18F-FAC PET Visualizes Brain-Infiltrating Leukocytes in a Mouse Model of Multiple Sclerosis

Abstract

Brain-infiltrating leukocytes contribute to multiple sclerosis (MS) and autoimmune encephalomyelitis and likely play a role in traumatic brain injury, seizure, and stroke. Brain-infiltrating leukocytes are also primary targets for MS disease-modifying therapies. However, no method exists for noninvasively visualizing these cells in a living organism. 1-(2'-deoxy-2'-¹⁸F-fluoroarabinofuranosyl) cytosine (¹⁸F-FAC) is a PET radiotracer that measures deoxyribonucleoside salvage and accumulates preferentially in immune cells. We hypothesized that ¹⁸F-FAC PET could noninvasively image brain-infiltrating leukocytes. Methods: Healthy mice were imaged with ¹⁸F-FAC PET to quantify if this radiotracer crosses the blood-brain barrier (BBB). Experimental autoimmune encephalomyelitis (EAE) is a mouse disease model with brain-infiltrating leukocytes. To determine whether ¹⁸F-FAC accumulates in brain-infiltrating leukocytes, EAE mice were analyzed with ¹⁸F-FAC PET, digital autoradiography, and immunohistochemistry, and deoxyribonucleoside salvage activity in brain-infiltrating leukocytes was analyzed ex vivo. Fingolimod-treated EAE mice were imaged with ¹⁸F-FAC PET to assess if this approach can monitor the effect of an immunomodulatory drug on brain-infiltrating leukocytes. PET scans of individuals injected with 2-chloro-2'-deoxy-2'-¹⁸F-fluoro-9-β-d-arabinofuranosyl-adenine (¹⁸F-CFA), a PET radiotracer that measures deoxyribonucleoside salvage in humans, were analyzed to evaluate whether ¹⁸F-CFA crosses the human BBB. Results:¹⁸F-FAC accumulates in the healthy mouse brain at levels similar to ¹⁸F-FAC in the blood (2.54 ± 0.2 and 3.04 ± 0.3 percentage injected dose per gram, respectively) indicating that ¹⁸F-FAC crosses the BBB. EAE mice accumulate ¹⁸F-FAC in the brain at 180% of the levels of control mice. Brain ¹⁸F-FAC accumulation localizes to periventricular regions with significant leukocyte infiltration, and deoxyribonucleoside salvage activity is present at similar levels in brain-infiltrating T and innate immune cells. These data suggest that ¹⁸F-FAC accumulates in brain-infiltrating leukocytes in this model. Fingolimod-treated EAE mice accumulate ¹⁸F-FAC in the brain at 37% lower levels than control-treated EAE mice, demonstrating that ¹⁸F-FAC PET can monitor therapeutic interventions in this mouse model. ¹⁸F-CFA accumulates in the human brain at 15% of blood levels (0.08 ± 0.01 and 0.54 ± 0.07 SUV, respectively), indicating that ¹⁸F-CFA does not cross the BBB in humans. Conclusion:¹⁸F-FAC PET can visualize brain-infiltrating leukocytes in a mouse MS model and can monitor the response of these cells to an immunomodulatory drug. Translating this strategy into humans will require exploring additional radiotracers.

Keywords: PET imaging; autoimmune disease; brain; leukocytes.

FIGURE 1.

¹⁸F-FAC crosses the healthy BBB in mice. (A) Representative sagittal ¹⁸F-FAC PET/CT image of a healthy C57BL/6 mouse. Brain is outlined in white. (B) Representative ¹⁸F-FAC autoradiography and hematoxylin and eosin (H&E) staining of a sagittal brain section from a healthy C57BL/6 mouse. Scale bar = 3 mm; c = cerebellum; n = neocortex. (C) Blood and brain ¹⁸F-FAC levels, quantified from PET images of healthy C57BL/6 mice (n = 5). (D) Normalized blood and brain ¹⁸F-FAC levels, quantified from extracted blood and brain (n = 6). *P < 0.05. ns = not significant.

FIGURE 2.

Brain-infiltrating leukocytes are present in a mouse model of EAE. (A) Time course of EAE symptoms in immunocompetent and immunocompromised mice (n = 4). (B) Hematoxylin and eosin and immunohistochemical stains of brain sections of mice before and after immunization. Images are at ×40 magnification, with ×1 shown in insets. Scale bar = 50 μm. (C) Immune cell populations isolated by fluorescence-activated cell sorting from immunocompetent EAE mouse brains (n = 3).

FIGURE 3.

Brain accumulation of ¹⁸F-FAC is higher in EAE mice than in control mice. Sagittal ¹⁸F-FAC PET/CT images and quantification of immunocompetent (A) and immunocompromised (B) mice before and after immunization. Brains are outlined in white. FAC block = coinjection of mice with ¹⁸F-FAC and nonradiolabeled FAC. n = 4 for all experiments. *P < 0.05. **P < 0.01. ns = not significant.

FIGURE 4.

¹⁸F-FAC accumulates in brain-infiltrating leukocytes in an EAE mouse model. (A) dCK immunostaining of brain sections of mice before and after immunization. Images are at ×40 magnification, with ×1 shown in insets. Scale bar = 50 μm. (B) ¹⁸F-FAC autoradiography images and hematoxylin and eosin staining of sagittal brain sections of immunocompetent mice before and after immunization. Images are at ×1 (top) and ×40 (bottom) magnification. Scale bar = 50 μm. c = cerebellum; n = neocortex; x = regions where tissue wrinkled. (C) Deoxycytidine (dC) accumulation in leukocyte populations isolated from immunocompetent EAE mouse brains (n = 2). ns = not significant.

FIGURE 5.

¹⁸F-FAC PET can monitor immunomodulatory drug treatments in an EAE mouse model. Hematoxylin and eosin stains of brain sections (A) and sagittal PET/CT images and quantification of ¹⁸F-FAC accumulation (B) in brains of mice before and after immunization and treated with vehicle or fingolimod. Hematoxylin and eosin images are at ×40 magnification, with ×1 shown in insets. Brains are outlined in white. Scale bar = 50 μm. **P < 0.01. ***P < 0.001.