Effects of therapeutic treatment with angiotensin II receptor blocker on intraspinal inflammatory cell phenotype following experimental spinal cord injury
By Yaseen Baseer, Carleton B Jones, Ph.D. and T Bucky Jones, Ph.D.
Arizona College of Osteopathic Medicine, Midwestern University, Glendale, Arizona
We have previously shown beneficial effects of manipulating the renin-angiotensin system (RAS) to improve functional outcome following spinal cord injury in rats. Furthermore, corresponding analyses of changes in gene expression at the injury site suggest that drug treatment altered several chemokines associated with infiltration of inflammatory cells. Compounds such as captopril, an angiotensin-converting enzyme (ACE) inhibitor, and losartan, an angiotensin II type 1 receptor (AT1R) blocker, have been associated with beneficial regulatory control of the neuroinflammatory response in models of CNS disorders (e.g., Alzheimer disease).
Within the injured spinal cord, macrophages express distinct functional phenotypes. M1 phenotypes exhibit tissue destructive properties while M2 phenotypes promote tissue repair. Although our previous work showed that treatment with captopril or losartan did not affect the overall macrophage response to injury at the injury epicenter, it is possible that the functional profile of the macrophages was altered by drug treatment. The aim of the present study was to extend these initial data and determine whether losartan treatment promoted deviation toward an M2 phenotype in macrophages either at the site of injury or rostral and caudal to the injury site. We used immunohistochemical techniques to analyze histopathological changes in response to spinal cord injury in rats treated with losartan and examined the functional phenotype of macrophages entering the spinal cord after compression injury.Tissues were stained with an antibody that specifically binds to mannose receptor, type C, a scavenger receptor associated with a reparative M2 macrophage phenotype. Rostral and caudal to the site of injury (lesion margins) we observed a decrease in the M2 response over time from 7 days post-injury to 28 days post-injury with an associated increase in the M1 response. These data suggest that the beneficial effects of RAS inhibition following SCI are not due to promotion of an intraspinal M2 phenotype.
We hypothesized that inhibition of RAS via administration of an angiotensin receptor blocker (ARB) would alter the phenotype of recruited macrophages and other immune cells in spinal-injured rats.
Anesthetized female Sprague-Dawley rats underwent a laminectomy at vertebral level T8 and were given SCI by lateral compression with modified forceps. Animals were treated daily with losartan (i.p.) and sacrificed at 7, 14, and 28 days post-injury.
Spinal cords were excised at the above mentioned days and stored at -80°C in a OCT compound before being cut on a cryostat at 10 mm intervals. 0.1 M phosphate buffer was used as our primary buffer solution, and biotinylated horse anti- mouse (BHAM) was used as a secondary antibody. Myelin was evaluated using Eriochrome Cyanine histochemistry and sections were stained macrophage and T-cells using the following specific antibody clones:
GFAP: astrocyte intermediate filament protein OX-42: recognizes most macrophages via the integrin alpha M antigen which participates in various adhesive interactions of monocytes, macrophages, and granulocytes. CD-206: mannose receptor type C present on both dendritic cells and macrophages. Selectively expressed on M2 macrophages and associated with initiation of tissue repair
Immunohistochemistry for proportional area of stained tissue was measured using NIH ImageJ software. Images were imported into ImageJ and converted to 8-bit grayscale. The cross-section of the spinal cord was outlined and a thresholding tool was used to highlight the positively stained tissue. To determine the proportional area, the area of positively stained tissue was measured and divided by the total cross-sectional area of the spinal cord. The epicenter section was defined by the tissue section exhibiting the least amount of spared myelin. Sections were analyzed at 2 mm increments up to 6 mm rostral and 6 mm caudal to the epicenter for each animal. Glial cells were assessed using three antibodies that recognize different antigens: GFAP, OX42 and CD206.
GFAP: There was a significant effect of distance from the epicenter at 7 and 14 dpi, suggesting that the magnitude of GFAP expression differs across the rostrocaudal extent of the lesion. There was a significant effect of treatment with losartan observed at 28 dpi. Treatment with losartan decreased the magnitude of GFAP staining rostral and caudal to the epicenter, suggesting that inhibition of AT1R altered lesion dynamics and the induction of the astroglial scar.OX-42: There was a significant effect of distance from the epicenter at 7, 14, and 28 dpi, suggesting that the macrophage response decreases as the distance from the epicenter increases. The overall macrophage response to injury was not affected by treatment with losartan, suggesting that beneficial effects on locomotor recovery were not due to changes in the magnitude of the inflammatory response to injury.
CD206: The relative expression of CD206 was similar at all rostrocaudal lesion locations at 7 and 28 dpi. Given that the overall magnitude of the macrophage response diminished at sites distal to the lesion epicenter, but expression of CD206 was maintained suggests that macrophages further from the injury epicenter are more likely to express an M2, or reparative phenotype. There was no effect of losartan on the proportion of M2 macrophages at any time point assessed.
These findings suggest an effect of losartan on the astroglial scar, but not on the macrophage response to injury. Thus, it is possible that the beneficial effects observed on locomotor recovery in this model, and neuroprotective effects described in other models of CNS injuries, are not due to modulation of neuroinflammation, but instead may be due to effects on endogenous glial cells.