A Pilot Study for the Neuroprotective Effect of Gongjin-dan on Transient Middle Cerebral Artery Occlusion-Induced Ischemic Rat Brain

Abstract

In this study, we investigated whether gongjin-dan improves functional recovery and has neuroprotective effects on reducing the infarct volume after transient middle cerebral artery occlusion (MCAo). Infarct volume was measured using TTC staining and glucose utilization by F-18 FDG PET. Functional improvement was evaluated with the Rota-rod, treadmill, Garcia score test, and adhesive removal test. At 14 days after MCAo, neuronal cell survival, astrocytes expansion, and apoptosis were assessed by immunohistofluorescence staining in the peri-infarct region. Also, the expression of neurotrophic factors and inflammatory cytokines such as VEGF, BDNF, Cox-2, TNF-α, IL-1β, and IL-1α was measured in ischemic hemisphere regions. The gongjin-dan-treated group showed both reduced infarct volume and increased glucose utilization. Behavior tests demonstrated a significant improvement compared to the control. Also in the gongjin-dan treated group, NeuN-positive cells were increased and number of astrocytes, microglia, and apoptotic cells was significantly decreased compared with the control group in the ischemic peri-infarct area. Furthermore, the expression of VEGF and BDNF was increased and level of Cox-2, TNF-α, IL-1β, and IL-1α was decreased. These results suggest that gongjin-dan may improve functional outcome through the rapid restoration of metabolism and can be considered as a potential neuroprotective agent.

Figure 1

Effect of gongjin-dan on functional recovery after MCAo. Behavior performances in the Garcia's score (a), adhesive removal (b), Rota-rod (c), and treadmill test (d) from 1 to 14 days after MCAo. Behavior tests were significantly different at each time point starting at 3 days after MCAo. Data are expressed as mean ± SD, *P < 0.05.

Figure 2

Effect of gongjin-dan on infarct volume, glucose metabolism, and regional cerebral blood flow (rCBF) after MCAo. Images shown in the PET images and TTC-stained section of control (a) and gongjin-dan treatment group (b) at 14 days. ROI by F-18 FDG uptake was revealed as differential uptake rations (DURs) (c). Infarct volume was then analyzed using Meta-Morph program (d). In gongjin-dan treatment group, infarct volume was decreased compared to the control group. Also glucose uptake was increased significantly compared with control group. rCBF was significantly increased compared with control group (e). These effects were observed for 3, 4, 5, 8, 9, and 12 days after MCAo. Data are expressed as mean ± SD, *P < 0.05.

Figure 3

Quantitative analysis of immunoreactivities of GFAP, NeuN, and OX-42 at days on peri-infarct area after MCAo. Histological analysis was shown into GFAP, NeuN, OX-42 staining at 14 days after MCAo. Quantitative immunoreactivities for groups are shown as bar graphs on the right side of each panel. The expression of GFAP in gongjin-dan group was significantly decreased compared with control group. NeuN-positive cells also showed difference in the control group. There were many NeuN-positive cells in gongjin-dan group compared to control group. The expression of OX-42 in gongjin-dan group was significantly decreased compared with control group. In the gongjin-dan, the number of activated microglial cells was decreased and the number of neuronal cells was increased in the peri-infarct area. Data are expressed as mean ± SD, *P < 0.05.

Figure 4

Effect of gongjin-dan on the number of apoptotic cells after MCAo. TUNEL staining was used to identify apoptotic cells. (a) Apoptotic cells were identified by immunofluorescence Cy3 (red). TUNEL positive cells were counted using Meta-Morph program (b, d). TUNEL positive cells show the quantitative analysis data (e). The number of TUNEL positive cells was significantly decreased in the drug treated group compared to the control group. Data are expressed as mean ± SD, *P < 0.05, Scale bars denote, 50 μm.

Figure 5

Expression of growth factors and inflammatory cytokines in the ischemic rat brain. BDNF, VEGF, Cox-2, TNF-α, IL-1β, and IL-1α were detected by ELISA at 14 days after MCAo. Protein levels of these factors show the quantitative analysis data (a–f). The expression of BDNF and VEGF was significantly increased compared with the control group at 14 days after MCAo (a, b). Also, inflammatory cytokines such as Cox-2, TNF-α, IL-1β, and IL-1α were significantly decreased compared with the control group (c, d, e, f). Data are expressed as mean ± SD, *P < 0.05.