SEM. * p < 0.05 compared to the MCAO groups, n = 6 in each group.

Figure 3

Effects of LC and ALC pretreatment on the viability of PC12 cells exposed to oxygen-glucose deprivation (OGD). (a) OGD decreased the viability of PC12 cells time-dependently. PC12 cells were treated with OGD for 30, 60, 90, and 120 min, and the cell viability was determined by MTT assay (n = 6); (b) Effect of LC and ALC on the viability of PC12 cells treated without OGD. Cells were pretreated with 50, 100, 200 or 400 μmol/L LC and ALC for 24 h, and the cell viability was determined by MTT assay (n = 6); (c) Dose-dependent increase of the cell viability by LC and ALC in PC12 cells treated with OGD. Cells were pretreated with 50, 100, 200 and 400 μmol/L LC or ALC for 24 h, followed by OGD exposure for 120 min, and the cell viability was determined by MTT assay (n = 6). Values are mean ± SEM. * p < 0.05 compared to the OGD groups.

Figure 4

Effects of LC and ALC pretreatment on apoptosis and necrosis of PC12 cells treated with OGD. PC12 cells were pretreated without or with LC (200 μmol/L)) and ALC (200 μmol/L) for 24 h before OGD exposure for 120 min. PC12 cells treated without OGD used as an normal control. (a) Cell apoptosis were examined by TUNEL fluorescence staining; (b) Cell necrosis were examined by SYTOX fluorescence staining; (c) Cell necrosis were examined by PI fluorescence staining. * p < 0.05 compared to the OGD groups. Original magnifications: 200×.

Figure 4

Effects of LC and ALC pretreatment on apoptosis and necrosis of PC12 cells treated with OGD. PC12 cells were pretreated without or with LC (200 μmol/L)) and ALC (200 μmol/L) for 24 h before OGD exposure for 120 min. PC12 cells treated without OGD used as an normal control. (a) Cell apoptosis were examined by TUNEL fluorescence staining; (b) Cell necrosis were examined by SYTOX fluorescence staining; (c) Cell necrosis were examined by PI fluorescence staining. * p < 0.05 compared to the OGD groups. Original magnifications: 200×.

Figure 5

Effects of LC and ALC pretreatment on the activity of SOD and ATPase, and the level of MDA in PC12 cells exposed to OGD. PC12 cells were pretreated without or with LC (200 μmol/L) and ALC (200 μmol/L) for 24 h before OGD exposure for 120 min. PC12 cells treated without OGD used as an normal control. (a) Super oxide dismutase (SOD) activity in PC12 cells; (b) The levels of MDA in PC12 cells; (c) The ATPase activity in PC12 cells. Values given are the mean ± SEM (n = 6). * p < 0.05 compared with OGD groups.

Figure 5

Effects of LC and ALC pretreatment on the activity of SOD and ATPase, and the level of MDA in PC12 cells exposed to OGD. PC12 cells were pretreated without or with LC (200 μmol/L) and ALC (200 μmol/L) for 24 h before OGD exposure for 120 min. PC12 cells treated without OGD used as an normal control. (a) Super oxide dismutase (SOD) activity in PC12 cells; (b) The levels of MDA in PC12 cells; (c) The ATPase activity in PC12 cells. Values given are the mean ± SEM (n = 6). * p < 0.05 compared with OGD groups.