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  • A Novel Fluorescent Imaging Technique for Assessment of Cerebral Vasospasm after Experimental Subarachnoid Hemorrhage

    Final Number:

    Diane Aum BS; Ananth K. Vellimana MBBS; Byung Hee Han PhD; Itender Singh Ph.D.; Eric Milner BS; James Nelson; Gregory J. Zipfel MD

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2017 Annual Meeting

    Introduction: Various techniques have been developed to study changes in cerebral vasculature in numerous neuropathological processes including subarachnoid hemorrhage (SAH). In the laboratory setting, one of the most widely employed techniques uses India ink-gelatin casting to visualize cerebral vasculature. This technique, however, presents numerous challenges due to its high viscosity, rapid solidification, and its impact on immunohistochemical analysis. To overcome these limitations, we developed a novel technique for assessing cerebral vasculature using cerebrovascular perfusion with ROX SE, a fluorescent labeling dye.

    Methods: The endovascular perforation method at the ACA-MCA bifurcation was utilized to induce SAH in mice. Three days after SAH, the mice were anesthetized and perfused with a phosphate-buffered saline solution containing ROX SE followed by paraformaldehyde. Vasospasm was assessed by measuring the MCA diameter. Immunohistochemistry was utilized to assess other neuropathological endpoints.

    Results: ROX SE perfusion resulted in qualitatively superior visualization of cerebral vasculature compared to India ink-gelatin-casted brains. It allowed fluorescent illumination of vessel walls and circumvented problems of ambiguous borders of overlaying vessels and incomplete perfusion. For assessment of vasospasm following SAH, both methods detected significant differences between SAH and sham surgery groups (ROXSE: 87.04.0 µm vs. 64.52.6 µm, p<0.05; India ink: 95.32.9 µm vs. 74.54.2 µm, p<0.05). There were no significant differences between the two methods in the ability to detect vasospasm. However, the ROX SE perfusion technique exhibited several advantages including lower inter-observer variability in vessel diameter measurements, and ability to perform subsequent immunohistochemical studies for assessment of other neuropathological endpoints such as neuroinflammation, BBB breakdown, and microthrombosis.

    Conclusions: The ROX SE perfusion technique can be successfully utilized to evaluate SAH-induced cerebral vasospasm with results comparable to India ink-gelatin casting. This technique also offers multiple advantages including better precision and the ability to use ROX SE-perfused tissue for histological studies to assess other pathophysiological process occurring after SAH.

    Patient Care: Aneurysmal SAH is associated with significant mortality (average case fatality rate of 50%) and morbidity (30% of survivors become functionally dependent).1 It is now understood that secondary brain injury following SAH is a result of a multitude of pathophysiological events, including microvascular autoregulatory dysfunction, microvessel thrombosis, neuroinflammation, and neuronal cell death.2 Our novel imaging technique provides an effective and cost-efficient method to assess both vasospasm and other neuropathological endpoints. Given the multifactorial etiology of secondary brain injury following SAH, it is apparent that assessment of both vasospasm and non-vasospasm components of secondary brain injury is needed to develop effective therapeutic strategies for SAH.

    Learning Objectives: 1. Demonstrate that ROX SE perfusion technique allows qualitatively and quantitatively similar vessel measurements to India ink-gelatin casting. 2. Demonstrate that ROX SE perfusion detects cerebral vasospasm after experimental SAH 3. Perform immunohistochemical assessment in perfused tissue. 4. Compare inter-observer variability of ROX SE vs. India ink-gelatin. 5. Examine cost-effectiveness of ROX SE perfusion technique.

    References: 1. Linn FH, Rinkel GJ, Algra A, van Gijn J. Incidence of subarachnoid hemorrhage: Role of region, year, and rate of computed tomography: A meta-analysis. Stroke. 1996;27:625-629 2. Pluta RM, Hansen-Schwartz J, Dreier J, Vajkoczy P, Macdonald RL, Nishizawa S, et al. Cerebral vasospasm following subarachnoid hemorrhage: Time for a new world of thought. Neurological research. 2009;31:151-158

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