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A photothrombosis-induced ischaemic infarct model for study of hind brain stroke

A.J. Craig, A. Krishnan and G.D. Housley, Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, University of New South Wales, NSW 2052, Australia.

The mortality rate following a cerebellar infarction is 23% greater than for an infarct in any other part of the brain (Macdonell et al., 1987). Moreover, vertebrobasilar stroke has a mortality rate of 85% (Kaye, 2011). Despite the high mortality and morbidity of hind brain strokes, there are limited animal models to aid investigations into novel therapeutic options. Currently, small animal models of stroke focus on middle cerebral artery occlusion or global ischaemia. Neither of these models supports an investigation of hind brain infarction. We therefore investigated whether photothrombosis may provide the necessary spatio-temporal control of infarct volume; further, we resolved to settle the controversy over whether this technique provides a thrombotic infarct, or whether, as recently suggested (Frederix et al., 2007; Kleinschnitz et al., 2008), the brain injury is independent of the platelet aggregation-mediated occlusion of cerebrovascular microvessels. Here we show in the mouse cerebellum that photothrombosis can induce hind brain ischaemia, based on real-time imaging of thrombis development, magnetic resonance imaging (MRI) of the progression of these cerebellar infarcts, and histological analysis of the thrombis in the brain tissue. The infarcts were created following a protocol approved by the University of New South Wales Animal Care and Ethics Committee.

Photothrombosis in the cerebellum was achieved by intravenous administration of rose bengal followed immediately by coherent illumination at 561nm of the region of interest within the cerebellum of the isoflurane anaesthetized mouse (after Watson et al., 1985). The progression of clot formation in the vermis region of the cerebellum was visualized in real-time (established in ∼10 minutes) by intravital multi-photon imaging of the platelet aggregation (using Cy7-labelled anti-CD42). Infarcts were subsequently examined by high-field (9.4T) T2 weighted MRI at days 1, 4, and 7 post-ischaemia in situ, subsequent to deeply anaesthetizing the mouse and intracardiac perfusion with paraformaldehyde. Following MRI, the tissue was paraffin embedded, sectioned, and stained with haematoxylin and eosin to enable histological verification of the infarct and surrounding regions.

The MRI scans showed acute infarction of ∼1mm3 that corresponded to the primary vascular occlusion that was imaged using intravital microscopy. The anti-CD42 platelet marker showed the clot was restricted to the same area. This confirmed that the posterior circulation targeted using photo-induced thrombosis provided exclusive vascular support to the infarcted brain region. Further, the MRI and histology indicated that ventral to this site, there was altered diffusion (MRI) and histology, consistent with a penumbrum, which reflected propagation of collateral ischaemic injury. The key control study delivered the equivalent photonic flux in the absence of rose bengal infusion. This brain tissue lacked platelet aggregation and an infarct. These data indicated that endothelial activation by photo-sensitizing the cells with rose bengal was required for site-specific ischaemic brain injury. This model will enable study of neuroprotective signal pathways in the hind brain in the mouse model.

Frederix K, Chauhan AK, Kisucka J, Zhao BQ, Hoff EI, Spronk HM, Ten Cate H & Wagner DD. (2007) Platelet adhesion receptors do not modulate infarct volume after a photochemically induced stroke in mice. Brain Research 1185, 239-245.

Kaye V. (2011) Overview of Vertebrobasilar stroke, ed. Campagnolo D.

Kleinschnitz C, Braeuninger S, Pham M, Austinat M, Nolte I, Renne T, Nieswandt B, Bendszus M & Stoll G. (2008) Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis. Stroke; A Journal of Cerebral Circulation 39, 1262-1268.

Macdonell RA, Kalnins RM & Donnan GA. (1987). Cerebellar infarction: natural history, prognosis, and pathology. Stroke; A Journal of Cerebral Circulation 18, 849-855.

Watson BD, Dietrich WD, Busto R, Wachtel MS & Ginsberg MD. (1985). Induction of reproducible brain infarction by photochemically initiated thrombosis. Annals of Neurology 17, 497-504.

AC is supported by a UNSW Vice Chancellor’s post-doctoral Fellowship and UNSW Goldstar funding.