Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

Rats were divided into two groups: vehicle (saline)-treated and soluble TNFp55 receptor (sTNFp55R, 50 μg/ml; Sigma-Aldrich Co., St. Louis, MO)-treated groups. The dosage of sTNFp55R was determined as the highest dose that induced SE of comparable severity in 100% of animals with 5% mortality in the preliminary study. Animals were anesthetized (Zolretil, 50 mg/kg, i.m.; Virbac Laboratories, France) and placed in a stereotaxic frames. For the osmotic pump implantation, holes were drilled through the skull to introduce a brain infusion kit 1 (Alzet, Cupertino, CA) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; - 3.5 mm depth; flat skull position with bregma as reference), according to the atlas of Paxinos and Watson [24]. The infusion kit was sealed with dental cement and connected to an osmotic pump (1007D, Alzet, Cupertino, CA). The pump was placed in a subcutaneous pocket in the dorsal region. Animals received 0.5 μl/hr of vehicle or compound for 1 week. Therefore, the dose of sTNFp55R was 0.6 μg/day per each animal. The compounds began to be immediately infused after surgery. Since the volume of vasogenic edema peaked at 2-3 days after SE in our previous studies [5‐8, 20], we chose this time point. Thus, our experimental schedules at least inhibit the function of TNF-α from 3 days prior to SE to 4 days after SE when the volume of vasogenic edema peaked.

Three days after surgery, rats were treated with pilocarpine (380 mg/kg, i.p.; Sigma-Aldrich Co., St. Louis, MO) at 20 min after methylscopolamine (5 mg/kg, i.p.; Sigma-Aldrich Co., St. Louis, MO). Using this treatment paradigm, behavioral seizures typically began within 20-40 min. Approximately 80% of pilocarpine treated rats showed acute behavioral features of SE (including akinesia, facial automatisms, limbic seizures consisting of forelimb clonus with rearing, salivation, masticatory jaw movements, and falling). We applied the 2 hr-SE rat model, because > 90% of the rats that we monitored in our previous studies [25] displayed spontaneous, recurrent seizures within 1-3 months after pilocarpine-induced status epilepticus. Diazepam (10 mg/kg, i.p.; Hoffman Ia Roche, Neuilly sur-Seine) was administered 2 hours after onset of SE and repeated, as needed. The rats were then observed 3 - 4 hours a day in the vivarium for general behavior and occurrence of spontaneous seizures. Non-experienced SE (non-SE) rats (showing only acute seizure behaviors during 10 - 30 min, n = 8) and age-matched normal rats were used as controls (n = 7).

At designated time points (non-SE: 12 hr, 1 day, 2 days, 3 days, 4 days and 1 week after SE; n = 5, for each time point), animals were perfused transcardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) under urethane anesthesia (1.5 g/kg, i.p.; Sigma-Aldrich Co., St. Louis, MO). The brains were removed, and postfixed in the same fixative for 4 hr. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, the entire hippocampus was frozen and sectioned with a cryostat at 30 μm and consecutive sections were contained in six-well plates containing PBS. For stereological study, every sixth section in the series throughout the entire hippocampus was used in some animals.

Free-floating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in rabbit anti-MPO IgG (1:100, Thermo fisher scientific) or rabbit anti-MIP-2 IgG (1:200, Invitrogen, Carlsbad, CA) in PBS containing 0.3% Triton X-100 (Sigma-Aldrich Co., St. Louis, MO) and 2% normal goat serum(Sigma-Aldrich Co., St. Louis, MO) overnight at room temperature. After washing three times for 10 min with PBS, the sections were incubated sequentially, in goat anti-rabbit or horse anti-mouse IgG (Vector, Burlingame, CA) and ABC complex (Vector, Burlingame, CA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 min each. To confirm vasogenic edema, some tissue sections were reacted for serum-proteins using horse anti-rat IgG (Vector, Burlingame, CA) as a primary antibody. The sections were visualized with 3,3'-diaminobenzidine (DAB, Sigma-Aldrich Co., St. Louis, MO) in 0.1 M Tris buffer and mounted on the gelatin-coated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Munchen-Hallbergmoos). For negative controls, rat hippocampal tissues were incubated with 1 μg of the antibody that was preincubated with 1 μg of purified peptide for 1 hr at room temperature or incubated with pre-immune serum instead of the primary antibody. For negative controls, tissues were incubated with pre-immune serum instead of primary antibody.

Sections were incubated with 3% bovine serum albumin in PBS for 30 min at room temperature. Sections were then incubated in a mixture of goat anti-TNF-α IgG (1:1000, R&D systems, Minneapolis, MN)/mouse anti-OX-42 IgG (1:100, Serotec, Cambridge, UK), mouse anti-GFAP IgG (1:1000, an astroglial marker, Millipore Corporation, Billerica, MA)/rabbit anti-TNFp55R IgG (1:1000, Abcam, Cambridge, UK), mouse anti-GFAP IgG/rabbit anti-TNFp75R IgG (1:1000, Abcam, Cambridge, UK), mouse anti-SMI-71 IgM (1:1000, Covance, Berkeley, CA)/rabbit anti-TNFp75R IgG, mouse anti-GFAP IgG/rabbit anti-NF-κB (p65-Ser276, p65-Ser311, p65-Ser529, and p65-Thr435) IgG (1:100, Abcam, Cambridge, UK), mouse anti-SMI-71 IgM/rabbit anti-p65-Thr435 NF-κB IgG, mouse anti-SMI-71 IgM/rabbit anti-GLUT-1 IgG (1:100, Abcam, Cambridge, UK), or mouse anti-GFAP IgG/rabbit anti-MIP-2 IgG (1:100) in PBS containing 0.3% triton X-100 overnight at room temperature. After washing three times for 10 minutes with PBS, sections were also incubated in a mixture of FITC- and Cy3-conjugated secondary antisera (Amersham, San Francisco, CA), diluted 1:200, for 2 hr at room temperature. The sections were washed three times for 10 min with PBS, and mounted on gelatin-coated slides. For nuclei counterstaining, we used Vectashield mounting medium with DAPI (Vector, Burlingame, CA). All images were captured using an AxioImage M2 microscope and AxioVision Rel. 4.8 software.

Fluoro-Jade B (FJB) staining was used to identify degenerating neurons in tissues obtained from non-SE and 3 days post-SE animals in every group. In our previous [18, 25] and preliminary data, neuronal damage was first detectable at 3 days after SE. Therefore, we determined 3 days after SE as the best time point to look FJB. Briefly, sections were rinsed in distilled water, and mounted onto gelatin-coated slides and then dried on a slide warmer. The slides were immersed in 100% ethanol for 3 min, followed by 70% ethanol for 2 min and distilled water for 2 min. The slides were then transferred to 0.06% potassium permanganate for 15 min and gently agitated. After rinsing in distilled water for 2 min, the slides were incubated for 30 min in 0.001% FJB (Histo-Chem Inc., Jefferson, AR), freshly prepared by adding 20 ml of a 0.01% stock FJB solution to 180 ml of 0.1% acetic acid, with gentle shaking in the dark. After rinsing for 1 min in each of three changes of distilled water, the slides were dried, dehydrated in xylene and coverslipped with DPX (Sigma-Aldrich Co., St. Louis, MO). For stereological study, every sixth section in the series throughout the entire PC was used (see below).

To measure vasogenic edema, the volume of anti-rat IgG positive region in PC was estimated according to the formula based on the modified Cavalieri method: V = Σa × t _nom × 1/ssf, where a is area of the region of the delineated subfield measured by AxioVision Rel. 4.8 software, t _nom is the nominal section thickness (of 30 μm in this study), and ssf is the fraction of the sections sampled or section sampling fraction (of 1/6 in this study). The subfield areas were delineated with a 2.5 × objective lens [5, 7, 8, 18, 25]. The volumes are reported as mm³. An optical fractionator was used to estimate cell numbers. The optical fractionator (a combination of performing counting with the optical dissector, with fractionator sampling) is a stereological method based on a properly designed systematic random sampling method that by definition yields unbiased estimates of population number. The sampling procedure is accomplished by focusing through the depth of the tissue (the optical dissector height, h; 15 μm in all cases for this study). The number of each cell type (C) in each of the subregions is estimated as: C = ΣQ ^- × t/h × 1/asf × 1/ssf, where Q ^- is the number of cells actually counted in the dissectors that fell within the sectional profiles of the subregion seen on the sampled sections, and Asf is the areal sampling fraction calculated by the area of the counting frame of the dissector, a(frame) (of 50 × 50 μm² in this study) and the area associated with each x, y movement, grid (x, y step) (of 250 × 250 μm² in this study) {asf = (a(frame)/a(x, y step))}. The immunoreactive cells were counted with a 40× objective lens. The immunoreactive cells were counted with a 40× objective lens. All immunoreactive cells were counted regardless the intensity of labeling. Cell counts were performed by two different investigators who were blind to the classification of tissues. SE-induced PC atrophy is evident [8], so changes in cell number may be caused by an alterations in the volume of the PC. Therefore, the total number of cells was corrected by multiplying with appropriate correction factors (CF) representing the degree of shrinkage (or swelling) compared with the Non-SE.

The fluorescence intensities of SMI-71/p65-Thr435 phosphorylation or GFPA/p65-Thr435 phosphorylation were measured using a computer-assisted image analysis program (The University of Texas ImageTool program V. 3.0 and AxioVision Rel. 4.8 software). After regions were outlined, 30 areas/rat (300 μm²/area) were randomly selected within the PC, and double immunofluorescent merge images were captured from the PC (15 sections from each animal). Merge images were digitally separated to red or green image, and converted to grayscale images, respectively (n = 36 per region examined, in non-SE, 12 hr post-SE and 1 day post-SE). The range of intensity values was obtained from the selected images. Based on the mean range of intensity values, each image was normalized by adjusting the black and white range of the image. Manipulation of the images was restricted to threshold and brightness adjustments to the whole image. Intensity measurements are represented as the mean number of a 256 gray scale (NIH Image 1.59 software and AxioVision Rel. 4.8 software). Values for background staining were obtained from the corpus callosum. Optical density values were then corrected by subtracting the average values of background noise obtained from 15 image inputs.

In non-SE induced animals of the saline-infused groups, TNF-α immunoreactivity was weakly detected in PC neurons (data not shown). In 12 hr-post SE animals of the saline-infused group, most of the activated microglia showed strong TNF-α immunoreactivity (Figure 1A). This expression pattern was maintained up to 1 week after SE. In non-SE-induced animals of the saline-infused groups, TNFp55R and TNFp75R immunoreactivities were also weakly observed in astrocytes (data not shown). In 12 hr-post SE animals of the saline-infused group, TNFp55R immunoreactivity was observed in astrocytes (Figure 1B). Unlike TNFp55R, TNFp75R immunoreactivity was detected in endothelial cells as well as astrocytes (Figures 1C-E). One day to 1 week after SE, both TNFp55R and TNFp75R immunoreactivities were significantly reduced in astrocytes, not in endothelial cells, due to massive astroglial loss (data not shown) [5, 7, 8, 20].

In our previous [5, 7, 8, 20] and preliminary data, vasogenic edema and neuronal damage were noticeable at 1 day and 3 days after SE, respectively. Therefore, we determined that 3 days after SE was the best time point to evaluate the effect of sTNFp55R infusion on both vasogenic edema and neuronal damages induced by SE. In saline-treated animals, the PC was stained diffusely with anti-rat IgG (Figure 2A). The volume of vasogenic edema was 17.1 ± 1.5 mm³ (Figure 2E). The number of FJB-positive neurons in the PC was 236,145 ± 49,469 (Figure 2B). In sTNFp55R-treated animals, SE-induced vasogenic edema was attenuated to 9.8 ± 0.7 mm³ (Figures 2C and 2E). In addition, the number of FJB-positive neurons in the PC was 89,138 ± 5,698 (Figures 2D-E). Thus, sTNFp55R infusion attenuated SE-induced vasogenic edema and neuronal damage compared to saline-infused animals (p < 0.05).

It is well established that TNF-α is one of the major stimuli toward phosphorylation of NF-κB. To confirm TNF-α-mediated signaling following SE, we performed an immunohistochemical study using five phospho-NF-κB antibodies. Compared to non-SE animals (data not shown), 12 hr-post SE animals of the saline-infused group showed p65-Ser276, p65-Ser311, p65-Ser529, and p65-Ser536 phosphorylation in astrocytes (not endothelial cells). sTNFp55R infusion effectively reduced p65-Ser276 and p65-Ser311 phosphorylation (p < 0.05, respectively), while it could not affect p65-Ser529 or p65-Ser536 phosphorylation (Figures 3 and 4A). In contrast, p65-Thr435 phosphorylation was increased in endothelial cells (not astrocytes) within the PC of saline-infused animals 12 hr after SE (Figure 5A). In addition, sTNFp55R infusion effectively alleviated SE-induced p65-Thr435 phosphorylation in endothelial cells, compared to saline infusion (p < 0.05, Figure 5B).

Previously, we reported that SMI-71 (an endothelial barrier antigen) immunoreactivity decreased in the PC 1 day after SE [5]. Similarly, in 1 day-post SE animals of the saline-infused group, loss of SMI-71 immunoreactivity was detected in layer III/IV of the PC as compared to non-SE animals (Figures 4B and 5C-E, p < 0.05). Thus, loss of SMI-71 immunoreactivity correlated with volume of vasogenic edema following SE. This reduction in SMI-71 was accompanied by increased p65-Thr435 phosphorylation (Figures 5C-D). Therefore, the degree of SMI-71 immunoreactivity was inversely correlated to p65-Thr435 phosphorylation with a linear coefficient of correlation of -0.6324 (p < 0.05; Figure 4C). In addition, sTNFp55R infusion effectively alleviated p65-Thr435 phosphorylation and preserved SMI-71 immunoreactivity in endothelial cells following SE, as compared to saline infusion (p < 0.05; Figures 4A-B, 5D and 5F).

Recent studies have reported that neutrophils infiltrate the brain under certain pathological conditions [26]. Indeed, we have reported massive neutrophil infiltration in layer III/IV of the PC 1 day after SE [20]. In the present study, 1 day-post-SE animals of the saline-infused group showed infiltration of MPO-positive neutrophils into the PC. Similarly, 1 day-post-SE animals of the sTNFp55R-infused group showed neutrophil infiltration into the PC 1 day after SE (Figure 6A). The number of neutrophils/area in the PC region (including the vasogenic edema region and the non-vasogenic edema region) of sTNFp55R-infused animals was significantly lower than that of the saline-infused group (Figure 6D, p < 0.05). However, there was no difference in neutrophil infiltration per unit area of vasogenic edema between the saline- and sTNFp55R-infused groups (Figure 6D). Furthermore, neutrophil infiltration showed a direct proportion to the area of vasogenic edema, with a linear coefficient of correlation of 0.8631 (p < 0.05, Figure 6E). Therefore, our findings indicate that SE-induced neutrophil infiltration into the PC may be correlated to TNF-α-mediated vasogenic edema formation.

MIP-2 is a powerful chemokine that contributes to recruitment of neutrophils [27]. MIP-2 is undetectable or present at low levels under physiological conditions, and shows transient increases under pathological conditions via TNF-α and/or interleukin-1β (IL-1β)-dependent mechanisms [14]. Thus, it would be plausible that TNF-α-mediated MIP-2 expression may provoke SE-induced neutrophil infiltrations. To confirm this hypothesis, we investigated MIP-2 expression in the PC. Consistent with our previous study [20], some MIP-2-positive astrocytes were observed in the core and periphery of the vasogenic edema lesions, but not in the non-vasogenic edema region (Figure 6B and 6C). Although the number of MIP-2 positive cells per unit area in the PC region of sTNFp55R-infused animals was significantly lower than that of the saline-infused group due to reduction of the area of vasogenic edema, there was no difference in the number of MIP-2 positive cells per unit area of vasogenic edema between sTNFp55R-infused animals and saline-infused animals (Figure 6F). Furthermore, the number of MIP-2-positive cells showed a direct proportion to the unit area of vasogenic edema with a linear coefficient of correlation of 0.682 (p < 0.05, Figure 6G). Therefore, together with reduction in neutrophil infiltration in the PC region of sTNFp55R-infused animals, our findings provide evidence that TNF-α may regulate SE-induced neutrophil infiltration at least in the PC via vasogenic edema formation and not via direct TNF-α-mediated MIP-2 expression in astrocytes.