The human hereditary retinal degeneration is one of the main cause of irreversible blindness in the world. the mechanisms leading to retinal photoreceptor degeneration are not entirely clear. However, microglia acting as innate immune monitors are found to be activated early in retinal degeneration in many retinitis pigmentosa animal models. These activated microglia are involved in phagocyte rod cell fragments of degenerated retina, and also produce high levels of cytotoxic substances such as pro-inflammatory cytokines and chemokines, which aggravate the death of adjacent healthy photoreceptor cells. It suggests that microglia activation plays an important role in photoreceptor degeneration. At the same time, a series of studies have confirmed that some drugs can prevent or reduce neuronal death and slow the occurrence and progression of retinal degeneration by interfering with abnormal activation of microglia. It is expected to be a new choice for the treatment of hereditary retinal degeneration.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
Retinal macrophages and (or) microglial cells play important roles in regulating inflammation, angiogenesis and tissue repairing, thus affect the development and prognosis of ischemic retinal disease, ocular immune diseases and ocular tumors. Reversing the polarization imbalance of these cells may provide new therapeutic strategies for ischemic retinal disease and ocular immune diseases. The duality of the polarization direction of these cells is still controversial in the inflammatory reaction and pathological angiogenesis of ischemic retinal disease. Meanwhile, the plasticity and diversity of the function need to be further studied and discussed.
Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=−4.17, −7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=−30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=−6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
Neural stem cell is a kind of stem cells that can differentiate into neural and glial cells. While Müller cells, the main endogenous neural stem cell in retina,have the features to reentry into the cell cycle and differentiate into neural cells after retinal damage. Although it is highly effective for retinal Müller cell differentiation spontaneously after retinal injury in vertebrates, this feature is rigorous restricted in mammals. Recently, some transcription factors,such as Ascl1, Sox2, Lin28, Atoh7, are sufficient to drive quiescent Müller cells back in proliferation to generate new retinal neurons. Moreover, combining Ascl1 expression with a histone deacetylase inhibitor can bypass the limitation and increase the generation of new neurons in the adult retina. These regenerated neurons integrate the existing neuronal network and are able to respond to light, indicating that they can likely be used to restore vision. While these results are extremely promising, the regenerative response is still limited, likely because the proliferative capacity of mammalian Müller cells is low compared to their zebrafish counterparts. It is indeed necessary to identify new factors increasing the efficiency of the regenerative response.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell. MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining. ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002). ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
Ischemic retinopathy, resulting in multiple lesions like microvasculature damage, inflammation and neovascularization, is a major contributor of vision damage. In these pathological changes, retinal glia cannot be ignored in the development of retinopathy. They constitute a highly versatile population that interacts with various cells to maintain homeostasis and limit disease. Therefore, glial activation and gliosis are strikingly ubiquitous responses to almost every form of retinal disease. Both of microglial cells and Müller cells are major intrinsic retinal glial cells and they are in close relationship, which means they can influence each other, make joint action or even become interdependent. They exhibit morphological and functional changes to have an impact on degree of retinal injury through different responses, which mediated by glial cells are important not only for course of disease progression, but also for the maintenance of neuronal and photoreceptor survival. Thus, defining the mechanisms that underlie communications between microglial cells and Müller cells could enable the development of more selective therapeutic targets, with great potential clinical applications.