A hematopoietic contribution to microhemorrhage formation during antiviral CD8 T cell-initiated blood-brain barrier disruption

<p>Abstract</p> <p>Background</p> <p>The extent to which susceptibility to brain hemorrhage is derived from blood-derived factors or stromal tissue remains largely unknown. We have developed an inducible model of CD8 T cell-initiated blood-brain barrier (BBB) disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide-induced fatal syndrome (PIFS) model results in severe central nervous system (CNS) vascular permeability and death in the C57BL/6 mouse strain, but not in the 129 SvIm mouse strain, despite the two strains' having indistinguishable CD8 T-cell responses. Therefore, we hypothesize that hematopoietic factors contribute to susceptibility to brain hemorrhage, CNS vascular permeability and death following induction of PIFS.</p> <p>Methods</p> <p>PIFS was induced by intravenous injection of VP2_121-130peptide at 7 days post-TMEV infection. We then investigated brain inflammation, astrocyte activation, vascular permeability, functional deficit and microhemorrhage formation using T2*-weighted magnetic resonance imaging (MRI) in C57BL/6 and 129 SvIm mice. To investigate the contribution of hematopoietic cells in this model, hemorrhage-resistant 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Gadolinium-enhanced, T1-weighted MRI was used to visualize the extent of CNS vascular permeability after bone marrow transfer.</p> <p>Results</p> <p>C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2_121-130peptide, however, C57BL/6 mice had increased astrocyte activation, CNS vascular permeability, microhemorrhage formation and functional deficits compared to 129 SvIm mice. The 129 SvIm mice reconstituted with C57BL/6 but not autologous bone marrow had increased microhemorrhage formation as measured by T2*-weighted MRI, exhibited a profound increase in CNS vascular permeability as measured by three-dimensional volumetric analysis of gadolinium-enhanced, T1-weighted MRI, and became moribund in this model system.</p> <p>Conclusion</p> <p>C57BL/6 mice are highly susceptible to microhemorrhage formation, severe CNS vascular permeability and morbidity compared to the 129 SvIm mouse. This susceptibility is transferable with the bone marrow compartment, demonstrating that hematopoietic factors are responsible for the onset of brain microhemorrhage and vascular permeability in immune-mediated fatal BBB disruption.</p

Keratocyte apoptosis and not myofibroblast differentiation mark the graft/host interface at early time-points post-DSAEK in a cat model.

To evaluate myofibroblast differentiation as an etiology of haze at the graft-host interface in a cat model of Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK).DSAEK was performed on 10 eyes of 5 adult domestic short-hair cats. In vivo corneal imaging with slit lamp, confocal, and optical coherence tomography (OCT) were performed twice weekly. Cats were sacrificed and corneas harvested 4 hours, and 2, 4, 6, and 9 days post-DSAEK. Corneal sections were stained with the TUNEL method and immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and fibronectin with DAPI counterstain.At all in vivo imaging time-points, corneal OCT revealed an increase in backscatter of light and confocal imaging revealed an acellular zone at the graft-host interface. At all post-mortem time-points, immunohistochemistry revealed a complete absence of α-SMA staining at the graft-host interface. At 4 hours, extracellular fibronectin staining was identified along the graft-host interface and both fibronectin and TUNEL assay were positive within adjacent cells extending into the host stroma. By day 2, fibronectin and TUNEL staining diminished and a distinct acellular zone was present in the region of previously TUNEL-positive cells.OCT imaging consistently showed increased reflectivity at the graft-host interface in cat corneas in the days post-DSAEK. This was not associated with myofibroblast differentiation at the graft-host interface, but rather with apoptosis and the development of a subsequent acellular zone. The roles of extracellular matrix changes and keratocyte cell death and repopulation should be investigated further as potential contributors to the interface optical changes

Methods of Corneal Optical Coherence Tomography (OCT) Analysis.

<p>(<b>A</b>) OCT image of an <i>in vivo</i> cat cornea 4 hours after Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK). Note the greater intensity of backscatter at the graft-host interface (solid arrows) relative to the adjacent stromata. The perpendicular lines superimposed over the OCT image indicate the location of the areas analyzed for backscatter intensity (white and black) and thickness (red, orange, and maroon). Four perpendicular lines (white and black), 20 pixels wide and located +/−100 and +/−200 pixels from the central specular reflection were used to generate a pixel brightness profile. As depicted along the black line, intensity line graphs generated from each of the four perpendicular lines were then used to identify and measure intensity over three defined corneal regions; a 10 pixel thick region of interface (yellow), a 40 pixel thick region of the adjacent host stroma (green), and a 40 pixel thick region of the adjacent donor stroma (blue). Total corneal (red), host stromal (orange), and donor stromal (maroon) thickness measurements were taken in the central cornea as depicted by the central line. (<b>B</b>) A representative plot of normalized backscattered light intensity through a cat cornea post-DSAEK at a single time-point. This profile was generated as described above. These graphs were constructed for each of the line locations. Note the intensity peak at the interface (yellow) in comparison to that of the adjacent host (green) and donor stroma (blue).</p

Interface Intensity and Corneal Thickness Post-DSAEK.

<p>(<b>A</b>) Corneal optical coherence tomography (OCT) images of an <i>in vivo</i> cat cornea pre-operatively and at early post-Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) time-points. Note the easily discernible post-operative interface between the host and donor stromata appears brighter than the adjacent tissue. The host stroma also has increased thickness immediately post-operatively, which improves with time. (<b>B</b>) Corneal OCT image-derived backscatter intensity at the graft-host interface (yellow) in comparison to the adjacent host stroma (green) and donor stroma (blue) at early post-DSAEK time points. Note that the graft-host interface was consistently brighter than the adjacent host stroma and adjacent donor stroma. This result was statistically significant at the majority of time-points; between interface and adjacent host stroma at days 0 (p = 0.0078), 2/3 (p = 0.0053), 4/5 (p = 0.0019) and 6/7 (p = 0.0003), and between interface and adjacent donor stroma at days 0 (p = 0.0078), 2/3 (p = 0.0005), and 6/7 (p = 0.0155). This difference was no longer observed at the 8/9 day time point. * = significant difference between mean interface and mean host stromal intensity. † = significant difference between mean interface and mean donor stromal intensity. (<b>C</b>) OCT image-derived corneal thickness measurements across early post-DSAEK time-points. Note the brisk increase in total thickness from pre-operative levels (Pre-Op) to immediate post-operative (0) levels associated with the addition of the donor tissue, and the subsequent gradual decline in total thickness to day 6/7.</p

Immunohistochemistry for detection of alpha-smooth muscle actin (α-SMA) and fibronectin post-Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK).

<p>Corneal sections were stained with antibodies against α-SMA (red) to label myofibroblasts, antibodies against fibronectin (green), and 4′6′-diamidino-2-phenylondole dihydrochloride (DAPI) (blue) was used to label cell nuclei. (<b>A–C</b>) Photomicrographs of <i>ex vivo</i> cat corneal sections of the graft-host interface (arrows) on post-operative days 0 (<b>A</b>), 4 (<b>B</b>), and 9 (<b>C</b>)(scale bar for A – C = 0.2 mm). (<b>D and E</b>) The graft-host interface on days 0 (<b>D</b>) and 9 (<b>E</b>) (scale bar for D & E = 0.2 mm). (<b>F</b>) The central stroma of an unoperated cat cornea demonstrated an absence of α-SMA and mild diffuse fibronectin staining. (<b>G and H</b>) Incisional paracenteses wounds on day 0 (<b>G</b>) and day 9 (<b>H</b>) (scale bar for F – H = 0.4 mm). (<b>I</b>) An incisional paracentesis wound on day 9 at high magnification (scale bar for I = 0.2 mm). Note the lack of α-SMA staining at the graft-host interface (<b>C and E</b>), but positive α-SMA staining at the incisional wound (<b>H and I</b>) on day 9. On day 0, 4 hours after DSAEK, fibronectin staining was present extracellularly along the interface and also appeared to co-localize with DAPI in the cells of the adjacent host stroma (<b>A and D</b>). On day 9 post-DSAEK, there was faint fibronectin staining near the host stromal cells, but the interface fibronectin staining is much fainter and more consistent with the unoperated control.</p