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神经损伤与再生  

参与局灶性脑缺血再灌注损伤修复的轴突导向因子1一文的图片
(Volume 8 Number 1 January 2013)


Cite this article:
Xiaodan Wang, Jinming Xu, Jieqin Gong, Hui Shen, Xiaoping Wang. Expression of netrin-1 and its receptors, deleted in colorectal cancer and uncoordinated locomotion-5 homolog B, in rat brain following focal cerebral ischemia reperfusion injury[J]. Neural Regeneration Research, 2013, 8(1): 64-69.

 


Quantitative analysis of experimental animals

In this study, a total of 44 Sprague-Dawley rats were divided into a sham surgery group (n = 8) and a model group (n = 36). The focal cerebral ischemia reperfusion model was established in the model group, in which four rats died of excessive anesthesia-induced respiratory arrest, external carotid artery, cervical vagus nerve injury, or perforated vessel rupture at the carotid artery bifurcation. Ultimately, 8 rats in the sham surgery group and 32 rats in the model group were analyzed.

 

Netrin-1 expression in rat brain after focal cerebral ischemia reperfusion

Immunofluorescence microscopy demonstrated that netrin-1 was not expressed in the cerebral cortex of sham-operated rats and model rats. At 1 day after focal cerebral ischemia reperfusion, weakly fluorescent netrin-1 positive cells were visible in the cerebral cortex and striatum around the infarct in the model rats, but netrin-1 expression was absent in the center of infarct site. Netrin-1 expression in brain tissue of model rats increased significantly with reperfusion time, with the fluorescence becoming more intense particularly in the cerebral cortex at the peripheral ischemic site. The expression of netrin-1 peaked at 14 days and then gradually decreased. Netrin-1 was localized in the neuronal cell body (Figure 1).

Figure 1  Netrin-1 expression around the ischemia site in the cerebral cortex of model rats (immunofluorescence staining; scale bar: 50 μm).

(A–D) 1, 7, 14 and 21 days after ischemia, respectively. Netrin-1 expression was detected with an Alexa Fluor 488-conjugated secondary antibody (green).

Double immunofluorescence staining showed that netrin-1 immunoreactive cells and neuronal nuclear antigen-labeled neurons were co-expressed in the brain tissues of model rats after ischemia and reperfusion[14]. The fluorescence intensities of glial fibrillary acidic protein and CD11b, markers of reactive astrocytes and activated microglia[15-16], were significantly enhanced in large cells at the peripheral infarct site. However, glial fibrillary acidic protein and CD11b did not overlap with netrin-1, indicating that netrin-1 was expressed in neurons but not in astrocytes and microglia. In addition, netrin-1 expression was found at the edge of small vessels around the infarct site (Figure 2).

Figure 2 Netrin-1 was co-expressed with NeuN, GFAP and CD11b around the infarct site in the cerebral cortex at 14 days after middle cerebral artery occlusion (immunofluorescence double staining; scale bars: 20 μm).

Netrin-1 positive cells and NeuN labeled neurons were co-expressed (A), but netrin-1 did not co-localize with GFAP labeled astrocytes (B) and CD11b labeled microglia (C). (C) Netrin-1 was expressed around the vessels (arrow). Netrin-1 fluorescence is shown in green.

NeuN, GFAP and CD11b were detected with Alexa Fluor 594-conjugated secondary antibodies (red). NeuN: Neuronal nuclear antigen; GFAP:glial fibrillary acidic protein.

 

Expression of the Netrin-1 receptor, DCC, was upregulated in rat brain after focal cerebral ischemia
Immunofluorescence microscopy demonstrated that DCC was not expressed in the cerebral cortex of sham-operated rats and model rats, while few DCC positive cells were visible in the subcortical white matter and the hippocampus. At 1 day after reperfusion, the number of DCC positive cells in the cerebral cortex around the infarct in model rats increased significantly, with the DCC fluorescence intensity and the number of positive cells peaking at 14 days and then gradually decreasing (Figure 3).

Figure 3 Expression of deleted in colorectal cancer at the peripheral infarct site in the cerebral cortex of model rats (immunofluorescence staining; scale bar: 50 μm).

(A–D) 1, 7, 14 and 21 days after middle cerebral artery occlusion, respectively. Deleted in colorectal cancer expression was detected with an Alexa Fluor 488-conjugated secondary antibody (green).

DCC expression was localized in the cell membrane and the protrusions. Double immunofluorescence staining detected the co-expression of DCC, neuronal nuclear antigen and glial fibrillary acidic protein in the cerebral cortex around the infarct site, which mainly localized to neurons, and to a lesser extent astrocyte protrusions (Figure 4).

Figure 4 The deleted in colorectal cancer (DCC) receptor was co-expressed with NeuN and GFAP around the infarct site in the cerebral cortex at 14 days after middle cerebral artery occlusion (immunofluorescence double staining; scale bars: 20 μm).

The DCC positive signals are distributed in the cell membrane and protrusions, and are co-expressed with NeuN labeled neurons and GFAP labeled astrocytes. DCC fluorescence is shown in green. NeuN and GFAP were detected with Alexa Fluor 594-conjugated secondary antibodies (red). DAPI labeled nuclei are shown in blue.

(A) NeuN; (B) DCC; (C) DAPI; (D) DCC+/NeuN+/DAPI; (E) GFAP; (F) DCC; (G) DAPI; (H) DCC+/GFAP+/DAPI. (A–D) The same field of view; (E–H) the same field of view. NeuN: Neuronal nuclear antigen; GFAP:glial fibrillary acidic protein.

Focal cerebral ischemia did not affect the expression of the netrin-1 receptor, UNC5B, in rat brain

Immunofluorescence microscopy demonstrated that UNC5B was expressed in the cerebral cortex of sham-operated rats, and in the cerebral cortex and basal ganglia of model rats. The number of UNC5B positive cells and their staining intensity in the model rats were similar to those in the control group. UNC5B was expressed at the cell membrane and the protrusions, similar to DCC (Figure 5).

Figure 5 Expression of the netrin-1 receptor, uncoordinated locomotion-5 homolog B, in the rat cerebral cortex. Uncoordinated locomotion-5 homolog B expression was detected with an Alexa Fluor 488-conjugated secondary antibody (green). DAPI labeled nuclei are shown in blue. Scale bars: 50 μm.

(A) Cerebral cortex in the sham operation group.

(B) Peripheral ischemia cerebral cortex at 14 days after reperfusion.
 
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