Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry

Animals

Male C57BL/6 (B6) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Animals had a weight of 25 to 30 g (10–14 week) when used for experiments. Experiments were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine.

Isolation of primary retinal cells

Mice were anesthetized and perfused through the heart with phosphate buffer serum (PBS, Mediatech, Manassas, VA) to remove blood from the eyes before organ collection. Mice were anesthetized by intramuscular injection of 0.15 ml of Triple Cocktail containing ketamine, xylazine, and acepromazine. This was followed by perfusion through the heart. Retinas were isolated and minced following by digestion in a solution containing 15 IU/ml papain and 15 μg/ml DNase (Worthington Biochemicals, Freehold, NJ) for 30 min at 37 °C. Tissue was dissociated by gentle pipetting and passed through a 40 μm cell strainer. Flow-through was mixed with Fetal bovine serum (FBS; HyClone Laboratories Inc. South Logan, UT) and washed. Tissue trapped by the strainer was digested with 1 mg/ml collagenase type I (Worthington Biochemicals) for 30 min at 37 °C to free endothelial cells. After dissociation and mixing with FBS, cells were washed once in DMEM (Mediatech) with 10% FBS for 5 min at 300 g at room temperature. Cells obtained after papain-DNase and collagenase treatments were pooled and counted. Viability of the cells was consistently greater than 90% as assessed by trypan blue exclusion. Cells were kept on ice in Ca²⁺, Mg²⁺-free PBS (Mediatech) until immunofluorescent labeling.

Flow cytometry

Suspensions of primary retinal cells from two to five mice were pooled and were incubated for 10 min with immunoglobulin constant Fragment (Fc)-receptor blocking antibody (Fc block: anti-CD16/CD32, clone 2.4G2; BD Biosciences, San Jose, CA) to reduce nonspecific binding of antibodies used for flow cytometry. Retinal cell suspensions were incubated with the following antibodies: allophycocyanin (APC)-conjugated or APC-Alexa Fluor 750-conjugated anti-CD11b (this marker identifies microglia in the normal retina; eBiosciences, San Diego, CA), APC-conjugated anti-CD31 (to detect endothelial cells; eBiosciences), APC-conjugated anti-CD105 (to detect endothelial cells; R&D Systems, Minneapolis, MN), APC-conjugated anti-Thy1 (to detect ganglion neurons; eBiosciences) or appropriate isotype control antibodies. For detection of intracellular markers, cells were fixed and permeabilized by using IntraPrep permeabilization reagent (Beckman Coulter, Hialeah, FL) following manufacturer’s instructions [17]. Thereafter, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-vimentin (to detect Müller cells; Santa Cruz Biotechnologies, Santa Cruz, CA), phycoerythrin (PE)-conjugated anti-glial fibrillary acidic protein (GFAP; this marker identifies astrocytes in the normal mouse retina; Santa Cruz), anti-rhodopsin mAb (to detect photoreceptors; 1D4 and C7, gifts from Dr. Krzysztof Palczewski, Case Western Reserve University, Cleveland, OH) followed by APC-conjugated donkey F(ab’)2 fragment anti-mouse IgG Ab (eBiosciences) or appropriate isotype control Abs. Cells were analyzed after fixation with 1% paraformaldehyde. In certain experiments, cells were jointly stained with PE-conjugated or FITC-conjugated anti-CD40 mAb (BD Biosciences), PE-conjugated or FITC-conjugated anti-ICAM-1 mAb (eBiosciences) or Alexa Fluor 647-conjugated anti-NOS2 Ab (Santa Cruz). Expression of CD40 or ICAM-1 was analyzed on gated CD11b⁺, CD31⁺, CD105⁺, GFAP⁺, rhodopsin⁺, vimentin⁺, or Thy-1⁺ cells. Expression of NOS2 was analyzed in permeabilized CD11b⁺ cells. The average levels of expression of CD40, ICAM-1, and NOS2 in various retinal cell subsets are expressed as corrected Mean Fluorescence Intensity (cMFI). This was obtained by subtracting fluorescence obtained with isotype control Ab from the fluorescence obtained with Abs against CD40, ICAM-1, or NOS2. Flow cytometry data acquisition was performed using a LSR II and running FACSDiva software (Becton Dickinson, San Jose, CA). FlowJo software (Tree Star Inc., Ashland, OR) was used for data analysis. In certain experiments, vimentin⁺, and rhodopsin high (rhodopsin^hi) events were sorted using a FACSAria (Becton Dickinson). Sorted events were analyzed by light microscopy.

Mouse endothelial cells were used to determine whether enzymatic treatment affects expression of surface markers. Mouse high endothelial venule cells c (mHEVc) [18] stably transfected with CD40-encoding plasmid [19] were detached by exposure to Versene (Invitrogen, Carlsbad, CA) for 5 min. Cell suspensions were then incubated with or without enzymatic cocktail as we have described in this paper. Cells were subsequently incubated with fluorescent antibodies and were subjected to flow cytometry analysis

Model of retinal ischemia and reperfusion

Retinal ischemia was induced as we previously described [1,16]. The anterior chamber of one eye was cannulated with a 30 gauge needle attached to a line infusing normal saline. Intraocular pressure (IOP) was measured by a handheld tonometer (TONO Pen; Medtronic Solan, Jacksonville, FL) and pressure in the eye was regulated to 80 mmHg to 90 mmHg with a pressure infuser (Infu-surg; Ethox Corp., Buffalo, NY) and maintained for 90 min. The other eye of the same animal was set up as a control. After ischemia, the needle was withdrawn, IOP was normalized, and reflow of the retinal circulation was documented visually. Mice were euthanized with triple cocktail one or seven days after I-R injury and retinal cell suspensions were processed for flow cytometry.

Statistical analysis

All results were expressed as the mean±standard error of the mean. Data were analyzed by two-tailed Student’s t test. Differences were considered statistically significant at p<0.05.

Detection of ICAM-1 and CD40 on various retinal cells by flow cytometry

Enzymatic digestion was performed to obtain retinal single cell suspensions. Pilot experiments were conducted to determine whether enzymatic treatment impairs expression of surface markers. Cell suspensions of CD40⁺ mouse endothelial cells (mHEVc) were treated with or without enzymatic cocktail as described in Methods followed by flow cytometric analysis of cell surface markers. The levels of expression (cMFI) of CD31, CD40 and ICAM-1 were not affected by enzymatic treatment. The levels of expression for these molecules were as follows: No enzymatic treatment: CD31=105±14, CD40=99±7, ICAM-1=82±4; Enzymatic treatment: CD31=100±1, CD40=82±8, ICAM-1=78±1 (n=3; p>0.5). Thus, the protocol of enzymatic digestion appeared unlikely to significantly affect expression of surface molecules.

Single-cell suspensions were obtained from exsanguinated retinas. Each retina yielded on average 2.01±0.20x10⁶ cells (n=15). We applied flow cytometry to identify various cell types in the normal mouse retina. We used a panel of antibodies against markers shown by immunofluorescence to be expressed by different cell subsets in the normal mouse retina: CD11b for microglia/macrophages [20], CD31 for endothelial cells [21], GFAP for astrocytes [21], rhodopsin for photoreceptors [22,23], Thy-1 for ganglion neurons [24], and vimentin for Müller cells [21]. We started by examining photoreceptors and Müller cells, two major cell types in the retina. Retinal cells were permeabilized to detect intracellular rhodopsin and vimentin. Staining with anti-rhodopsin or anti-vimentin antibodies revealed expression of these intracellular markers as shown in Figure 1A. Regardless of the anti-rhodopsin antibody used (1D4 or C7), the expression of rhodopsin was bimodal (Figure 1A). Sorting of rhodopsin^hi events revealed that they consisted of rod-like structures of 1 μm in diameter (not shown), morphological features consistent with outer segments, the portion of the photoreceptors that expresses high levels of rhodopsin. Flow cytometry also examines two forms of light scatter: forward scatter (FSC) and side scatter (SSC), parameters that indicate the size and cell granularity respectively. Figure 1B reveals the FSC and SSC pattern of rhodopsin⁺ and vimentin⁺ retinal cells. Similarly, gating on CD11b⁺, CD31⁺, GFAP⁺, and Thy-1⁺ cells revealed the various patterns of FSC and SSC for each cell population (Figure 1C). The phenotypic composition of the cells released from the retina after enzymatic digestion is shown in Figure 2. Results were similar regardless of whether surface markers were assessed on permeabilized versus non-permeabilized cells (not shown). These studies also include staining with a mAb against CD105, a marker for endothelial cells.

ICAM-1 is an adhesion molecule that plays a pivotal role in recruitment of leukocytes to the retina and the development of vascular histopathology [2,5]. In addition, recent studies identified CD40 as an important regulator of retinal inflammation and neurovascular degeneration [16]. We used flow cytometry to examine the expression of these molecules on various retinal cells. Cell suspensions were stained with anti-ICAM-1 or anti-CD40 mAb plus either antibodies against CD11b, CD31, CD105, GFAP, rhodopsin, Thy-1, or vimentin. Varying levels of ICAM-1 were detected on all the subsets of retinal cells examined (Figure 3). ICAM-1 expression did not appear to be homogenous within rhodopsin⁺ and Thy-1⁺ cell populations. CD40 was detected only on CD11b⁺ (microglia/macrophages), CD31⁺(endothelial), Thy-1⁺(ganglion), and vimentin⁺ cells (Müller) cells (Figure 4). Despite the typical low levels of expression, CD40 is an important mediator of inflammation [15]. These results indicate that flow cytometry allows quantitation of surface molecules involved in retinal inflammatory disorders.

ICAM-1 upregulation after retinal ischemia–reperfusion

Next, we examined if flow cytometry can quantify upregulation of ICAM-1. Endothelial and Müller cells are major components of the blood-retinal barrier [25] and thus, are likely to be involved in the adhesion-dependent recruitment of leukocytes to the retina. Ischemia-Reperfusion (I-R) of the retina results in upregulation of ICAM-1 mRNA [1]. However, it is not known if ICAM-1 protein levels increase in retinal endothelial and Müller cells after I-R. We used a well established model of I/R-induced retinal injury (transient elevation of IOP) [1] to examine this possibility. Retinal cells from ischemic and nonischemic eyes were stained with isotype control antibodies or with an anti-ICAM-1 mAb plus either anti-CD105 or anti-vimentin antibodies. CD105⁺ and vimentin⁺ cells from ischemic retinas upregulated ICAM-1 compared to cells from control retinas (Figure 5; p=0.03; n=3). These results indicate that flow cytometry allows measuring upregulation of a surface molecule involved in retinal inflammatory processes.

Upregulation of NOS2 in retinal microglia after retinal ischemia/reperfusion

NOS2 plays an important role in neurovascular degeneration in retinopathies including those mediated by acute ischemia and diabetes [1,7,9,26,27]. I/R-induced retinal injury results in NOS2 upregulation in microglia at 5 to 7 days post-I/R, time points where NOS2⁺ peripheral blood neutrophils (that can also express CD11b) are no longer detected in the retina [27]. We used retinas at seven days postischemia to examine whether flow cytometry can detect upregulation of NOS2. Retinal cells from ischemic and nonischemic eyes were stained with an anti-CD11b mAb followed by permeabilization and staining with an anti-NOS2 antibody. As shown in Figure 6, NOS2 was upregulated within CD11b⁺ cells from ischemic retinas (p<0.01; n=3). These results indicate that flow cytometry allows quantitation of an intracellular molecule involved in retinal inflammatory processes.