Expression of cell adhesion molecules in the injured brain after mTBI.

<p>In these experiments, the depth of brain deformation was set at 1 mm, and the images shown were acquired at 6 h post-mTBI. (A, B) Double immunostaining of injured brains with anti-P-selectin (SELP) antibody and an antibody to von Willebrand factor (vWF), a marker for endothelial cells. Note that SELP is expressed both ipsilaterally and contralaterally (Ipsi/Contra) on the endothelium of pial (arrows) and intraparenchymal microvessels. SELP was not expressed in the intact rat brain (data not shown). SAS and CVI are the subarachnoid space and the cistern of velum interpositum, respectively. (C, D) Double immunostaining of injured brains with anti-intercellular adhesion molecule 1 (ICAM1) antibody and an antibody to vWF. Note that ICAM1 is expressed on the endothelium of the ipsilateral pial (arrows), but not intraparenchymal, microvessels. ICAM1 is also not expressed on the endothelium of the contralateral pial microvessels. Bars: panels A–D, 50 μm.</p

Changes in mRNA levels of proinflammatory mediators in the cerebral cortex and hippocampus at 6 and 24 h after mTBI as assessed by real-time RT-PCR.

<p>In these experiments, the depth of brain deformation was set at 1.5 mm. The production of proinflammatory mediators in the ipsilateral (I-Cx) versus contralateral (C-Cx) cortex, and the ipsilateral (I-Hip) versus contralateral (C-Hip) hippocampus is shown. Peptidylprolyl isomerase A (PPIA)/cyclophilin A was used for the normalization of the data. The F and the corresponding <i>p</i> values for each proinflammatory mediator are indicated. *<i>p</i><0.05, **<i>p</i><0.01 for the ipsilateral versus contralateral Cx or Hip. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for the difference in expression of proinflammatory mediators at 6 versus 24 h post-mTBI. (<i>n</i> = 4–6 rats per group)</p

Changes in protein levels of proinflammatory mediators in the hippocampus at 6 and 24 h after mTBI as assessed by ELISA.

<p>The severity of injury was gradually increased by increasing the depth of brain deformation in 0.5-mm increments from 0.5 to 1.5 mm. The production of proinflammatory mediators in the ipsilateral (I-Hip) and contralateral (C-Hip) hippocampi, as well as the hippocampus from sham-injured rats (S), is shown. *<i>p</i><0.05, **<i>p</i><0.01 for the ipsilateral versus contralateral Hip. The F and the corresponding <i>p</i> values for each proinflammatory mediator are indicated. ^#<i>p</i><0.05, ^##<i>p</i><0.01 for the difference in expression of proinflammatory mediators from sham-injured rats. ^††<i>p</i><0.01 for the difference in expression of proinflammatory mediators at 6 versus 24 h post-mTBI. (<i>n</i> = 3–8 rats per group)</p

The influx of monocytes into the injured brain after mTBI.

<p>In these experiments, the depth of brain deformation was set at 1 mm, and the images shown were acquired at 24 h post-mTBI. Double immunostaining of injured brains was performed with anti-CD68 antibody, a monocyte marker, and an antibody to von Willebrand factor (vWF), a marker for endothelial cells. (A, B) Similar to severe TBI, in mTBI, monocytes did not appear in the injured brain until 24 h after the impact. However, only a small number of monocytes invaded the injured brain parenchyma at 24 h post-mTBI. Note that at this time point after injury, these inflammatory cells were predominantly located close to intraparenchymal blood microvessels. (C, D) In contrast to brain parenchyma, a large number of monocytes crossed the pial microvessels and invaded the subarachnoid space (SAS) near the injury site. Note that some of these monocytes appeared to subsequently move along the perivascular space to enter the brain parenchyma (arrows). (E–G) An influx of monocytes into the ipsilateral cistern of velum interpositum (CVI) also occurred at 24 h post-mTBI. Bars: panels A, B, E, 100 μm; panels C, D, F, G, 50 μm.</p

Changes in concentration of neutrophil chemoattractant CXCL1 in peripheral blood plasma and serum at 6 and 24 h after mTBI as assessed by ELISA.

<p>In these experiments, the depth of brain deformation was set at 1 mm. Note that sham-injured rats had a high serum level of CXCL1 (~10-fold higher than that observed in plasma), which did not change in response to injury. Other neutrophil chemoattractants investigated (CXCL2, CXCL3, and CCL2) could not be detected in peripheral blood plasma with the immunoassays used in this study. The F and the corresponding <i>p</i> values are indicated. *<i>p</i><0.05 for the difference from sham-injured rats. ^†<i>p</i><0.05 for the difference in CXCL1 levels at 6 versus 24 h post-mTBI. (<i>n</i> = 3–9 rats per group)</p

Changes in mRNA levels of proinflammatory mediators in the cerebral cortex and hippocampus at 6 h after mTBI as assessed by real-time RT-PCR.

<p>The severity of injury was gradually increased by increasing the depth of brain deformation in 0.5-mm increments from 0.5 to 1.5 mm. The production of proinflammatory mediators in the ipsilateral (I-Cx) versus contralateral (C-Cx) cortex, and the ipsilateral (I-Hip) versus contralateral (C-Hip) hippocampus is shown. Peptidylprolyl isomerase A (PPIA)/cyclophilin A was used for the normalization of the data. The F and the corresponding <i>p</i> values for each proinflammatory mediator are indicated. *<i>p</i><0.05, **<i>p</i><0.01 for the ipsilateral versus contralateral Cx or Hip. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for the difference in expression of proinflammatory mediators in response to a 0.5-mm increase in brain deformation. (<i>n</i> = 4–6 rats per group)</p

The sequences of primers and TaqMan probes, and the predicted sizes of PCR products.

<p>The sequences of primers and TaqMan probes, and the predicted sizes of PCR products.</p

The synergistic interactions between TNF-α and AVP in the choroid plexus epithelium as assessed by the level of activation of JNK and its target transcription factors c-Jun and ATF2.

<p>The activation of the JNK signaling cascade in the Z310 cell line was assessed after 1-h incubation with either TNF-α (5 ng/ml) or a combination of TNF-α (5 ng/ml) and AVP (1 nM). In non-radioactive JNK activity assays, 200 ng of recombinant (rec) human c-Jun protein was used as a substrate for JNK. The extent of activation of c-Jun and ATF2 was assessed by Western blotting (30 µg of total protein per lane was loaded). The representative immunoblots are shown based on three independent experiments. The ratios of optical density of bands for phosphorylated proteins over the optical density of bands for the total (phosphorylated and non-phosphorylated) proteins are shown.</p

Post-traumatic synthesis of proinflammatory mediators by the lateral ventricle choroid plexus (CP) as assessed by real-time RT-PCR.

<p>A comparison of AVP-deficient Brattleboro rats (<i>Avp</i>^di/di) with their parental Long-Evans strain (WT). The production of proinflammatory mediators in the ipsilateral (Ipsi CP) and contralateral (Contr CP) CPs is shown. Cyclophilin A (Cycl-A) was used for the normalization of the data. *<i>p</i><0.05, **<i>p</i><0.01 for the ipsilateral versus contralateral CP. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for <i>Avp</i>^di/di versus WT rats (<i>n</i> = 6–9 per rat strain per time point).</p

The synergistic interactions between TNF-α and AVP (A), and IL-1β and AVP (B) in the choroid plexus epithelium as assessed by real-time RT-PCR.

<p>Changes in production of CXCL1 in the choroid plexus epithelial cell line Z310 were assessed after 1-h incubation with either the cytokine (TNF-α or IL-1β) alone or a combination of the cytokine (TNF-α or IL-1β) and AVP. The exposure of Z310 cells to AVP alone at a concentration ranging between 0.1 nM and 1 µM resulted in only small increases in CXCL1 synthesis (a maximum increase observed was 6-fold relative to control with AVP at 100 nM). *<i>p</i><0.05, **<i>p</i><0.01 for treatment versus control. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for TNF-α + AVP versus TNF-α alone or for IL-1β + AVP versus IL-1β alone (<i>n</i> = 3–4 per group).</p