Isolation and Ex Vivo Characterization of the Immunophenotype and Function of Microglia/Macrophage Populations in Normal Dog Retina

Abstract

Microglia are the primary resident immune cells of the retina and are involved in the pathogenesis of various retinal diseases. In this study, we optimized experimental conditions to isolate microglia from canine retinas and characterized ex vivo their immunophenotype and function using flow cytometry (FACS). The most suitable protocol included a mechanical dissociation of the retina and an enzymatic digestion using DNAse and collagenase. Extraction was carried out by density gradient centrifugation, and retinal microglia accumulated on distinct interfaces of 1.072 and 1.088 g/mL of a Percoll gradient. Immunophenotypical characterization was performed with monoclonal antibodies CD11b, CD11c, CD18, CD45, CD44, B7-1 (CD80), B7-2 (CD86), CD1c, ICAM-1 (CD54), CD14, MHCI, MHCII, CD68, CD3, CD4, CD8α, and CD21. The most prevalent microglia population in the normal canine retina is CD11b^highCD45^low. Functionally, retinal microglia exhibited phagocytosis and reactive oxygen species (ROS) generation activities. To conclude, ex vivo examinations of retinal microglia are feasible and possibly reflect the in vivo conditions, avoiding artifacts observed in tissue culture. The established method will be relevant to examine microglia from diseased canine retinas in order to elucidate their roles in degenerative processes.

43.1 Introduction

Microglia are important resident immune cells of the retina and central nervous system (CNS). They are particularly sensitive to changes in the surrounding environment, becoming readily activated in host response to infection or injury (reviewed by [1]). Microglia occur in different isoforms and respond to pathological events by progressing from a resting ramified state to an active state with retraction of processes [2]. In retina, these active sentinels have essential roles in controlling development, aging, and function by secreting growth factors and inflammatory cytokines to promote either neuroprotection or neuronal damage. They also have been implicated in the pathogenesis of various retinal diseases [3-5].

Microglia isolation and purification ex vivo is complex; difficulties include contamination with macrophages, a relatively small number of microglia present in tissues, and absence of specific markers differentiating microglia from other blood derived mononuclear cells [6, 7]. However, ex vivo analysis has the great advantage to more closely reflect in vivo conditions compared to results obtained using cell culture systems. Previous studies established microglia isolation protocols in mouse [6] and rat [8] CNS, canine spinal cord [9], and canine brain that was either normal [7] or infected with canine distemper virus [10]. Retinal microglia have been isolated and characterized in humans [11] and rats [12] using Percoll density gradient centrifugation and FACS analysis, but these experimental tools have not yet been applied to dog retinas.

With the goal of characterizing microglia immunophenotypes and function in different retinal diseases, we have developed a protocol for ex vivo isolation of microglia from canine retinas.

43.2 Materials and Methods

43.2.1 Dogs

Normal retinas from mixed-breed dogs were examined to define optimal experimental conditions for microglia isolation and characterization. The ages were 7 (dog #1, female), 20 (dog #2, female), 25 (dog #3, female), and 35 weeks (dog #4 and #5, males). The research was conducted in full compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

43.2.2 Ex vivo isolation of canine retinal microglia

Following an optimized protocol developed for brain [7], dogs were given 12,000 units of heparin intravenously and euthanized by pentobarbital overdose. Immediately after death, perfusion was performed with 1 L of normal saline solution via left ventricle of the heart. Sufficient perfusion was indicated by water-like fluid leaving the right atrium and the absence of blood in the retinal veins, as assessed by indirect ophthalmic examination. Following perfusion, eyes were removed and both neuroretinas separated and pooled. After mincing through a stainless-steel sieve, mechanically dissociated cells were centrifuged and then enzymatically digested for 30 min at 37 °C with type II collagenase (5.7 mg/g retina; Roche Diagnostics) and DNAse I (500 units/g retina; Sigma-Aldrich). A Percoll gradient was established in a 15 mL Falcon tube with 2 mL of Percoll (GE-Amersham Biosciences) diluted in Hanks' buffer at 1.124 g/mL, subsequently overlayed with 2 mL Percoll of 1.088 g/mL, 2 mL of 1.072 g/mL, and finally 2 mL of 1.030 g/mL containing the cell solution. After centrifugation microglia were collected from the interfaces of the 1.072 (majority of cells) and 1.088 g/mL (less cells) layers. Microglia were adjusted to a concentration of 2 × 10⁵ cells in 50 mL, immunostained, and analyzed immediately by FACS.

The above-described protocol was applied for dog #1, while for dogs #2 and #3 neither perfusion nor DNAse and collagenase digestion were performed. The cells of dogs #4 and #5 were isolated using two successive Percoll gradients as previously done for microglia isolation from rats [8] and dogs [7, 9]; an initial gradient consisting of two densities and a major gradient with five densities, including an additional density of 1.060 g/mL. Microglia from dog #5 were collected separately from Percoll densities 1.060 and 1.072 g/mL and, as the number of cells was lower, no functional analyses were performed.

43.2.3 Monoclonal antibodies (mAb) and immunophenotyping

Microglia characterization was performed with mAbs binding the epitopes B7-1 (CD80), B7-2 (CD86), CD11b, CD11c, CD18, CD1c, ICAM-1, CD3, CD4, CD8α, CD21, and MHC class II (dilution 1:5; Leukocyte Antigen Biology Laboratory, University of California, Davis), CD14 (1:10; Dako), CD44 (1:10; Serotec), CD45 (1:10; Serotec), MHC class I (1:20; Veterinary Medical Research & Development), and CD68 (1:10; Santa Cruz Biotechnology). Secondary antibodies (dilution 1:100) were goat anti-mouse (Jackson Immuno Research Laboratories), rabbit anti-rat (Serotec) for CD44, and streptavidin-conjugated fluorescein-isothiocyanate (Serotec) for CD45. Cell preparations with secondary Abs only served as negative controls. Cells were measured immediately after incubation and washing using a FACSCalibur™ (BD Biosciences) flow cytometer and analyzed with the CellQuest™ software. Microglia were identified based on the parameters size (FSC), complexity (SSC), and relative expression of CD11b^high and CD45^low, as previously shown [6, 11, 12].

43.2.4 Phagocytosis assay

Directly FITC-labeled Staphylococcus aureus (Life Technologies) were suspended in PBS, adjusted to a concentration of 10⁸ bacteria/mL, and incubated with either PBS (non-opsonized bacteria) or pooled dog serum (opsonized bacteria). Non-opsonized and opsonized bacteria suspensions were added to the microglia (1:100) and incubated for 60 min at 37 °C and 5% CO₂ [7]. PBS served as negative control. Phagocytosis was measured immediately using a FACSCalibur™ flow cytometer.

43.2.5 ROS generation test

Production of reactive oxygen species (ROS) by microglia was measured as previously described [7, 10]. Briefly, 2 × 10⁶ microglia/mL were stimulated with either PBS or phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich), followed by 15 μg/mL dihydrorhodamine (DHR) 123 (Marker Gene Technologies Inc.). A tube containing only microglia was used as negative reference. ROS generation was measured immediately using a FACSCalibur™ flow cytometer.

43.3 Results

We developed a protocol for ex vivo analysis of canine retinal microglia based on previous reports for brain and spinal cord [7, 9]. Five young dogs were examined and retinal cells collected under slightly different conditions to achieve the highest purified microglia populations. Retinal microglia showed a characteristic immunophenotype of CD11b^highCD45^low (Fig. 43.1a). In normal dogs, retinal microglia represented a population of relatively small cells with low complexity (Fig. 43.1b, gate R1). All 17 surface markers could be detected in the microglia of the 5 dogs; the % of positive cells is presented in Table 43.1. Dogs #1-3 showed low contamination of the retinal microglia cell yield with lymphocytes (cells positive to CD3, CD4, or CD21 <2.4%), while dogs #4 and #5 (density 1.060) showed low expression of the microglia markers and high expression of the lymphocyte markers.

Fig. 43.1.

Retinal microglia have a characteristic immunophenotype of CD11b^highCD45^low. The histograms show expression intensities (mean fluorescent channel numbers) of CD11b (a1) and CD45 (a2) measured in two fluorescence channels (FL-H) compared to negative controls. (b) The isolated retinal microglia form a population of relatively small cells (forward scatter, FSC-H) with low complexity (side scatter, SSC-H). Only cells within the gate R1 were further analyzed.

Table 43.1. Retinal microglia expression (in %) of 14 microglia and 3 (CD3, CD4, CD21) lymphocyte surface molecules. Values of 5 normal dogs are shown; in dog #5 cells were collected from Percoll densities 1.060* and 1.072** g/mL and evaluated separately.

	Dog
Antibody	#1	#2	#3	#4	#5*	#5**	Mean
CD11b	7.3	24.5	11.5	3.1	6.6	18.5	11.9
CD11c	6.8	23.3	12.7	2.4	6.7	39	15.2
CD18	10.8	26.5	24.9	3.2	3	26.9	15.9
CD45	6.9	3	8.1	1.5	4.5	26.7	8.5
CD44	55.8	69.6	93.4	47.8	70.8	52.4	65
B7-1	11.6	30.8	10	5.9	3.7	13	12.5
B7-2	13.0	6.9	8.9	4.6	3.8	23.3	10.1
CD1c	31.5	37.3	9.7	3.7	3.9	33.5	19.9
ICAM-1	34.2	39.3	33.2	6.5	2.7	36.4	25.4
CD14	24.5	19.1	11.6	1.8	3	27.4	14.6
MHCI	67.1	12.6	24.2	33	23.8	35.1	32.6
MHCII	9.1	32.1	23.2	3.8	2	26.1	16.1
CD68	3.3	0.3	1.9	2.3	1.9	17.8	4.6
CD8α	7.4	21.2	31.5	1.8	4.9	25	15.3
CD3	0.6	1.4	0.9	2.6	5.6	1.3	2.1
CD4	1.1	1.1	1.4	2.2	19.1	1.7	4.4
CD21	1.3	0.8	2.5	1.7	5.6	2.2	2.4

Phagocytosis assay and ROS generation test were performed in dogs #1-4. As duplicates showed high consistency, the median was used for analysis. Canine retinal microglia performed phagocytosis after adding both, opsonized (x̅ = 64%) and non-opsonized (x̅ = 60%) S. aureus (Fig. 43.2). In all dogs, the % of phagocytosing microglia increased with opsonized compared to non-opsonized bacteria.

Fig. 43.2.

Retinal microglial phagocytosis after incubation with (a) PBS (negative control), (b) non-opsonized, and (c) opsonized FITC-labeled S. aureus. The x-axis shows fluorescence intensity and the y-axis cell complexity.

ROS generation test demonstrated that canine retinal microglia were able to produce ROS. However, no increase in intensity and % of ROS generating microglia was noted after addition of PMA compared to PBS.

43.4 Discussion and Conclusions

In the present study, we developed an ex vivo extraction protocol to examine the microglia cell population from dog retinas. A total of 13 different markers known to be expressed on microglia (CD11b, CD11c, CD18, CD45, CD44, B7-1, B7-2, CD1c, ICAM-1, CD14, MHCI, MHCII, CD68) were analyzed by FACS. Additionally, CD3, CD4, CD21, and CD8α were tested to verify contamination with lymphocytes although CD8α has been shown to also be expressed by mouse microglia [13, 14]. Our data showed CD8α-positive microglia and suggested that this marker is present in canine retinal microglia.

The use of two successive Percoll gradients (dogs #4 and #5) considerably decreased the number of isolated microglia and did not result in an increased purity of the cell yield. Isolation of microglia from canine brain and spinal cord required an initial gradient in particular to remove myelin [7, 9]. As the mammalian retina is devoid of oligodendrocytes and the axons of retinal ganglion cells are not myelinated where they course through the retina, an initial gradient was not necessary. Cells from the Percoll densities 1.088 and 1.072 g/mL were highly positive to microglia markers, while cells from other interfaces (i.e. 1.060 of dog #5) were predominantly lymphocytes.

Based on these results, the optimal experimental conditions for microglia extraction and testing ex vivo seem to include perfusion, mechanical dissociation and enzymatic (DNAse and collagenase) digestion, separation with one Percoll gradient, and collection from the Percoll interfaces 1.072 and 1.088 g/mL.

Functionally, canine retinal microglia from all tested dogs exhibited phagocytosis activities, and had the capability to generate ROS. They could be triggered to ingest S. aureus bacteria, but not to produce more ROS with the trigger PMA. ROS generation has also been described in macrophages [15] and brain microglia [7, 10].

This method to analyze microglia ex vivo will be useful in future studies to evaluate and compare the immunophenotype and the function of different microglia populations associated with photoreceptor degeneration in canine models, e.g. rcd1, XLPRA1, XLPRA2, which carry mutations in genes known to cause human inherited blindness. Indeed, this ex vivo method will be valuable in examining the effects of therapeutic strategies on microglia populations, as it might better reflect in vivo conditions compared to tissue cultures, where artifacts are often observed.