Early retinal inflammatory biomarkers in the middle cerebral artery occlusion model of ischemic stroke

Abstract

Purpose: The transient middle cerebral artery occlusion (MCAO) model of stroke is one of the most commonly used models to study focal cerebral ischemia. This procedure also results in the simultaneous occlusion of the ophthalmic artery that supplies the retina. Retinal cell death is seen days after reperfusion and leads to functional deficits; however, the mechanism responsible for this injury has not been investigated. Given that the eye may have a unique ocular immune response to an ischemic challenge, this study examined the inflammatory response to retinal ischemia in the MCAO model.

Methods: Young male C57B/6 mice were subjected to 90-min transient MCAO and were euthanized at several time points up to 7 days. Transcription of inflammatory cytokines was measured with quantitative real-time PCR, and immune cell activation (e.g., phagocytosis) and migration were assessed with ophthalmoscopy and flow cytometry.

Results: Observation of the affected eye revealed symptoms consistent with Horner's syndrome. Light ophthalmoscopy confirmed the reduced blood flow of the retinal arteries during occlusion. CX3CR1-GFP reporter mice were then employed to evaluate the extent of the ocular microglia and monocyte activation. A significant increase in green fluorescent protein (GFP)-positive macrophages was seen throughout the ischemic area compared to the sham and contralateral control eyes. RT-PCR revealed enhanced expression of the monocyte chemotactic molecule CCL2 early after reperfusion followed by a delayed increase in the proinflammatory cytokine TNF-α. Further analysis of peripheral leukocyte recruitment by flow cytometry determined that monocytes and neutrophils were the predominant immune cells to infiltrate at 72 h. A transient reduction in retinal microglia numbers was also observed, demonstrating the ischemic sensitivity of these cells. Blood-eye barrier permeability to small and large tracer molecules was increased by 72 h. Retinal microglia exhibited enhanced phagocytic activity following MCAO; however, infiltrating myeloid cells were significantly more efficient at phagocytizing material at all time points. Immune homeostasis in the affected eye was largely restored by 7 days.

Conclusions: This work demonstrates that there is a robust inflammatory response in the eye following MCAO, which may contribute to a worsening of retinal injury and visual impairment. These results mirror what has been observed in the brain after MCAO, suggesting a conserved inflammatory signaling response to ischemia in the central nervous system. Imaging of the eye may therefore serve as a useful non-invasive prognostic indicator of brain injury after MCAO. Future studies are needed to determine whether this inflammatory response is a potential target for therapeutic manipulation in retinal ischemia.

Figure 1

Blood flow reduction through the ophthalmic artery via middle cerebral artery occlusion. The middle cerebral artery (MCA) and the ophthalmic artery in the mouse and occlusion following suture insertion. A: A representative image showing the gross observation of acute sensitivity in the ipsilateral eye during MCA occlusion. B: Ophthalmoscopic imaging depicts blood flow to the contralateral (left) and ipsilateral (right) retina during MCA occlusion.

Figure 2

Acute expression of inflammatory mediators in the ipsilateral eye after MCAO. Quantitative real-time PCR analysis shows a significant relative fold increase in interleukin-1 beta (IL-1β) (A) and tumor necrosis factor (TNF) (B) mRNA expression in the ipsilateral eye 8 h after stroke compared to sham surgery. At 24 h, expression of the inflammatory signaling genes monocyte chemoattractant protein-1 (CCL2) (C) and myeloperoxidase (MPO) (D) is increased following ischemia. For all experiments, n = 4/group. Error bars show mean ± standard error of mean (SEM). Abbreviations: GAPDH = glyceraldehyde 3-phosphate dehydrogenase, SH = sham, ST = stroke. *p<0.05; **p<0.01; ***p<0.001.

Figure 3

Differential cytokine expression and vascular permeability at 72 h. Quantitative real-time PCR analysis shows no difference in interleukin-1 beta (IL-1β) (A) and tumor necrosis factor (TNF) (B) mRNA expression in the ipsilateral eye after stroke compared to sham at 72 h. Expression of the inflammatory signaling genes monocyte chemoattractant protein-1 (CCL2) (C) and myeloperoxidase (MPO) (D) continued to be increased at 72 h following ischemia. For all quantitative real-time PCR experiments, n = 5/group. Vascular permeability in the eye was measured following injection of low molecular weight (sodium fluorescein (NaF), E) and high molecular weight (Evans Blue, F) dye tracers at 72 h in the stroke and sham groups (n = 10/group). Error bars show mean ± standard error of mean (SEM). Abbreviation: GAPDH = glyceraldehyde 3-phosphate dehydrogenase, SH = sham, ST = stroke, NaF. *p<0.05; **p<0.01; ***p<0.001.

Figure 4

Increased number of microglia/macrophages in the retina at 72 h. A: Representative light depicting a significant increase in CX3CR1-GFP-positive microglia and macrophages in the ischemic ipsilateral eye at 72 h compared to the contralateral side. B: Fluorescence ophthalmoscopic images depicting a significant increase in CX3CR1-GFP-positive microglia and macrophages in the ischemic ipsilateral eye at 72 h compared to the contralateral side. The green fluorescence signal intensity was measured in the sham and stroke groups at 72 h after reperfusion (n = 4/group, C). Error bars show mean ± standard error of mean (SEM). Abbreviations: SH = sham, ST = stroke. *p<0.05; **p<0.01; ***p<0.001.

Figure 5

Changes in the number of resident retinal microglia and infiltrating bone marrow–derived myeloid cells over 7 days after MCAO. Mice were subject to 90 min of occlusion followed by 72 h or 7 days of reperfusion and analyzed with flow cytometry (n = 5/group). A: A representative dot plot shows resident microglia (CD45^intCD11b⁺) and infiltrating leukocyte (CD45^hi) populations in the ipsilateral sham and stroke eyes at 72 h. B: A representative dot plot shows the identification of monocyte (Ly6C⁺Ly6G^-) and neutrophil (Ly6C⁺Ly6G⁺) subsets of infiltrating myeloid (CD45^hiCD11b⁺) cells. C: The absolute number of resident microglia was quantified in the sham and stroke groups. D: The absolute number of infiltrating leukocytes was quantified in the sham and stroke groups. E: Pie charts depict the cellular composition of the infiltrating myeloid (CD45^hiCD11b⁺) cell fraction in sham (0 h) and at 72 h and 7 days. Error bars show mean ± standard error of mean (SEM). *p<0.05; **p<0.01; ***p<0.001.

Figure 6

Phagocytic activity of resident microglia and infiltrating myeloid cells in the ischemic retina. A: The mean side scatter intensity was quantified at 72 h after sham or stroke. B: The mean fluorescence intensity of CD11b⁺ microglia was quantified at 72 h after sham or stroke. The phagocytic potential of retinal myeloid cells after middle cerebral artery occlusion (MCAO) was assessed using ex vivo fluorescent bead assay, and the percentage of phagocytic myeloid cells was measured using flow cytometry. C: A representative histogram shows the relative phagocytic activity of resident microglia after sham (black) and stroke (blue) and infiltrating CD45^hiCD11b⁺ myeloid cells (red). Positive gating was determined using fluorescence minus one (FMO) control (shaded gray). D: The percentages of bead-positive CD45^intCD11b⁺ microglia versus CD45^hiCD11b⁺ myeloid cells were quantified at three time points. Error bars show mean ± standard error of mean (SEM). Abbreviations: MFI = mean fluorescence intensity, a.u.i. = arbitrary units of intensity, SH = sham, ST = stroke. *p<0.05; **p<0.01; ***p<0.001.