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Brain to spleen stem cell migration
cell therapies in stroke and other neurological disorders with pathologies characterized by aberrant inflammation. The spleen is a major contributor to the peripheral inflammatory response observed following stroke.13,14 Acting as a reservoir for leukocytes, the spleen is the pri- mary disseminator of inflammatory cells in response to injury.15 This splenic response, paired with the compro- mised BBB following stroke, contributes to the infiltration of pro-inflammatory mediators into the brain and wors- ened outcomes.16-18 We have previously reported that human bone marrow mesenchymal stromal cells (hBMSC) delivered intravenously preferentially migrate to the spleen, dampening systemic inflammation.19 These findings support the therapeutic potential of targeting the peripheral inflammatory response via the spleen to abro- gate neuroinflammation, in addition to implicating stem
cells as inflammation-homing biologics.
In light of the spleen and peripheral inflammation being
principal culprits in neuroinflammatory-induced cell death processes20,21 the recently characterized cerebral lymphatic system opens a new avenue of research in stem cell ther- apies for neurological disorders.22 Cognizant that the spleen is a major destination for lymphatic drainage, the cerebral lymphatic system could serve as an efficient route for brain-to-spleen stem cell migration. To date, this notion of intracerebrally transplanted stem cells migrating remotely away from the implantation sites in ischemic regions, albeit outside the brain, has not been investigated. Here, we report for the first time that stem cells can migrate from the cerebrum to the periphery via lymphatic vessels, likely amplified by stroke-induced local and peripheral inflammation. This line of investigation advances the concept of targeting the source of the periph- eral inflammatory response by harnessing lymphatic ves- sel-directed migration of stem cells. The present study also provides valuable data toward a novel understanding of how intracerebral transplantation of stem cells functions to repair the damaged brain through peripheral effectors.
Methods
Animals and housing
All experiments were approved by the Institutional Animal Care and Use Committee of the University of South Florida, Morsani College of Medicine and were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals. All experiments were carried out on 2-month old Sprague– Dawley male rats (Harlan Laboratories, Indianapolis, IN, USA) and rats were either exposed to sham (n=6) or stroke surgery, with the latter further classified as mild (n=9) or severe (n=9) based on the severity of the stroke as evidenced by pathological outcomes. There were six animals in the sham-treated group, nine in the mild stroke group, and nine in the severe stroke group across all treat- ments, and all animals were treated with hBMSC.
Stroke surgery
Animals underwent middle cerebral artery occlusion surgery as described in our previous study.23 Sham surgery involved exposing and isolating the common carotid and internal carotid arteries before closing the incision. Severe stroke was induced by 60 min intraluminal filament occlusion of the right internal carotid artery with simultaneous ligation of the contralateral (left) common
carotid artery for 30 min. In contrast, mild stroke was induced by 60 min occlusion of the right internal carotid artery, without ligat- ing the left common carotid artery.
Cell preparation
hBMSC and bEnd.3-expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) were purchased from the American Type Culture Collection (ATCC, VA, USA). The immor- talized BV-2 murine microglial cells24 were maintained in Dulbecco modified Eagle medium (Gibco, MA, USA). Immortalized bEnd.3 and BV-2 cells were used to better manage the growth of these cells in culture over longer periods of time.25 For transplantation preparation, hBMSC density was adjusted to 7.5×106 cells in 216 mL of phosphate-buffered saline. For cell migration, the cell densi- ty was adjusted to 1×106 cells in 5 mL fluorescent medium (ThermoFisher, MA, USA). Thereafter, cells were incubated with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlo- rate (DiD, Invitrogen, OR, USA) for 30 min, to aid the visualiza- tion of hBMSC migration.
Transplantation
One hour after middle cerebral artery occlusion surgery, rats were anesthetized and hBMSC were injected intracerebrally into the striatum and cortex of the ischemic hemisphere over 10 min as previously described.26 Within a single needle pass, two deposits of hBMSC were made in the striatum (DV=5.0 mm and DV=4.0 mm) and one deposit of hBMSC was made in the cortex (DV=3.0 mm) for a total of three separate deposits (AP=+0.5 mm, ML=+2.8 mm, DV=5.0 mm, 4.0 mm, and 3.0 mm).26 Each deposit contained 1×105 cells/3 mL phosphate-buffered saline for a total of 3×105 cells suspended in 9 mL phosphate-buffered saline for the entire transplant regimen per animal.
Brain and organ harvesting, fixation, and sectioning
Rats were euthanized under deep anesthesia on day 1, day 3, or day 7 after transplantation for ex vivo imaging analysis, as described in our past study.21
Measurement of infarct area
Hematoxylin and eosin staining was performed to confirm the core infarct injury of our stroke model as shown in our previous studies.24,27
Immunohistochemistry assays
Human nuclei (HuNu) and OX6 staining was performed as described in our previous study.21 Details of the immunohisto- chemistry assays are described in the Online Supplementary Material.
Cell migration assay
The cell migration assay was performed using a FalconTM FlouroBlokTM 96-well HTS insert system with 3.0 mm pores (Life Science, NC, USA). BV2 and bEnd.3 cells were fed with fresh growth medium at the bottom of the lower chamber in a 96-well plate. There were three groups in the bottom cell: BV2, bEnd.3, and BV2+bEnd.3. The cell density was adjusted to 2×104 cells in 200 mL growth medium/well overnight. Cells were treated with different doses of tumor necrosis factor-alpha (TNF-a; 0 ng/mL, 25 ng/mL, 50 ng/mL, or 100 ng/mL) in an incubator overnight. The details of the cell migration assay are described in the Online Supplementary Material.
Statistical analysis
All data are expressed as the mean ± standard error of mean and statistically evaluated using one-way or two-way analysis of vari-
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