Journal: Investigative Ophthalmology & Visual Science

Article Title: Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection

doi: 10.1167/iovs.18-24086

Figure Lengend Snippet: (A) Photomicrographs of Gr-1 (A1), RIP3 (A2), and DAPI staining in the MCMV-injected eye of an IS Rip3 +/+ mouse at day 7 p.i. As shown in the merged image (A3), a few Gr-1–stained neutrophils were observed in the inner retina but no RIP3 staining was detected in Gr-1–stained cells. (B) Photomicrographs of CD3 (B1), RIP3 (B2), and DAPI staining in the MCMV-injected eye of an IS Rip3 +/+ mouse at day 7 p.i. As shown in the merged image (B3), a few CD3-stained T cells were found in the inner retina and some also stained positive for RIP3. (C) Photomicrographs of GFAP (C1), RIP3 (C2), and DAPI staining in the MCMV-injected eye of an IS Rip3 +/+ mouse at day 7 p.i. As shown in the merged image (C3), many RIP3-stained cells in the inner retina of injected eyes also stained positive for GFAP, a marker for glia/Müller cells. Arrows indicate RIP3-stained glia/Müller cells in the outer nuclear layer.

Article Snippet: Mouse anti-GFAP (glial fibrillary acidic protein, specific for glia/Müller cells), FITC-labeled anti-CD11b (specific for macrophages/microglia), FITC-labeled anti-CD3 (specific for CD3 + T cells), and FITC-labeled anti-Gr-1 (specific for neutrophils) were all from BD Biosciences (San Jose, CA, USA).

Techniques: Staining, Injection, Marker

Journal: Journal of Neuroinflammation

Article Title: Transgenic inhibition of astroglial NF-κB restrains the neuroinflammatory and neurodegenerative outcomes of experimental mouse glaucoma

doi: 10.1186/s12974-020-01930-1

Figure Lengend Snippet: Experimental modeling of ocular hypertension-induced glaucoma in GFAP-IκKβ mice and controls. a Intraocular pressure elevation was induced in GFAP-IκKβ mice and transgenic controls (IκKβ f/f mice wild-type for cre ) by microbead injections into the anterior chamber of one eye. Fellow eyes received similar injections of physiological saline. Intraocular pressure curves were obtained over the experimental period of 12 weeks. Blue arrows show the time points for microbead (or saline) injections. Microbead injections resulted in a significant increase in intraocular pressure (*** P < 0.001) with no significant difference between GFAP-IκKβ mice and IκKβ f/f controls ( P = 0.45). Immunolabeling of retinal whole mounts ( b ) and a histological section ( c ; RGCL, retinal ganglion cell layer; INL, inner nuclear layer) demonstrate cre-recombinase (red) expression in GFAP+ astroglia (green) after tamoxifen-injection in GFAP-IκKβ mice (not in IκKβ f/f controls). In order to confirm astroglia-targeted deletion of IκKβ , we also analyzed IκKβ expression and downstream activity (by analysis of p65 phosphorylation) in retina and optic nerve samples obtained from transgenic mice and controls. Panels d and e show IκKβ, or phospho-p65 (red), immunolabeling of GFAP+ astroglia (green) in retinal whole mounts obtained from ocular hypertensive eyes of GFAP-IκKβ mice or IκKβ f/f controls. Despite prominent localization of IκKβ (and phospho-p65) immunolabeling to GFAP+ astroglia in control mice, astroglial IκKβ (and phospho-p65) labeling was decreased in GFAP-IκKβ mice (scale bar, 100 μm). Yellow and red boxed areas on merged images in panels d and e are shown in higher magnification. Histological sections of the optic nerve (panel f ) and retina (panel g ) similarly showed decreased immunolabeling of GFAP+ astroglia for phospho-p65 (and decreased colocalization of GFAP and phospho-p65 immunolabeling) in ocular hypertensive GFAP-IκKβ mice relative to ocular hypertensive IκKβ f/f controls (scale bar, 100 μm). Red or yellow boxed areas in panel g are shown in higher magnification. Note that although no phospho-p65 immunolabeling was detectable in GFAP+ astroglia in the RGC layer (corresponding to astrocytes), GFAP− neurons (white arrows) exhibited phospho-p65 immunolabeling in the GFAP-IκKβ retina. The GFAP+ cells in the inner nuclear layer (corresponding to Müller glia; translucent arrows) also immunolabeled for phospho-p65. As presented in panel h , for testing p65 phosphorylation by quantitative Western blot analysis, astroglia were isolated from retina and optic nerve (ON, including the optic nerve head) samples by immunomagnetic cell selection. Immunoblots of enriched astroglia proteins exhibited immunoreactivity for GFAP (red), an astroglia marker, but were negative for neuron markers (NeuN or NFP, green). When the immunoblots of astroglia proteins were probed with a phosphorylation site-specific antibody to NF-κB subunit p65 (green), band intensities (normalized to β-actin bands, red) indicated over four-fold increased expression of phospho-p65 with ocular hypertension (OHT) in IκKβ f/f control mice (* P = 0.005, and P = 0.004 for retina and optic nerve samples, ON, respectively). However, both the basal expression in normotensive (NT) eyes and the ocular hypertension-induced upregulation of phospho-p65 were not detectable in the retina or optic nerve astroglia obtained from GFAP-IκKβ mice. Presented immunoblots and the quantitative data (mean ± SD) represent a minimum of 10 mice per group in triplicated analyses

Article Snippet: Alternatively, a monoclonal antibody to ACSA-1 (GLAST) was used to select retinal Müller glia (1:10; Miltenyl Biotech, catalog number: 130-095-822).

Techniques: Transgenic Assay, Saline, Immunolabeling, Expressing, Injection, Activity Assay, Phospho-proteomics, Control, Labeling, Western Blot, Isolation, Selection, Marker

Journal: Journal of Neuroinflammation

Article Title: Transgenic inhibition of astroglial NF-κB restrains the neuroinflammatory and neurodegenerative outcomes of experimental mouse glaucoma

doi: 10.1186/s12974-020-01930-1

Figure Lengend Snippet: Effects of astroglial IκKβ deletion on neuroinflammatory responses of the ocular hypertensive mouse retina. In order to determine the inflammatory status of retina ( a ) and optic nerve ( b ) tissues, cytokine titers were analyzed by ELISA. We detected significantly reduced production of pro-inflammatory cytokines in ocular hypertensive GFAP-IκKβ eyes compared to ocular hypertensive controls (IκKβ f/f mice wild-type for cre ). Bar graphs show fold decrease in ocular hypertension (OHT)-induced pro-inflammatory cytokine production with GFAP-IκKβ. Data (mean ± SD) from retina and optic nerve samples are presented by separate graphs (represents a minimum of 4 mice per group; ** P < 0.001, * P < 0.05). c Isolated samples of retinal astroglia and microglia (by immunomagnetic cell selection) also presented reduced titers of TNF-α (a major pro-inflammatory cytokine relevant to glaucomatous neurodegeneration) in ocular hypertensive GFAP-IκKβ than ocular hypertensive IκKβ f/f controls. However, there was no significant difference between the TNF-α titers in normotensive (NT) samples from GFAP-IκKβ or IκKβ f/f mice ( P > 0.05). Reduced production of TNF-α with GFAP-IκKβ was more significant in astroglia (** P < 0.001) than microglia (* P = 0.02). When the isolated samples of retinal Müller glia were similarly analyzed, no significant difference was detectable in the ocular hypertension-induced TNF-α production of Müller glia between GFAP-IκKβ mice and IκKβ f/f controls ( P = 0.06). d Astroglial pro-inflammatory phenotype was also studied by immunohistochemical analysis. Presented are TNF-α immunolabeling of retinal tissue sections (scale bar, 100 μm), and red or yellow boxed areas are shown in higher magnification. TNF-α immunolabeling (red) of GFAP+ astroglia (green) was prominently higher in ocular hypertensive IκKβ f/f retina (white arrows) than normotensive controls. However, astroglial TNF-α immunolabeling was not detectable in the RGC layer (corresponding to astrocytes), but still detectable in the inner nuclear layer (corresponding to Müller glia; translucent arrows) of ocular hypertensive GFAP-IκKβ retinas. Blue indicates nuclear DAPI staining. RGCL, and INL mark retinal ganglion cells layer, and inner nuclear layer, respectively

Article Snippet: Alternatively, a monoclonal antibody to ACSA-1 (GLAST) was used to select retinal Müller glia (1:10; Miltenyl Biotech, catalog number: 130-095-822).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Selection, Immunohistochemical staining, Immunolabeling, Staining

Journal: PLoS Pathogens

Article Title: A Mouse Model for Chikungunya: Young Age and Inefficient Type-I Interferon Signaling Are Risk Factors for Severe Disease

doi: 10.1371/journal.ppat.0040029

Figure Lengend Snippet: IFN-α/βR −/− mice inoculated via the ID route with 20 PFU were sacrificed at D3 pi and immunostaining was performed on brain cryosections. Nuclei appear in blue and CHIKV in red. Basal lamina (collagen IV) is stained in purple (A) or green (B), and astrocytes and glia limitans (GFAP) appear in green (A and C). Leptomeningeal cells (arrows) display a strong immunolabeling for CHIKV while brain microvessells (arrowheads) and glial cells do not (A). Virchow-Robin spaces showed immunostaining for CHIKV (B) as well as ependymal cells (C) and choroid plexuses (D). Bar is 10 μm.

Article Snippet: The following antibodies were used: polyclonal rabbit anti-collagen IV (Chemicon, Temecula CA, 1:200), polyclonal chicken anti-vimentin (Abcam, Cambridge, UK, 1:200), monoclonal mouse anti-GFAP (BD pharmingen 1:1,000 or 1:5,000 to only see the glia limitans ), monoclonal rat anti-macrophage antigen F4/80 (Abcam, 1:100), polyclonal rabbit anti-PECAM1/CD31 (Abcam, 1:400), human serum anti-CHIKV were obtained and characterized by the Centre National de Référence des Arbovirus as positive for anti-CHIKV IgM and IgG.

Techniques: Immunostaining, Staining, Immunolabeling

Journal: Neurology International

Article Title: Hypomyelinating Leukodystrophy 10 (HLD10)-Associated Mutations of PYCR2 Form Large Size Mitochondria, Inhibiting Oligodendroglial Cell Morphological Differentiation

doi: 10.3390/neurolint14040085

Figure Lengend Snippet: Cells expressing the R119C or R251C proteins, but not the wild type proteins, exhibit decreased expression levels of oligodendroglial cell differentiation marker proteins. ( A , B ) The lysates of the respective FBD-102b cells were immunoblotted with an antibody against oligodendroglial cell differentiation marker protein MBP or CNPase, oligodendroglial cell lineage marker Sox10. Additionally, antibodies against actin, ATP1A1, ErbB4, and pan-ErbB were utilized as the control protein markers. Their expression levels are shown statistically compared to their respective controls. (**, p < 0.01; n = 3 blots for MBP, CNPase, and actin, and n = 4 blots for ATP1A1, ErbB4, and pan-ErbB).

Article Snippet: Mouse monoclonal anti-myelin basic protein specific for myelin-forming glial cells (MBP, Cat. No. 836506; immunoblotting [IB], 1/500) from BioLegend (San Diego, CA, USA); mouse monoclonal anti-2′,3′-cyclic nucleotide 3′-phospho-diesterase specific for myelin-forming glial cells (CNPase, Cat. No. 5664; IB, 1/500) from Cell Signaling Technology (Danvers, MA, USA); mouse monoclonal alpha 1 sodium potassium ATPase (ATP1A1; Cat. No. sc-21712; IB, 1/100) as the general control membrane protein marker, mouse monoclonal oligodendroglial cell-rich ErbB4 (Cat. No. sc-8050; IB, 1/100), mouse monoclonal pan-ErbB receptors (Cat. No. sc-101; IB, 1/100) as the membrane protein, mouse monoclonal anti-myelin-forming glial cell lineage-specific Sox10 (Cat. No. sc-365692; IB, 1/500), and mouse monoclonal anti-lysosomal-associated membrane protein 1 (LAMP1, Cat. No. sc-20011; immunofluorescence [IF], 1/200) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-actin (control actin, Cat. No. M177-3; IB, 1/40,000) and mouse monoclonal anti-endoplasmic reticulum (ER)-resident Lys-Asp-Glu-Leu oligopeptide antigen (KDEL, Cat. No. M181-3; IF, 1/500) from MBL (Aichi, Japan); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, Cat. No. 610823; IF, 1/500) and mouse monoclonal anti-mitochondrion-specific mitochondrial heat shock protein family D member 1 (HSPD1, Cat. No. 611562; IF, 1/5000) from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Expressing, Cell Differentiation, Marker, Control