CB₂ receptor-mediated microglial migration.

<p>A) Human microglia were loaded onto the upper chemotaxis chambers with lower chambers filled with media (C) or CCL2 (3 to 100 ng/ml) and incubated for 3h. B) Human microglia were loaded onto upper chemotaxis chambers and pretreated with CB antagonists (SR141716A or SR144528, 3 µM) for 30 min followed by WIN55,212-2 (1 µM) treatment for 3h prior to be assembled with lower chambers filled with media (C) or CCL2 (100 ng/ml) and incubated for 3h. Migrated microglial cells were quantified by Diff-Quik staining. *p<0.05, **p<0.01 vs. control (C); ^††p<0.01 vs. CCL2; ^§§ p<0.01 vs. WIN55,212-2+CCL2.</p

Antioxidant gene expression is upregulated in the brains of mice with herpes encephalitis.

<p>Balb/c mice were infected intranasally with 2×10⁵ PFU of HSV-1 strain 17 syn+ (n = 7). An equal volume of saline was delivered to control mice (n = 6). At 7 d p.i. mice were anaesthetized, dissected and 2 mm-thick sections of subcortical brain tissue were collected for mRNA extraction, cDNA synthesis and semi-quantitiative PCR analysis of HO-1 (*p = 0.01), Gpx1 (*p = 0.001), and Nrf2 expression.</p

Blockade of gp120-induced suppression of DAT activity.

<p>Human mesencephalic neuronal/glial cultures were untreated (C) or pretreated with <b>A</b>) WIN55,212-2 or <b>B</b>) JWH015 at the stated concentrations for 3h prior to gp120 (10 nM) treatment for 5 days followed by ³H-DA addition for 10 min as a measurement of ³H-DA uptake for DAT activity. Data are mean ± SEM of triplicates of 3-4 separate experiments using different brain tissue specimens. **p<0.01 vs. control; ††p<0.01 vs. gp120. </p

Plasma cell recruitment and retention in the brain following MCMV infection.

<p><b>A</b>. Single cell suspensions of infiltrating brain leukocytes from MCMV-infected mice were obtained and analyzed at the indicated time points. Contour plots depicting percentages of CD38⁺ and CD138⁺ double positive cells from the gated CD45hi population are shown. These cells also stained negative for CD3 and CD19. <b>B</b>. Data showing the mean (±SEM) absolute number of cells within the infiltrating CD45^(hi)CD3⁻CD19⁻ population pooled from 3 independent experiments. <b>C</b>. Immunofluorescent staining showing the distribution of CD138⁺ plasma cells in both the ventricles and brain parenchyma at 30 d p.i. (magnification 20×).</p

Increased brain ROS levels during herpes encephalitis.

<p>Balb/c mice were infected intranasally with 2×10⁵ PFU of HSV-1 strain 17 syn+ (n = 10). An equal volume of saline was delivered to control mice (n = 8). At 7 d p.i. whole brains were pooled, mononuclear cells were isolated and analyzed via flow cytometry using fluorescent-conjugated antibodies, CD11b-APC and CD45-APC-Cy7. CD11b⁺, CD45^hi macrophages/neutrophils were gated for further analysis of intracellular ROS via detection of DCFH-DA (20 µM). <b>A</b>) DCFH-DA fluorescence spectrum in CD11b⁺, CD45^hi cells from saline (blue) and HSV-infected (red) mice. Non-DCFH-loaded control is black. Composite (<b>B</b>) and individual (<b>C</b>) ROS data are presented as fold induction of HSV-infected mice (n = 5) over controls (n = 5). *p<0.05.</p

Blockade of gp120-induced apoptosis of dopaminergic neurons in human mesencephalic neuronal/glial cultures.

<p><b>A</b>) Human mesencephalic neuronal/glial cultures were left untreated (C) or pretreated with WIN55,212-2 (0.3 μM) or the inactive enantiomer WIN55,212-3 (0.3 μM) for 3h prior to gp120 (10 nM) treatment for 5 days; apoptosis was measured by cell death ELISA. <b>B</b>) Human mesencephalic neuronal/glial cultures were either untreated (C), pretreated with WIN55,212-2 (0.3 μM) for 3h or anti-CXCR4 Ab for 1h prior to gp120 (10 nM) treatment or treated with heat-inactivated gp120 for 5 days. After fixation and permeabilization, dopaminergic neurons were identified by immunostaining for TH and apoptotic cells were assessed by cell counting with propidium iodide staining. <b>C</b>) Human mesencephalic neuronal/glial cultures were untreated (C) or pretreated with JWH015 (0.1 to 3 µM) for 3h prior to gp120 (10 nM) treatment for 5 days; apoptosis was measured by cell death ELISA. Data are mean ± SD of triplicates and representative of 2-4 separate experiments using different brain tissue specimens. **p<0.01 vs. control (C); ††p<0.01 vs. gp120; p< 0.05 between (WIN55,212-2+gp120) vs. (WIN55,212-3+gp120) (in A).</p

Blockade of human microglial migration towards supernatants from human mesencephalic neuronal/glial cultures.

<p><b>A</b>) Human mesencephalic neuronal/glial cultures were pretreated with WIN55,212-2 (0.1 to 1 μM) for 3h followed by either no treatment or gp120 (10 nM) treatment for 72h. Supernatants were then collected for the chemotaxis assay. Highly purified human microglia were added to upper chambers with the lower chambers filled with supernatants from the human mesencephalic neuronal/glial cultures. After 3 h of incubation, human microglia that had migrated from upper chambers into lower chambers were quantified by Diff-Quik staining. <b>B</b>) Antibodies for chemokines CCL2, CX3CL1 and CXCL10 were added to collected supernatants for 30 min prior to chemotaxis assay. Data are mean ± SEM of triplicates of 3 separate experiments using different brain tissue specimens. **p<0.01 vs. control, ††p<0.01 vs. gp120.</p

Reduced GSH production in MNCs exposed to HSV-stimulated microglia.

<p>Reduced GSH production in MNCs exposed to HSV-stimulated microglia.</p

Blockade of gp120-induced production of CCL2, CX3CL1, IL-1β and CXCL10 in human mesencephalic neuronal/glial cultures.

<p>Human mesencephalic neuronal/glial cultures were pretreated with WIN55,212-2 (0.3 to 3 μM) or WIN55,212-3 (3 μM) for 3h prior to gp120 (10 nM) treatment for 72h followed by supernatant collection for measurement of <b>A</b>) CCL2, <b>B</b>) CX3CL1, <b>C</b>) IL-1β and <b>D</b>) CXCL10 production by ELISA. Data are mean ± SEM of triplicates of 2 separate experiments using different brain tissue specimens. *p<0.05, **p<0.01vs. control (C); ^††p<0.01 vs. gp120.</p

Involvement of cannabinoid receptors in gp120-induced suppression of DA uptake and apoptosis.

<p>Human mesencephalic neuronal/glial cultures were untreated (C) or pretreated with SR141716A (CB₁ antagonist) or SR144528 (CB₂ antagonist) (1 μM) for 30 min prior to WIN55,212-2 (0.3 μM) or JWH015 (CB₂ agonist) (1 μM) addition for 3h followed by gp120 (10 nM) treatment for 5 days to measure <b>A</b>) ³H-DA uptake and <b>B</b>, <b>C</b>) apoptosis by cell death ELISA. Data are mean ± SEM of triplicates of 2-4 separate experiments using different brain tissue specimens. **p<0.01 vs. control; ††p<0.01 vs. gp120; §§p<0.01 vs. WIN55,212-2+gp120 (in A, B) or JWH015+gp120 (in C); p<0.05 between (SR144528+JWH015+gp120) vs. (SR141716A+JWH015+gp120) (in C).</p