Fig. 66.2 Expression of GFAP in Müller cells. (a–d) The GFAP staining intensity of RCS rat Müller cells increased with the progression of retinal degeneration (PND15, 30, 60, 120, respectively). Note the staining in the vitreal endfeet compared to control rat retinas at similar ages (e–h). Scale bars 20 μm

66.3.3Effect of Mixed Retinal Cells of RCS Rats on Normal Müller Cells

After one day of co-culturing, few Müller cells expressed GFAP, and no ERK positive cells were found. At 3 d and 7 d after co-culture, the number of GFAP and ERK positive cells increased dramatically, in contrast GFAP and ERK expression was not found in control cultures. Western-blot test confirmed that after 3 days the quantity of GFAP and ERK protein expressed by Müller cells (GFAP: 0.59±0.16; ERK: 0.59±0.14) was significantly higher (P<0.05) than in control cultures (GFAP: 0.39±0.11; ERK: 0.25±0.06) (Fig. 66.5).

66.4 Discussion

As a chronic retinal degenerative disease, retinitis pigmentosa, which features a progressive loss of photoreceptors, also shows remarkable changes of other retinal neurons, such as bipolar and retinal ganglion cells. However, Müller cell changes have not been characterised morphologically. Müller cells are connected to almost all the retinal neurons by their specialized morphology, which is the foundation of normal retinal function (Reichenbach et al., 1995; Newman 1996). Müller cells are involved in many retinal physiological activities including glycometabolism, blood regulation, neurotransmitter cycling, homeostasis and control of neuronal excitability (Newman and Zahs 1998; Stevens et al., 2003; Bringmann et al., 2004). Therefore, when Müller cells are activated, the changed function may dramatically affect the retinal pathophysiologic processes.

Fig. 66.5 Expression GFAP (green) and ERK (red) in response to co-cultures of RCS retinal tissue. (a–b) Each set of four micrographs shows the labeling for GFAP (upper left) ERK (upper right), DAPI (lower left) and the merged images (lower right) after 7 days RCS co-culture (a) and control co-culture (b). Scale bar 20 μm. (c) Western-blot confirmed the increase expression of GFAP and ERK in Müller cells at different co-cultured days. Lanes 1, 3 and 5 represent Müller cells co-cultured with controls after 1, 3, and 7 days respectively; lanes 2, 4, and 6 show expression of Müller cells co-cultured with mixed RCS rat retinal cells after 1, 3, and 7 days respectively. After 3 days coculture the quantity of GFAP and ERK protein expressed by Müller cells was significantly higher (P < 0.05) than in control cultures (n = 3 samples)

At an early stage of embryonic development, the immature Müller cells play an important role in the formation of retinal neural circuitry by supplying framework for neuronal migration. When mature Müller cells are activated, they may resume this kind of function (Willbold et al., 1997; Stier and Schlosshauer 1998). We found that Müller cells showed remarkable change in the progress of RCS rat retinal degeneration. In response to the simulation induced by retinal degeneration, Müller cells develop morphologically more rapidly than cells in normal animals, which may be considered as the beginning of compensation. With the progress of retinal degeneration, RCS rat Müller cells become hypertrophic and disorganized and as degeneration proceeds part of their processes extend into the subretinal space. This invasion of the subretinal space might be a major component of the ‘glial seal’ and an attempt at retinal reconstruction. Studies on AMD (age-related macular degeneration) and retinal detachment showed that when activated, Müller cells began to proliferate, and may extend their processes into other layers of the retina including the subretinal space (Steven and Geoffrey 2003; Sullivan et al., 2003; Robert et al., 2003). The formation of the glial seal blocks the connection between RPE and photoreceptors thus exacerbating the disease process. In this study, we have seen both

the initial proliferation of Müller cells and the formation of a similar glial seal composed of Müller cell processes (immunostaining) in RCS rats with advanced retinal degeneration.

Our Western blot analysis confirmed that the expression of proteins related to Müller cell activation are indeed upregulated. GFAP and ERK are activation-related proteins of Müller cell. Under many pathological conditions, the expression of GFAP in Müller cells increases, thus establishing GFAP as a marker of Müller cell activation (Malhotra et al., 1990; Vijay 2007). In other animal models such as glaucoma, retinal detachment and diabetes retinopathy, Müller cells were activated at early stage of injury by showing an increased expression of GFAP (Francke et al., 2001; Fletcher et al., 2005; Xue et al., 2006). ERK is also a marker of glial cell proliferation and differentiation (Yang et al., 2008). In the retina, ERK is prominently expressed in Müller cells, and in many retinal disease models its increased expression is coincident with an increase in GFAP, thus suggesting that Müller cells initiate proliferation and differentiation (Scott et al., 2001; Masumi et al., 2002; Gulgun et al., 2003). The significant increase of GFAP and ERK protein seen in this study supports the data showing proliferation of Müller cells and their activated in response to the progression of retinal degeneration in the of RCS rats.

To determine whether the Müller cell activation is a primary reaction or a secondary response to the retinal degeneration, we co-cultured mixed retinal cells of RCS rats with Müller cells from control animals. The results showed a significant increase of GFAP and ERK expression in response to the degenerative PND30 RCS rat retina. From that under the effect of mixed retinal cells of RCS rats, this suggests that normal Müller cells are activated by a diffusible factor(s) in the RCS rat retina.

We have shown by both in vivo and in vitro techniques that Müller cells were activated in the progress of RCS rats retinal degeneration and underwent dramatic changes in morphology and protein expression. Determining the factors that initiate this process and ways to control this process may significantly improve the rehabilitation of the degenerating retina.

Acknowledgments The authors thank Dr. T. Fitz Gibon for comments on earlier drafts of the paper; Yu Xiao Zeng, Dong Ning Liu, Li Feng Chen, Yan Hua Wang for excellent technical support. This work was supported by the National Basic Research Program of China (Grant No. 2007CB512203), and the funds from a Nature Science Foundation of China (Grant No. 30772371).

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Chapter 67

Effect of 3 -Daidzein Sulfonic Sodium on the

Anti-oxidation of Retinal Ischemia/Reperfusion Injury in Rats

Huang Zhihua, Li Liangdong, Li Xiao, Cheng Fang, and Zeng Jing

Abstract

Objective: To research the 3 -daidzein sulfonic sodium’s (DSS) effect on antioxidation of retinal ischemia/reperfusion (RI/R) injury in rats.

Methods: RI/R in rats was made by reperfusion for 1 h after occlusion of common carotid artery (CCA) for 1 h and the rats were divided into four groups randomly, including sham control group, model group of RI/R injury, low-dosage DSS group and high-dosage DSS group. 1 ml/kg NS was injected through sublingual vein after CCA was dissociated in sham group. Other groups were treated with normal saline, low-dosage DSS (1 mg/kg) and high-dosage DSS (2 mg/kg) through sublingual vein respectively before occlusion of CCA. After occlusion of CCA for 1 h and reperfusion for 1 h, blood was put out and separated into the serum. Then detect the contents of MDA and NO, the activity of SOD and GSH-PX.

Result: (1) The content of MDA was increased in model group (P<0.05), and lowdosage DSS can reverse it (P<0.05). (2) The content of serum NO in model group was lower than in sham group (P<0.001). The content of serum NO in model group is lower than in low-dosage DSS group (P<0.05) and high-dosage DSS group (P<0.01) respectively. (3) The activity of GSH-PX in high-dosage group was higher than in model group (P<0.05). (4) The activity of SOD in low-dosage group was higher than in model group (P<0.05).

Conclusion: DSS can decrease the cellular damage and protect the optical function in RI/R injury through elevating the content of NO, the activity of SOD and GSH-PX, and increasing anti-oxidation.

Z. Jing (B)

Preclinical Medical College, Gannan Medical University, Ganzhou, Jiangxi Province 341000, China

e-mail: zengjing61@hotmail.com

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_67,C Springer Science+Business Media, LLC 2010