elimination-serotonergic-neurons-stereotaxic-injection-57

Elimination of Serotonergic Neurons by Stereotaxic Injection of 5,7-Dihydroxytryptamine in the Dorsal Raphe Nuclei of Mice

Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of ...

Tongji University

Local and chronic inflammation induced by amyloid-β (Aβ) plays a central role in the development of age-related macular degeneration. The retina is an immune-privileged site due to local tissue barrier. Yet, the manner by which immune cells pass through this barrier and accumulate in the retina remains unclear. Matrix metalloproteinases (MMPs) induce barrier disruption via proteolysis of epithelial tight junction (TJ) proteins. We hypothesized that Aβ-induced MMP secretion causes disruption of epithelial barrier integrity. To test this hypothesis, human adult retinal pigment epithelial (haRPE) cells were exposed to Aβ, and the expression of MMP-2 and MMP-9 was detected using gelatin zymography. To demonstrate the key role of MMPs in modulating epithelial barrier structure, the MMP agonist 4-aminophenylmercuric acetate (APMA), an MMP inhibitor (GM6001) and siRNA against MMP-9 were employed for comparison. We found that MMP-9, secreted by Aβ- or APMA-stimulated cells, mediated low transepithelial electrical resistance (TER) and high transepithelial permeability by disrupting TJ proteins. However, these alterations were reduced by the MMP inhibitor GM6001 or by silencing of the MMP-9 gene. Our findings suggest that the degradation of TJ proteins such as zonula occludens-1, occludin and F-actin by MMP-9 secreted by Aβ-stimulated cells constitutes an important mechanism in the breakdown of the barrier which contributes to chronic inflammation in the retina of age-related macular degeneration.

Amyloid-β-induced matrix metalloproteinase-9 secretion is associated with retinal pigment epithelial barrier disruption.

Chronic inflammation is implicated in the pathogenesis of AMD. The source of chronic inflammation is often attributed to the progressive activation of immune cells over time. However, recent studies have shown that senescent cells can alter tissue microenvironment via secretion of growth factors, proteases, and inflammatory cytokines and might be an additional source of chronic inflammation. We hypothesized that altered secretory pattern in Aβ-induced senescent RPE cells may contribute to compromised RPE barrier integrity and chronic inflammation in AMD.

Aβ-induced senescent retinal pigment epithelial cells create a proinflammatory microenvironment in AMD.

Drusen are considered a hallmark characteristic of age-related macular degeneration (AMD). In our previous study, we found that amyloid-β (Aβ) peptide, a component of drusen, induced the cells of the retinal pigment epithelium (RPE; RPE cells) to enter senescence; however, its effects in vivo remain unknown. Thus, the present study was carried out to explore the in vivo effects of Aβ peptide on RPE cell senescence and senescence-associated inflammation in C57BL/6 mice. C57BL/6 mice received a subretinal injection of Aβ(1-42) peptide; on day 7 post-injection, the mice were anesthetized and subjected to whole-body perfusion with 4% paraformaldehyde (PFA) in PBS and the whole eyes were then enucleated. Retinal function was assessed by electroretinography (ERG), and the morphological characteristics of the retina were examined by light and electron microscopy. Fundus autofluorescence (FAF) was examined by confocal scanning laser ophthalmoscopy (cSLO). The expression of p16INK4a, a marker of cellular senescence, was examined by immunofluorescence staining and western blot analysis. The RPE-choroid was analyzed for cytokine expression by RT-PCR. In Aβ(1-42)-injected mice, scotopic ERG responses declined. Degenerative alterations, including the disruption of the inner segment (IS)/outer segment (OS) junction and extensive vacuolation and thickness of Bruch's membrane (BrM) were observed under a a light microscope. The accumulation of vacuoles and the loss of basal infoldings in the RPE were identified using an electron microscope. FAF and p16INK4a expression increased in Aβ(1-42)-injected mice. In addition, Aβ(1-42) upregulated interleukin (IL)-6 and IL-8 gene expression in the RPE-choroid. In conclusion, our results confirm the effects of Aβ(1-42) peptide on RPE senescence in vivo. The Aβ-injected mice developed AMD-like ocular pathology. It is thus suggested that RPE cell senescence is a potential mechanistic link between inflammation and retinal degeneration.

Subretinal injection of amyloid-β peptide accelerates RPE cell senescence and retinal degeneration.

To investigate the alterations of retinal tissue induced by OAβ(1-42) subretinal injection in normal C57BL/6N mice.

[Alterations of retinal tissue induced by amyloid β(1- 42) subretinal injection in mice].

Our previous study indicated that Aβ-induced Retinal Pigment Epithelial (RPE) cell senescence may be associated with chronic inflammation in age-related macular degeneration (AMD). The present study was designed to explore whether Aβ deposition and RPE senescence could be found in the senescence-prone mouse strain 8 (SAMP8), which is an animal model for AMD.

Detecting Aβ deposition and RPE cell senescence in the retinas of SAMP8 mice.

Neuron regeneration from pluripotent stem cells (PSCs) differentiation or somatic cells trans-differentiation is a promising approach for cell replacement in neurodegenerative diseases and provides a powerful tool for investigating neural development, modeling neurological diseases, and uncovering the mechanisms that underlie diseases. Advancing the materials that are applied in neural differentiation and trans-differentiation promotes the safety, efficiency, and efficacy of neuron regeneration. In the neural differentiation process, matrix materials, either natural or synthetic, not only provide a structural and biochemical support for the monolayer or three-dimensional (3D) cultured cells but also assist in cell adhesion and cell-to-cell communication. They play important roles in directing the differentiation of PSCs into neural cells and modeling neurological diseases. For the trans-differentiation of neural cells, several materials have been used to make the conversion feasible for future therapy. Here, the most current applications of materials for neural differentiation for PSCs, neuronal trans-differentiation, and neurological disease modeling is summarized and discussed.

Lining Cao

Institute for Regenerative Medicine,
Shanghai East Hospital,
Frontier Science Center for Stem Cell Research,
School of Life Sciences and Technology,
Institute for Regenerative Medicine, Shanghai East Hospital, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology

Tongji University

Elimination of Serotonergic Neurons by Stereotaxic Injection of 5,7-Dihydroxytryptamine in the Dorsal Raphe Nuclei of Mice