Table 4-1. Comparison of physiological parameter among the groups
Sham Control FA60 FA80 FA100 DFA
10 min before MCAo
Blood sugar (mg/dl) 103.0±23.1 115.2±23.1 110.2±33.2 123.0±29.1 88.7±23.6 116.2±35.8 pH 7.31±0.03 7.32±0.04 7.31±0.02 7.33±0.04 7.31±0.03 7.31±0.03 pO2 (mmHg) 106.0±14.4 95.8±10.9 105.7±11.3 105.5±9.7 98.2±9.9 95.0±8.9 pCO2 (mmHg) 27.0±8.4 30.2±3.5 24.0±5.7 30.0±8.1 30.2±5.5 30.8±2.7 MABP (mmHg) 79.2±13.3 81.8±10.2 77.7±8.5 87.2±12.5 74.2±5.0 76.8±9.9 Heart rate (beats/min) 347±68 350±42 393±34 318±50 345±45 324±51 90 min after MCAo
Blood sugar (mg/dl) 112.7±30.8 101.2±15.2 114.2±15.5 113.7±24.2 103.5±33.4 117.5±20.7 pH 7.33±0.04 7.36±0.03 7.36±0.03 7.36±0.03 7.35±0.05 7.34±0.05
pO2 (mmHg) 92.2±11.3 96.7±11.0 97.0±11.5 102.3±8.3 90.3±8.0 92.5±14.3
Data were presented as Mean ± SD. Sham: sham group; Control: control group; FA60: FA60 group; FA80: FA80 group; FA100: FA100 group; FA60: FA100 group; DFA: DFA group;
MK: MK group; MCAo: middle cerebral artery occlusion; MABP: mean arterial blood pressure.
Table 4-1. 續
Sham Control FA60 FA80 FA100 DFA
pCO2 (mmHg) 27.8±7.7 27.1±8.0 23.1±4.9 20.7±3.4 26.7±5.1 22.0±5.0 MABP (mmHg) 82.0±9.6 84.3±9.9 82.3±13.0 87.2±14.3 81.3±16.5 75.2±10.4 Heart rate (beats/min) 340±65 397±89 363±31 407±62 392±72 352±50 10 min after reperfusion
Blood sugar (mg/dl) 105.8±32.9 99.2±19.8 108.3±25.8 115.7±24.8 125.2±20.9 124.0±33.4 pH 7.34±0.02 7.36±0.03 7.38±0.05 7.36±0.03 7.35±0.04 7.37±0.05 pO2 (mmHg) 95.0±6.5 90.8±5.9 100.8±11.4 101.7±10.9 95.8±8.50 92.0±7.2 pCO2 (mmHg) 26.6±6.1 24.3±6.5 20.4±3.3 19.6±5.4 24.3±4.9 21.1±4.4 MABP (mmHg) 81.2±12.0 84.3±14.0 88.5±10.2 80.3±14.1 88.5±17.8 77.8±8.0 Heart rate (beats/min) 342±52 436±61 373±51 394±76 414±56 362±56
4-1-2. FA 對再灌流 24 小時後腦梗塞面積之影響
中大腦動脈阻塞90 分鐘，再灌流 24 小時各實驗組形成腦梗塞(Figure 4-1A)。我們分析發現 control、 FA60、 FA80、 FA100及 DFA 組之梗塞 面積百分比均顯著大於sham 組(p < 0.001, p < 0.001, p < 0.01, p < 0.05, p
< 0.01, respectively)。進一步分析發現 FA80、FA100及DFA 組之梗塞面積 百分比顯著小於control 組(all p < 0.001)及 FA60組(p < 0.05, p < 0.01, p <
0.05, respectively) (Figure 4-1B)。
Figure 4-1. Effect of ferulic acid on cerebral infarct in ischemia-reperfusion injured rats. (A) Focal cerebral infarct developed after the middle cerebral artery had been occluded for 90 min followed by 24 h of reperfusion in Sprague-Dawley rats. The 2,3,5-triphenyl-tetrazolium chloride stain of the infarct area showed a white color, whereas the non-infarct area was reddish-purple color. Sham: sham group; Control:
control group; FA60: FA60 group; FA80: FA80 group; FA100: FA100 group;
DFA: DFA group. Scale bar= 1 cm. (B) Comparison with the percentage of infarct area among the sham, control, FA60, FA80, FA100 and DFA groups was measured at 24 h of reperfusion. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the sham group; ###p < 0.001 compared with the control group; @p < 0.05, @@p < 0.01 compared with the FA60 group.
(A)
4-1-3. FA 對再灌流 24 小時後神經學行為缺陷之影響
老鼠於中大腦動脈阻塞90 分鐘，再灌流 24 小時形成程度不等之神經 學缺陷。control (13.5 ± 0.8)、FA60 (11.5 ± 1.5)、 FA80 (8.5 ± 0.8)、 FA100 (8.3 ± 0.5)及 DFA (9.8 ± 1.2) 組之神經學缺陷分數顯著大於 sham (1.2 ± 0.4) 組 (all p < 0.001)。然而，FA80、 FA100及DFA 組之神經學缺陷分 數顯著低於control 組 (all p < 0.001)。另外，FA80及FA100組之神經學缺 陷分數亦顯著低於FA60 組 (p < 0.001, p < 0.001, respectively)。
4-1-4. FA 對再灌流 2 小時後 oxidized hydroethidine 螢光染色陽性細胞表現 之影響
所有本實驗偵測之免疫陽性(immunoreactive; positive)細胞或血管均 (B)
在腦冠切片正方形虛線內計數定量(counts/1mm2, Figure 4-2)。再灌流 2 小時，control 組於皮質與紋狀體梗塞區之 oxidized hydroethidine 表現均 顯著高於sham 組 (both p < 0.001)；而 FA100 組於皮質與紋狀體梗塞區 之oxidized hydroethidine 表現均顯著低於 control 組(both p < 0.01; Figure 4-3, Table 4-2)。
Figure 4-2. Representative photograph showed the brain coronal section located on the posterior bregma 0.92 mm position. The square dotted line within the cerebral infarct zone indicates the region for calculating immunoreactive (positive) cells (vessels). cortex: the ischemic core of cortex; striatum: the ischemic core of striatum. The length and width of square dotted line= 1 mm.
Figure 4-3. Effect of ferulic acid on oxidized hydroethidine fluorescent stain positive cells at 2 h of reperfusion. Sham: sham group; Control:
control group; FA100: FA100 group; Cortex: the ischemic core of cortex;
Striatum: the ischemic core of striatum. Arrows indicated oxidized hydroethidine positive cells. Scale bar= 40 μm.
4-1-5. FA 對再灌流 2 小時後 ICAM-1、MPO 及 NF-κB (p50)陽性細胞(血管) 表現之影響
再灌流2 小時於皮質梗塞區，ICAM-1 免疫陽性血管數於 sham、control 及 FA100 組表現，彼此之間並無統計學上的差異(Data not shown)。而在 再灌流2 小時紋狀體梗塞區，control 及 FA100 組之 ICAM-1 免疫陽性血 管數表現顯著大於sham 組 (p < 0.001 and p < 0.05, respectively; Figure 4-4, Table 4-2)，然而 FA100 組之 ICAM-1 免疫陽性血管數表現顯著低於
control 組 (p < 0.01; Figure 4-4, Table 4-2)。而在 MPO 的表現，於皮質 及紋狀體梗塞區中，FA100 和 control 組彼此之間並無統計學上的差異 (Data not shown)；另外在 NF-κB (p50)表現方面，sham、control 及 FA100 組 在 皮 質 及 紋 狀 體 梗 塞 區 彼 此 之 間 並 無 統 計 學 上 的 差 異(Data not shown)。
Figure 4-4. Effect of ferulic acid on the intercellular adhesion molecule-1 (ICAM-1) immunoreative vessels at 2 h and 24 h of reperfusion. Sham:
sham group; Control: control group; FA100: FA100 group; Cortex: the ischemic core of cortex; Striatum: the ischemic core of striatum; 2 h: at 2 h of reperfusion; 24 h: at 24 h of reperfusion. Arrows indicated ICAM-1 immunoreactive vessels. Scale bar= 40 μm.
4-1-6. FA 對再灌流 24 小時後 ICAM-1、MPO 及 NF-κB (p50)陽性細胞(血 管)表現之影響
在皮質梗塞區中，再灌流 24 小時 control 及 FA100 組之 ICAM-1 免 疫 陽 性 血 管 數 表 現 顯 著 大 於 sham 組 (p < 0.001 and p < 0.05, respectively)，同一時間，FA100 組之 ICAM-1 免疫陽性血管數表現則顯 著低於control 組(p < 0.001; Figure 4-4, Table 4-2)。另外在紋狀體梗塞區 中，control 及 FA100 組之 ICAM-1 免疫陽性血管數表現顯著大於 sham 組(p < 0.001 and p < 0.05, respectively)；縱使如此，FA100 組之 ICAM-1 免疫陽性血管數表現仍低於control 組(p < 0.01; Figure 4-4, Table 4-2)。
MPO 之分析，我們發現 control 及 FA100組於皮質梗塞區之MPO 表現顯 著高於sham 組(p < 0.001 and p < 0.01, respectively)；然而 FA100 組之 MPO 表現則顯著低於 control 組(p < 0.001; Figure 4-5, Table 4-2)。另外 在紋狀體梗塞區，control 及 FA100組之MPO 表現彼此之間並無統計學上 的差異(Data not shown)。此外在 NF-κB (p50)免疫陽性細胞之表現，
control 及 FA100組於皮質梗塞區之NF-κB (p50)免疫陽性細胞表現顯著高 於sham 組(p < 0.001 and p < 0.01, respectively)；而 FA100組之NF-κB (p50) 免疫陽性細胞表現仍顯著低於 control 組(p < 0.001; Figure 4-5, Table 4-2。另外我們發現 control 及 FA100組於紋狀體梗塞區之 NF-κB (p50)免 疫陽性細胞表現顯著高於sham 組(p < 0.001 and p < 0.01, respectively)；
進一步分析得知FA100組之NF-κB (p50)免疫陽性細胞表現仍低於 control 組(p < 0.01; Figure 4-5, Table 4-2)。
Figure 4-5. Effect of ferulic acid on the nuclear factor-κB (NF-κB) and myeloperoxidase (MPO) immunoreative cells at 24 h of reperfusion. Sham:
sham group; Control: control group; FA100: FA100 group; Cortex: the ischemic core of cortex; Striatum: the ischemic core of striatum; Arrows indicated NF-κB and MPO immunoreactive cells, respectively. Scale bar=
40 μm.
Table 4-2. The expression of oxidized hydroehtidine, ICAM-1, MPO and NF-κB immunoreactive (positive) cells (vessels) (counts/1mm2)
2h 24h
Data were presented as Mean ± SD. Oxidized hydroethidine: oxidized hydroethidine positive cells; ICAM-1: ICAM-1 immunoreactive vessels; MPO: MPO immunoreactive cells; NF-κB: NF-κB immunoreactive cells; Cortex: the ischemic core of cortex; Striatum:
the ischemic core of striatum; Sham: sham group; Control: control group; FA100:FA100
group; 2 h: at 2 h of reperfusion; 24 h: at 24 h of reperfusion. †p<0.05, ††p<0.01,
†††p<0.001 compared with the sham group; **p<0.01, ***p<0.001 compared with the control group.
Table 4-2. 續
2h 24h MPO
Cortex
Sham 0.5±0.8
Control 183.8±21.2†††
FA100 77.0±52.9††***
NF-κB Cortex
Sham 1.3±2.0
Control 267.2±44.0†††
FA100 108.5±59.4††***
Striatum
Sham 0.3±0.5
Control 307.0±94.6†††
FA100 142.5±52.1††**
4-2.第二階段實驗結果 4-2-1.生理指標
於腦缺血前10 分鐘及腦缺血後 90 分鐘測量生理指標包括有 blood gas(pH、pO2 及 pCO2)及 blood sugar，結果得知 sham 組、control 組及 FA 組相較，彼此之間生理指標並無統計學上的差異(p>0.05; Table 4-3)。
Table 4-3. Physiological parameters
Data were presented as Mean ± SD. BS: blood sugar; sham: sham group; control: control group; FA: ferulic acid-treated group; 10 min before ischemia: 10 min before cerebral ischemia; 90 min after ischemia: 90 min after cerebral ischemia; 2 h: sacrificed at 2 h of reperfusion; 10 h: sacrificed at 10 h of reperfusion; 24 h: sacrificed at 24 h of reperfusion;
36 h: sacrificed at 36 h of reperfusion.
10 min before ischemia
90 min after ischemia
Table 4-3. 續
ICAM-1 mRNA 於皮質梗塞區的表現，發現 sham 組、control 組及 FA 組相較，彼此之間並無統計學上的差異(p>0.05; Figure 4-6)。而 ICAM-1 mRNA 於紋狀體梗塞區的表現，得知 control 組同側(ipsilateral hemisphere) 梗塞區顯著高於sham 組(2.1-fold, p<0.05; Figure 4-6)；另外 FA 組同側梗 塞區ICAM-1 mRNA 表現則顯著低於 control 組(0.7-fold, p<0.05; Figure 4-6)。而在 control 組中，我們也合理地發現同側梗塞區 ICAM-1 mRNA
的表現顯著高於對側區(1.7- fold, p<0.05; Figure 4-6)。接著分析 Mac-1 mRNA 的表現，在皮質梗塞區表現和 ICAM-1 mRNA 類似，sham 組、
control 組及 FA 組相較，彼此之間並無統計學上的差異(p>0.05; Figure 4-6)；在紋狀體同側梗塞區方面，sham 組和 control 組並無統計學上的差 異(p>0.05; Figure 4-6)，而 FA 組之 Mac-1 mRNA 表現則顯著低於 control 組(0.5-fold, p<0.05; Figure 4-6)。此外，control 組與 FA 組於皮質及紋狀 體梗塞區之IL-1β 和 TNF-α mRNA 表現之比較均無統計學上的差異(Data not shown)。
Figure 4-6. The expression of ICAM-1 and Mac-1 mRNA in the ischemic areas of the cortex and striatum were detected at 2 h of reperfusion. (A) Representative RT-PCR signals showed the expression of ICAM-1 and Mac-1 mRNA in the chosen areas of cortex and striatum. C: ischemic cortex; S: ischemic striatum; ipsi: ipsilateral; cont: contralateral; sham:
sham group; control: control group; FA: ferulic acid-treated group.
GAPDH mRNA was used as the internal control. The relative expression of (B) ICAM-1 and (C) Mac-1 mRNA was quantified in the chosen striatum of the sham, control and FA groups. Data were presented as mean ± SD.
*p<0.05 compared with the sham group; #p<0.05 compared with the control group; &p<0.05 compared with the ipsilateral hemisphere. (n=4) (A)
4-2-3. FA 對再灌流 2、10、24 及 36 小時後 Mac-1、4-HNE 及 8-OHdG 陽
Densitometry of ICAM-1 gene expression relative to GAPDH (%)
& #
*
(B)
sham control FA
Densitometry of Mac-1 gene expression relative to GAPDH (%)
0
Mac-1 抗原表現於活化白血球及微膠細胞中。在半陰影區及皮質、紋
control 組及 FA 組之 4-HNE 陽性細胞則顯著呈現(both p<0.01)；縱使如 此，FA 組之 4-HNE 陽性細胞表現仍低於 control 組(p<0.05; Figure 4-9;
Table 4-4)。在半陰影區及梗塞核心區中，分析 8-OHdG 陽性細胞得知，
再灌流 2 小時 8-OHdG 陽性細胞少量呈現於 sham 組、control 組及 FA 組，隨後再灌流10、24 及 36 小時，相較於 sham 組，8-OHdG 陽性細胞 則顯著呈現於 control 組及 FA 組(both p<0.01)，而在上述各個相同時間 點，FA 組與 control 組相較，顯著降低 8-OHdG 陽性細胞之表現(p<0.05; (both p<0.01)，而 FA 組之 TUNEL 陽性細胞則顯著低於 control 組(p<0.05;
Figure 4-11; Table 4-4)。在皮質及紋狀體梗塞區中，再灌流 24 及 36 小時，
control 組及 FA 組之 TUNEL 陽性細胞數均顯著高於 sham 組(both p<0.01)，然而，FA 組之 TUNEL 陽性細胞仍顯著低於 control 組(p<0.05;
Figure 4-11; Table 4-4)。
Figure 4-7. Representative photograph showed the brain coronal section (2,3,5-triphenyl tetrazolium chloride stain; TTC stain) located on the posterior bregma 0.92 mm position. The dotted line squares indicated the areas for evaluating positive cells. P: penumbra area of the cortex; C:
ischemic core area of the cortex; S: ischemic core area of the striatum.
Dotted square=1 mm2
Figure 4-8. Representative photographs depicted the expression of Mac-1 in the penumbra and ischemic core areas at 10, 24, and 36 h of reperfusion. P:
penumbra area of the cortex; C: ischemic core area of the cortex; S: ischemic core area of the striatum; N: negative control of stain; 10 h: sacrificed at 10 h of reperfusion; 24 h: sacrificed at 24 h of reperfusion; 36 h: sacrificed at 36 h of reperfusion; sham: sham group; control: control group; FA: ferulic acid-treated group. Arrows indicated Mac-1 positive cells. Scale bar=100 μm
Figure 4-9. Representative photographs depicted the expression of 4-HNE in the penumbra and ischemic core areas at 10, 24 and 36 h of reperfusion.
P: penumbra area of the cortex; C: ischemic core area of the cortex; S:
ischemic core area of the striatum; N: negative control of stain; 10 h:
sacrificed at 10 h of reperfusion; 24 h: sacrificed at 24 h of reperfusion; 36 h: sacrificed at 36 h of reperfusion; sham: sham group; control: control group; FA: ferulic acid-treated group. Arrows indicated 4-HNE positive cells. Scale bar=100 μm
Figure 4-10. Representative photographs depicted the expression of 8-OHdG in the penumbra and ischemic core areas at 10, 24 and 36 h of reperfusion. P: penumbra area of the cortex; C: ischemic core area of the cortex; S: ischemic core area of the striatum; N: negative control of stain;
10 h: sacrificed at 10 h of reperfusion; 24 h: sacrificed at 24 h of reperfusion; 36 h: sacrificed at 36 h of reperfusion; sham: sham group;
control: control group; FA: ferulic acid-treated group. Arrows indicated 8-OHdG positive cells. Scale bar=100 μm
Figure 4-11. Representative photographs depicted the distribution of TUNEL positive cells in the penumbra and ischemic core areas at 10, 24 and 36 h of reperfusion. P: penumbra area of the cortex; C: ischemic core area of the cortex; S: ischemic core area of the striatum; 10 h: sacrificed at 10 h of reperfusion; 24 h: sacrificed at 24 h of reperfusion; 36 h: sacrificed at 36 h of reperfusion; sham: sham group; control: control group; FA:
ferulic acid-treated group. Arrows indicated TUNEL positive cells. Scale bar=100 μm
4-2-5. FA 對再灌流 10 小時後 active caspase 3 及 active caspase 3-NeuN 陽 性細胞表現之影響
在active caspase 3 單獨染色中，再灌流 10 小時 active caspase 3 陽性 細胞稀少出現於sham 組之半陰影區中；同一時間，與 sham 組比較，active caspase 3 陽性細胞於 control 組及 FA 組之半陰影區則顯著呈現(both p<0.01)，而 FA 組於半陰影區之 active caspase 3 陽性細胞則顯著低於 control 組(p<0.05; Figure 4-12; Table 4-5)。進一步分析 active caspase 3-NeuN 雙重染色，我們發現 NeuN 陽性細胞大量出現於 sham 組之半陰 影區中，而 active caspase 3 陽性細胞同時染上 NeuN 則表現於 control 組 及FA 組中，其 active caspase 3-NeuN 雙重染色陽性細胞所呈現之分布數 量型態與active caspase 3 單獨染色中所呈現相同(Figure 4-12)。
4-2-6. FA 對再灌流 36 小時後 NeuN 陽性細胞表現之影響
NeuN 陽性細胞大量呈現於 sham 組中，而缺血區中之神經元則呈皺 縮及三角型狀(Figure 4-13)。相較於 sham 組之半陰影區，control 組之 NeuN 陽性細胞數顯著下降(p<0.01)，相反地，FA 組相較於 control 組則 顯著上升NeuN 陽性細胞之表現(p<0.05; Figure 4-13; Table 4-5)。在皮質 及紋狀體梗塞區中，與sham 組相較，control 組及 FA 組顯著降低 NeuN 抗原呈現(both p<0.01)，而 FA 組之 NeuN 陽性細胞數則顯著高於 control 組(p<0.05; Figure 4-13; Table 4-5)。
Figure 4-12. Representative photographs depicted active caspase 3 and NeuN expression in the penumbra area at 10 h of reperfusion. Sham: sham group; control: control group; FA: ferulic acid-treated group; I: active caspase 3 single staining; II: active caspase 3-NeuN double staining; N:
negative control of stain. Arrows in I and II showed active caspase 3-labeled cells and active caspase 3-NeuN double-labeled cells, respectively. Scale bars=100 μm
Figure 4-13. Representative photographs depicted NeuN expression in the penumbra and ischemic core areas at 36 h of reperfusion. P: penumbra area of the cortex; C: ischemic core area of the cortex; S: ischemic core area of the striatum; N: negative control of stain; sham: sham group; control:
control group; FA: ferulic acid-treated group. Scale bar= 100 μm
Table 4-4. The effects of ferulic acid on Mac-1, 4-HNE, 8-OHdG and TUNEL positive cells (counts/1 mm2)
2 h (n=6) 10 h (n=5) 24 h (n=6) 36 h (n=3)
Data were presented as Mean ± SD. Mac-1: Mac-1 positive cells; 4-HNE: 4-HNE positive cells; 8-OHdG: 8-OHdG positive cells; TUNEL: TUNEL positive cells;
penumbra: penumbra area of the cortex; cortex: ischemic core area of the cortex; striatum:
ischemic core area of the striatum; sham: sham group; control: control group; FA: ferulic acid-treated group; 2 h: sacrificed at 2 h of reperfusion; 10 h: sacrificed at 10 h of reperfusion; 24 h: sacrificed at 24 h of reperfusion; 36 h: sacrificed at 36 h of reperfusion.
*p<0.01 compared with the sham group; #p<0.05 compared with the control group.
Table 4-4. 續
Table 4-4. 續
Table 4-5. The effects of ferulic acid on active caspase 3 and NeuN-labeled cells (counts/1 mm2)
10 h (n=5) 36 h (n=3) Active caspase 3
penumbra
sham 0.1 ± 1.1
Data were presented as Mean ± SD. Active caspase 3: active caspase 3-labeled cells;
Data were presented as Mean ± SD. Active caspase 3: active caspase 3-labeled cells;