Introduction: The immune system’s response to tissue injuries has emerged as a pivot event in the healing response. Regenerative medicine is hence increasingly investigating how an initial pro-inflammatory response converts to a pro-repair response. Implantation of bioscaffolds exploits this mechanism and allows tissue to regrow in large defects. Even the brain, which is known to not regrow tissue, can regenerate lost tissue after implantation of an inductive bioscaffold, consisting of extracellular matrix (ECM) formulated as a hydrogel (1). However, little is known about how immune cells respond to the implantation of a bioscaffold into the brain. We therefore here aimed to characterize the acute spatio-temporal dynamics of immune cell infiltration into the brain and a bioscaffold after its implantation into a sub-acute stroke tissue cavity. To visualize infiltrating immune cells, they were systemically tagged using perfluorocarbon nanoemulsions for detection by 19F MRI, while placing their distribution into anatomical context using 1H MRI.
Methods:
Animals. Sprague-Dawley rats (12 weeks old) underwent 70 minutes of right middle cerebral artery occlusion to model stroke. Twelve days post-stroke animals were injected with perfluorcarbon (PFC) nanoemulsions (360 mg/mL, V-Sense, CelSense, Pittsburgh, PA) through the tail vein to label circulating immune cells. 19F MRI acquisition parameters were previously optimized for the fast imaging with steady-state precession (FISP) sequence(2). A baseline 19F (TR=4.8 ms, TE=2.4 ms, matrix 96x96, FOV 40 x 40 mm, slice thickness 1.5 mm, acquisition time 60 minutes) and geometrically identical T2-weighted (RARE, TR=5000 ms, TE=40 ms, matrix 192x192, slice thickness 1.5 mm, acquisition time 60 minutes) 1H MRI scan was acquired to verify the delivery of PFC, as evidenced by “hot” blood vessels. Thirteen days post-stroke ECM hydrogel (4 mg/mL) was injected into the stroke cavity. 19F MR images were acquired at 1, 2, 3, 4, 5, 6, 9, 18, and 24 hours post-implantation. 19F spins were quantified for the peri-infarct area and the ECM hydrogel using VoxelTracker (CelSense). After scanning, rats were perfused. Fluorescence immunohistochemistry on brain tissue quantified the number of macrophages, lymphocytes and neutrophils that invaded the peri-infarct area and the ECM hydrogel. Co-localization with the red fluorescence of the PFC afforded a quantitation of the number of PFC-labeled and -unlabeled cells.
Results: Baseline 19F/1H MR images revealed the administration of PFC nanoemulsion into the blood stream with no PFC leaking into the stroke lesion (12 days post-stroke). Upon implantation of an ECM hydrogel into the stroke cavity, a major signal increase in 19F was evident in the peri-infarct area, as well as the ECM hydrogel (Figure 1A). Quantitation of the signal over time indicated an increased signal first in the peri-infarct tissue at 6 hours post-implantation, followed by a gradual invasion into the ECM hydrogel peaking at 12 hours post-implantation (Figure 1B). Histological verification of the PFC-labeled cells distribution validated that the 19F signal reflects the infiltration of immune cells (Figure 2). Immunohistochemistry further indicates that only a very small proportion of immune cells (1-2%) in the peri-infarct area were PFC negative, whereas up to 20% of immune cells in the ECM were PFC negative. The highest proportion of infiltrating cells consisted of neutrophils followed by macrophages in the peri-infarct area, as well as the ECM hydrogel. Only a small proportion (<2%) were lymphocytes in the peri-infarct area. No lymphocytes were present in the ECM hydrogel.
Conclusion: PFC nanoemulsion detection using 19F MRI provides a unique tool to characterize the spatio-temporal dynamics of labelled-immune cell infiltration into brain tissue and bioscaffolds. This approach will be essential to improve our understanding of how the immune system mediates bioscaffold-induced tissue regeneration.

Acknowledgements

No acknowledgement found.