Table 1.
Primary and secondary antibodies used for immunohistochemistry analysis.
Fig 1.
Chemotherapy results in a high blood cells chimerism in GFP+/-→ WT mice.
Blood chimerism was assessed by flow cytometry 8 weeks following transplantation of GFP+/- bone marrow-derived cells in wild-type (WT) C57BL/6 recipient mice conditioned with busulfan and cyclophosphamide (n = 15) and compared with WT (n = 6) and GFP+/- mice (n = 9). (A) Representative flow cytometry contour plots showing the gating strategy for blood leukocytes (i.e., CD45+) (continuous rectangle), granulocytes (CD45+/CD11b+/Ly6G+) (dashed rectangle), inflammatory monocytes (CD45+/CD11b+/CD115+/Ly6Chi) (continuous circle) and patrolling monocytes (CD45+/CD11b+/CD115+/Ly6Clow) (dashed circle). (B) Representative flow cytometry contour plots illustrating the strategy used to determine GFP expression in each of these leukocyte populations. (C) Histogram illustrating the percentage of CD45+/GFP+ cells compared to total leukocytes (CD45+) in WT, GFP+/- and GFP+/-→WT mice and (D) histogram showing the evaluation of GFP expression in the different blood cell populations of the myeloid lineage in all three groups of mice. Results are from two independent experiments. Statistical analyses were performed using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test. *, P≤0.05 compared to GFP+/- mice.
Fig 2.
Kinetics of infiltrating blood myeloid leukocytes in the CNS during HSE.
Brain leukocytes of GFP+/-→WT mice were analysed by flow cytometry prior to and on days 4, 6, 8 and 10 following intranasal infection with 1.2x106 PFU of HSV-1. (A) Flow cytometry contour plots illustrating the gating strategy used for brain leukocytes differentiation in mice sacrificed on day 6 following infection. CD45 marker was used to discriminate microglia (i.e., CD45low) (continuous rectangle) from infiltrating leukocytes (i.e., CD45hi) (dashed rectangle). Among CD45hi/CD11b+/GFP+ infiltrating myeloid cells (dashed circle), neutrophils were selected based on the expression of the granulocyte marker Ly6G whereas monocyte subsets were discriminated according to Ly6C expression level into Ly6Chi inflammatory monocytes and Ly6Clow patrolling monocytes. (B) Infiltrating leukocytes, (C) neutrophils, (D) inflammatory monocytes and (E) patrolling monocytes percentages with respect to total brain leukocytes were evaluated during HSE. Histograms represent data obtained with n = 5 to 6 mice per time point. (F) Percentage of body weight changes in GFP+/-→WT mice following infection with HSV-1 (n = 9 mice). Statistical analyses were performed using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test. *, P<0.05; **, P<0.01; ***, P<0.001 compared to non-infected group.
Fig 3.
Intranasal infection with HSV-1 induces blood leukocytes infiltration in the CNS of chimeric C57BL/6 mice.
Representative micrographs illustrating the localization of GFP+ infiltrating cells in the olfactory bulb, the interbrain and the hindbrain of GFP+/-→WT mice. Sixteen week-old chimeric mice were infected with HSV-1 by the intranasal route and sacrificed prior to (negative control) and on days 4, 6, 8 and 10 post-infection (5 mice per group). (A) Brain slices of 25-μm thick were processed for immunohistochemistry staining with a primary polyclonal goat anti-GFP and a secondary Alexa 488-conjugated chicken anti-goat antibodies (green). In non-infected mice (NI), no GFP+ cells could be found in the brain parenchyma. Following infection, peripheral leukocytes infiltrated the CNS and could be detectable mainly in the olfactory bulb, the interbrain and the hindbrain. Scale bar 100 μm. (B) Representative micrographs illustrating the localization of HSV-1 particles in different regions of the brains of GFP+/-→WT mice on day 6 post-infection. Brain slices were processed for immunohistochemistry analysis with a primary polyclonal rabbit anti-HSV-1 antibody and a secondary Alexa 594-conjugated goat anti-rabbit antibody (red), followed by nuclear staining with DAPI (blue). Viral antigens were detected in the olfactory bulb, the interbrain and the hindbrain. Scale bar 50 μm.
Fig 4.
Evaluation of blood-brain barrier integrity in chimeric C57BL/6 mice following HSV-1 infection.
Micrographs illustrating the detection of albumin in the brain parenchyma of non-infected (NI) animals and mice sacrificed on days 6 and 8 following HSV-1 infection. Brain slices were processed for immunohistochemistry with primary goat anti-albumin and secondary Alexa 594-conjugated chicken anti-goat (red) antibodies. Pictures selected showed no immuno-reactivity for albumin in non-infected mice in the olfactory bulb and hindbrain. Positive control corresponds to a slice of an ischemic brain tissue obtained in a mouse model of stroke. Scale bar 400 μm.
Fig 5.
Blood-derived GFP+ monocytes that infiltrate the brain parenchyma following HSV-1 infection express the microglia marker Iba1.
GFP+/-→WT mice were infected with HSV-1 via the intranasal route and sacrificed prior to and on days 4, 6, 8 and 10 following infection. (A) The location and phenotype of engrafted GFP+ cells were assessed in brain sections by double immunostaining with goat anti-GFP and rabbit anti-Iba1 followed by secondary antibodies, Alexa 488-conjugated chicken anti-goat (green) and Alexa 594-conjugated chicken anti-rabbit (red), respectively. Nuclear staining with DAPI is shown in blue. Scale bar 25 μm. Higher magnification of brain sections illustrating the ramified (B) and amoeboid (C) forms of infiltrating monocytes (GFP+) that expressed the microglia marker Iba1 on days 4 and 6 post-infection, respectively. Panels (D) and (E) show resident microglia (Iba1+/GFP-) with ramified and amoeboid morphologies related to resting and activated states, respectively, on day 6 following infection. Scale bar 25 μm.
Fig 6.
Resident microglia and monocyte-derived macrophages express the CD68 and MHC II activation markers in response to HSV-1 infection.
(A) Brain sections of infected GFP+/-→WT mice were stained for microglial cells with rabbit anti-Iba1 and for lysosomal activation marker with rat anti-CD68 followed by secondary Alexa 594-conjugated goat anti-rabbit (red) and Cy5-conjugated goat anti-rat (white) antibodies, respectively. Nuclear staining with DAPI is shown in blue. Pictures were taken from brain sections of mice sacrificed on day 6 or 8 following infection. (B) Staining of brain sections for microglial cells with rabbit anti-Iba1 and for cell surface activation marker with rat anti-MHC II followed by secondary antibodies, Alexa 594-conjugated goat anti-rabbit (red) and Cy5-conjugated goat anti-rat (white), respectively. Both resident microglia (Iba1+/GFP-) (white circles) and blood monocyte-derived macrophages (GFP+/Iba1+) (white rectangles) expressed the lysosomal marker CD68 (Fig 6A) and the cell surface marker MHC II (Fig 6B). Scale bar 50 μm.