Redefining the Koizumi model of mouse cerebral ischemia: A comparative longitudinal study of cerebral and retinal ischemia in the Koizumi and Longa middle cerebral artery occlusion models

Abstract

Cerebral and retinal ischemia share similar pathogenesis and epidemiology, each carrying both acute and prolonged risk of the other and often co-occurring. The most used preclinical stroke models, the Koizumi and Longa middle cerebral artery occlusion (MCAO) methods, have reported retinal damage with great variability, leaving the disruption of retinal blood supply via MCAO poorly investigated, even providing conflicting assumptions on the origin of the ophthalmic artery in rodents. The aim of our study was to use longitudinal in vivo magnetic resonance assessment of cerebral and retinal vascular perfusion after the ischemic injury to clarify whether and how the Koizumi and Longa methods induce retinal ischemia and how they differ in terms of cerebral and retinal lesion evolution. We provided anatomical evidence of the origin of the ophthalmic artery in mice from the pterygopalatine artery. Following the Koizumi surgery, retinal responses to ischemia overlapped with those in the brain, resulting in permanent damage. In contrast, the Longa method produced only extensive cerebral lesions, with greater tissue loss than in the Koizumi method. Additionally, our data suggests the Koizumi method should be redefined as a model of ischemia with chronic hypoperfusion rather than of ischemia and reperfusion.

Keywords: Brain; ischemia; magnetic resonance angiography; mice; retina.

Figure 1.

Survival proportions and animal weight after middle cerebral artery occlusion by filament insertion through the common carotid artery (CCA), external carotid artery (ECA), and the ECA or CCA sham-operated (CCA sham and ECA sham) animals. Statistical differences using a mixed model ANOVA and Tukey post hoc test for ECA to ECA sham group (†) P < 0.05; (†††) P < 0.001; CCA to CCA sham group (###) P < 0.001. Error bars represent standard deviation.

Figure 2.

Intraoperative laser Doppler flowmetry shows incomplete reperfusion in the Koizumi (CCA) middle cerebral artery occlusion method. Middle cerebral artery (MCA) perfusion measured with laser Doppler flowmetry during MCA occlusion (MCAO) by filament insertion through the common carotid artery (CCA), the external carotid artery (ECA), and sham surgeries (CCA sham and ECA sham). Statistical differences using mixed model ANOVA and Tukey post hoc test for the CCA to ECA group (***) P < 0.001; CCA to CCA sham group (###) P < 0.001; ECA to ECA sham group (†††) P < 0.001; CCA sham to ECA sham group (‡‡‡) P < 0.001. Error bars represent standard deviation.

Figure 3.

High-resolution MR angiography defines the Koizumi (CCA) middle cerebral artery occlusion method as a model of ischemia with chronic hypoperfusion. Maximum intensity projections (MIP) of MR angiography scans (a) and measured ipsilateral (b) and contralateral (c) arterial vasculature volume before and after middle cerebral artery occlusion (MCAO) by filament insertion through the common carotid artery (CCA; a-upper row) or external carotid artery (ECA; a-bottom row) show animals at baseline (BL) and on days 2 (D2), 9 (D9), and 35 (D35) after MCAO. Pre-surgery (BL) angiograms: yellow arrow – ophthalmic artery (OA); blue arrow – middle cerebral artery (MCA); green arrow – pterygopalatine artery (PPA). On D2, the MIP show hypoperfusion of the ipsilateral (IL) hemisphere in the CCA group. On D9, the attenuation of blood flow persists in the IL internal carotid artery (ICA), distal portion of the MCA, PPA, OA, and the segment between the anterior cerebral artery (ACA) and MCA, with prominent hyperperfusion of the contralateral (CL) ICA and the segments between the posterior cerebral artery (PCA) and MCA, and MCA and ACA. On D35, undetectable signal intensity of the IL PPA, OA, and ICA, with increased signal intensity of the CL ICA and the CL part of the circle of Willis. Tortuosity of the IL MCA (lateral view of CCA D35). On D2, the ECA-MCAO group shows lower signal intensity of the IL MCA with hypoperfused segment between the IL ACA and MCA, and no changes in the PPA or OA. On D9, the perfusion of the IL hemisphere is reestablished and slightly hyperperfused. On D35, the perfusion in the ECA-MCAO group shows baseline levels. Looping and kinking of the distal portion of the IL MCA (lateral view of ECA D35). Red arrows represent hypoperfused arteries or arterial tortuosity and path alteration. Statistical differences using mixed model ANOVA and Tukey post hoc test for the CCA to ECA group (***) P < 0.001; CCA sham to ECA sham group (‡) P < 0.05 (‡‡) P < 0.01. Error bars represent standard deviation.

Figure 4.

The Longa (ECA) middle cerebral artery occlusion method exacerbates the ischemic lesion and edema formation in the acute phase and tissue loss in the chronic phase. Ischemic lesion volume (a), normalized ipsilateral (b) and contralateral (d) hemisphere volume, and tissue loss (c) after middle cerebral artery occlusion (MCAO) by filament insertion through the common carotid artery (CCA), the external carotid artery (ECA), and in sham-operated (CCA sham and ECA sham) animals, measured before (baseline - BL) and on day 2 (D2), 9 (D9), and 35 (D35) after MCAO. Representative magnetic resonance T2 images of the mouse brain at baseline and after CCA or ECA MCAO at the measured time points (e). Statistical differences for the normalized ipsilateral and contralateral hemisphere volume and tissue loss using the mixed model ANOVA and Tukey post hoc test, and for the ischemic lesion volume mixed model ANOVA and Sidak post hoc test: CCA to ECA group (***) P < 0.001; CCA to CCA sham group: (###) P < 0.001; ECA to ECA sham group (†††) P < 0.001, (†) P < 0.05. Data presented as median with interquartile range.

Figure 5.

The Longa (ECA) middle cerebral artery occlusion method aggravates the neurological status in the acute phase of stroke. The neurological score (a), posture, flexion and forelimb placing (b), and motility and gait disturbances (c) before (baseline, BL) and 2 (D2), 9 (D9) and 35 (D35) days after middle cerebral artery occlusion (MCAO) by filament insertion through the common carotid artery (CCA), the external carotid artery (ECA), and in sham-operated (CCA sham and ECA sham) animals. Statistical differences using mixed model ANOVA and Tukey post hoc test for the CCA to ECA group (***) P < 0.001; CCA to CCA sham group (###) P < 0.001; ECA to ECA sham group (†††) P < 0.001. Data presented as median with interquartile range.

Figure 6.

The Koizumi (CCA) middle cerebral artery occlusion method induces transient retinal thickening. The average retinal thickness (a), and retinal thickness in 3 points inferiorly and 3 superiorly from the optic nerve head (b) measured on a mid-sagittal T2-weighted anatomical magnetic resonance imaging scan bisecting the center of the eye and the optic nerve (c) after middle cerebral artery occlusion by filament insertion through the common carotid artery (CCA), the external carotid artery (ECA), and in sham-operated (CCA sham and ECA sham) animals. Statistical differences using mixed model ANOVA and Tukey post hoc test for the CCA to ECA group (***) P < 0.001, (*) P < 0.05; CCA to CCA sham group: (#) P < 0.05. Data presented as median with interquartile range.

Figure 7.

Histological and immunofluorescent staining reveals cell loss and thinning in the Koizumi (CCA) middle cerebral artery occlusion method. Average retinal thickness measured on the hemalaun/eosin-stained sections (a) 35 days after middle cerebral artery occlusion by filament insertion through the common carotid artery (CCA), the external carotid artery (ECA), and in sham-operated (CCA sham and ECA sham) animals, shows thinning of the ipsilateral (IL) retina in the CCA animals. Representative section of the contralateral (CL) retina of the CCA group 35 days after MCAO displays normal morphology (b). The IL retina in the CCA group (c) displays disrupted retinal morphology, a pronounced cell loss in the ganglion cell layer (GCL), thinning of the internal plexiform layer (IPL), cell loss in the internal nuclear layer (INL), and thinning of the outer plexiform layer (OPL). Immunofluorescent staining of retinal cross-sections 35 days after MCAO confirms NeuN-positive cell loss in the ganglion cell layer of the CCA IL retinas (d, e). Magnification 20× scale bar = 100 μm. Statistical differences using mixed model ANOVA and Tukey post hoc test for the CCA to ECA group (***) P < 0.001. Data presented as median with interquartile range.