Luoyutong Treatment Promotes Functional Recovery and Neuronal Plasticity after Cerebral Ischemia-Reperfusion Injury in Rats

Abstract

Luoyutong (LYT) capsule has been used to treat cerebrovascular diseases clinically in China and is now patented and approved by the State Food and Drug Administration. In this retrospective validation study we investigated the ability of LYT to protect against cerebral ischemia-reperfusion injury in rats. Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion followed by reperfusion. Capsule containing LYT (high dose and medium dose) as treatment group and Citicoline Sodium as positive control treatment group were administered daily to rats 30 min after reperfusion. Treatment was continued for either 3 days or 14 days. A saline solution was administered to control animals. Behavior tests were performed after 3 and 14 days of treatment. Our findings revealed that LYT treatment improved the neurological outcome, decreased cerebral infarction volume, and reduced apoptosis. Additionally, LYT improved neural plasticity, as the expression of synaptophysin, microtubule associated protein, and myelin basic protein was upregulated by LYT treatment, while neurofilament 200 expression was reduced. Moreover, levels of brain derived neurotrophic factor and basic fibroblast growth factor were increased. Our results suggest that LYT treatment may protect against ischemic injury and improve neural plasticity.

Figure 1

Schematic representation of the experimental procedures. (Luoyutong: LYT; middle cerebral artery occlusion: MCAO; 2,3,5-triphenyltetrazolium chloride: TTC; microtubule associated protein: MAP-2; myelin basic protein: MBP; brain derived neurotrophic factor: BDNF; basic fibroblast growth factor: b-FGF; neuron-specific nuclear protein: NeuN; neurofilament 200: NF200).

Figure 2

LYT (Luoyutong) decreases infarct volume and improves neurological function in rats following ischemic reperfusion. (a) Longa's score, 0–12 neurological score test, and modified foot fault test of MCAO (middle cerebral artery occlusion), LYTM (medium dose (0.4 g/kg) of LYT), LYTH (high dose (0.8 g/kg) of LYT), and CS (Citicoline Sodium) 3 days after MCAO. (b) Longa's score, 0–12 neurological score test, and modified foot fault test of MCAO, LYTM, LYTH, and CS 14 days after MCAO. (c) Infarct volume (%) of MCAO, LYTM, LYTH, and CS 3 days after MCAO. (d) Infarct volume (%) of MCAO, LYTM, LYTH, and CS 14 days after MCAO; ^∗ P < 0.05 versus MCAO and ^∗∗ P < 0.01 versus MCAO.

Figure 3

LYT (Luoyutong) reduces neuronal apoptosis and decreases the level of caspase-3. (a) Neuronal apoptosis in the ipsilateral cortex was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling and 4′ 6-diamidino-2-phenylindole double staining 14 days after ischemic reperfusion. (b) Expression of activated caspase-3 was detected by Western blot 14 days after ischemic reperfusion. n = 3. ^∗ P < 0.05 versus Sham and ^# P < 0.05 versus MCAO.

Figure 4

LYT (Luoyutong) induced upregulation of synaptophysin expression after MCAO (middle cerebral artery occlusion). Western blot detection and quantitative analysis of (a) synaptophysin expression 3 days after MCAO and (c) synaptophysin expression 14 days after MCAO. (b) Representative immunofluorescence images showing colocalization of synaptophysin (red) and NeuN (neuron-specific nuclear protein) (green) in the cortex. Blue DAPI staining indicates the nuclei. n = 3. ^∗ P < 0.05 versus Sham.

Figure 5

LYT (Luoyutong) induced upregulation of MAP-2 (microtubule associated protein) expression after MCAO (middle cerebral artery occlusion). Western blot detection and quantitative analysis of (a) MAP-2 expression 3 days after MCAO and (c) MAP-2 expression 14 days after MCAO. (b) Representative immunofluorescence images showing colocalization of MAP-2 (red) and NeuN (neuron-specific nuclear protein) (green) in the cortex. Blue DAPI staining indicates the nuclei. n = 3. ^∗ P < 0.05 versus Sham, ^∗∗ P < 0.01 versus Sham, and ^# P < 0.05 versus MCAO.

Figure 6

LYT (Luoyutong) induced upregulation of MBP (myelin basic protein) expression after MCAO (middle cerebral artery occlusion). Western blot detection and quantitative analysis of (a) MBP expression 3 days after MCAO and (c) MBP expression 14 days after MCAO. (b) Representative immunofluorescence images showing colocalization of MBP (red) and NF200 (neurofilament 200) (green) in the cortex. Blue DAPI staining indicates the nuclei. n = 3. ^∗∗ P < 0.01 versus Sham and ^# P < 0.05 versus MCAO.

Figure 7

LYT (Luoyutong) induced upregulation of BDNF (brain derived neurotrophic factor) expression after MCAO (middle cerebral artery occlusion). Western blot detection and quantitative analysis of (a) BDNF expression 3 days after MCAO and (c) BDNF expression 14 days after MCAO. (b) Representative immunofluorescence images showing colocalization of BDNF (red) and NeuN (neuron-specific nuclear protein) (green) in the cortex. Blue DAPI staining indicates the nuclei. n = 3. ^∗∗ P < 0.01 versus Sham and ^# P < 0.05 versus MCAO.

Figure 8

LYT (Luoyutong) induced upregulation of b-FGF (basic fibroblast growth factor) expression after MCAO (middle cerebral artery occlusion). Western blot detection and quantitative analysis of (a) b-FGF expression 3 days after MCAO and (c) b-FGF expression 14 days after MCAO. (b) Representative immunofluorescence images showing colocalization of b-FGF (red) and NeuN (neuron-specific nuclear protein) (green) in the cortex. Blue DAPI staining indicates the nuclei. n = 3. ^∗∗ P < 0.01 versus Sham and ^# P < 0.05 versus MCAO.