Materials and methods
Mice
Adult, male C57BL/6J wildtype (WT) mice were purchased from Taconic Ltd. (Ry, Denmark) and transferred to the Biomedical Laboratory, University of Southern Denmark, Odense, Denmark. Mice were housed in groups and maintained on a 12 h light/dark cycle and allowed free access to food and water ad libitum. All experiments were approved by the Danish Animal Health Care Committee.
TNF inhibitor treatment (Drug treatment)
Mice were treated every third day with XPro1596 (100 mg/ml Xencor Inc., Steed et al. 2003) diluted in saline (N=8), etanercept (50 mg/ml Enbrel, Amgen-Wyeth, Thousand Oaks, CA, USA, Murray and Dahl, 1997) in saline (N=8) or saline vehicle (0.9% physiological saline solution) (N=8). Drugs were administrated subcutaneously at a dose of 10 mg/kg. To investigate the short-term and long-term effects of XPro1595 and etanercept on cell proliferation, mice were treated with XPro1595 or etanercept for two weeks (short-term survival) or 2 months (long-term survival) (Figure 6). Treatment dose of XPro1595 and etanercept and time points of administration were determined based on previous publications (Brambilla et al 2011, Novrup et al. 2014, Taoufik et al. 2011, Zalevsky et al. 2007).
Fifty mg/kg of the thymidine analogue BrdU (5’-Bromo-2’-Deoxyuridine; Sigma, Denmark) diluted in sterile saline was administrated 24 hours (at day 2) after the first injection of XPro1595 or etanercept in short-term treated and long-term treated mice in order to investigate the effects XPro1595 or etanercept on proliferation.
All experiments were performed blinded to XPro1595, etanercept or saline treated mice.
Figure 6: Timeline of drug (XPro1595, etanercept and BrdU) treatments.
Dissection of thoracic and abdominal aortas
Fourteen days treated or 2 months treated mice were deeply anaesthetized with an overdose of pentobarbital (200 mg/ml) containing lidocaine (20 mg/mL; Glostrup Apotek, Glostrup, Denmark). A cut was made at abdomen and cut through the diaphragm and ribs and rib cage was removed so the heart was accessible to provide drainage for blood. Blood was collected for cytokine profile analysis and immediately transcardially perfused with 5mL of cold 0.15M Soerensen’s phosphate buffer (SB) followed by 20 mL cold 4% paraformaldehyde (PFA) in SB. While grasping the anterior end of aorta the aorta were carefully isolated from surrounded tissue using a scissor run closed under the aorta. A cut was made at the descending part of thoracic aorta, the thoracic aorta, abdominal aorta, the intestine with superior and inferior mesenteric arteries were removed and post-fixated in 4% PFA and transferred to 1XPBS solution with 0.05% azid after 24 hours post fixation.
Dissection of mesenteric arteries
A section of the intestine with associated mesenteric arteries and veins were cut and removed using sterile scissors and forceps. Hereafter excised section was transferred to a petri dish containing 1X physiological saline solution (PSS) solution (Appendix I) and the intestine was pinned flat using needles. Under a dissection microscope (Olympus SZX16) the second order arteries close to the first order superior mesenteric artery were isolated carefully by clearing away from surrounded fat tissue and connective tissue. The dissected second order arteries were cut using scissors and forceps and transferred in 1XPBS with 0.05% azid.
Tissue processing for histology analysis
Aortas and mesenteric arteries were cut into three pieces of 2mm, processed and paraffin-embedded according to standard procedure. Finally, the aortas and mesenteric arteries were cut into 5μm-thick microtome sections. Sections were allowed to air dry 24 hours at room temperature (RT) and afterwards placed one hour at 60ºC before further stainings.
Hematoxylin and eosin staining
For visualization of general morphology of thoracic aortas, abdominal aortas and mesenteric arteries after exposure to XPro1595 or etanercept, the tissues were stained with H&E staining. Prior to staining the paraffin-embedded sections were deparaffinised 3 x 5 min in xylene, 2 x 3 min in 99% ethanol, 2 x 3 min in 96% ethanol and 2 x 3 min in 70 % ethanol. Hereafter, the sections were rinsed in distilled water and counterstained in concentrated Mayer’s Hematoxylin solution (Sakura Finetik) for 10 minutes. Afterwards the sections were rinsed in running tap water for 10 minutes and distilled water for 2 min and briefly immersed in 0,2 % eosin solution (diluted in 70% ethanol) (Sakura Finetik) for 30 sec. Finally, the sections were dehydrated in 70% ethanol (rapidly 4-5 dips) followed by 3-4 dips in 96% ethanol, 10-15 dips in 96% ethanol, 3 x 50 dips in 99% ethanol and cleared in 3 x 5 min in xylene and mounted with Depex (Merck, Germany). Digital images were acquired choosing the bright field (BF) mode at 10x magnification on an Olympus BX51 microscope fitted with an Olympus DP26 digital camera. The setup was connected to a PC with the CellSens software.
Millers elastic staining
To confirm elastic fibers of 14 days treated or 2 months treated mice, sections were stained with Miller’s elastic staining using Elastic Van Gieson Stain Kit (Miller) (Atom Scientific, RRSK11). The sections were deparaffinised as described earlier. Hereafter sections were rinsed in 96% ethanol before placed in Millers Elastin Stain for 3 hours at RT. After three hours staining sections were washed in distilled water and the intensity of staining of the elastic fibers were checked microscopically before sections were counterstained in Van Gieson Stain for 3 min. Van Giesonstains collagen red. Afterwards sections were allowed to blot dry for 10 min at RT. Finally, sections were dehydrated quickly in 70% EtOH, 96% EtOH, 99% EtOH and 2 min in Tissue clear (Sakura Finetek, USA Inc., Torrance, CA, 1466) and mounted with Pertex (CellPath Ltd, Newtown, Powys, UK, SEA-0100-00A. Images of Miller’s staining were imported into ImageJ program and the area of lumen and external elastic lamina were drawn. The exact area of the internal elastic lamina and external elastic lamina was calculated by subtracting the values of area the lumen from the area from the area of external elastic lamina (Figure 7). We used 4 to 6 aortas of each mouse to measure the area of the internal and external elastin fibers and a mean value of 4 or 6 aortas of each mouse were calculated. Eight mice from each treatment group were used in this study.
Figure 7: Picture of thoracic aorta stained with Miller’s staining. The area of the internal elastic lamina and external elastic lamina defining the area of the media. M= media area.
Immunohistochemistry for incorporated cell proliferation marker BrdU
To investigate the effects of treatment on cell proliferation an immunostaining of incorporated BrdU using anti-BrdU antibody was performed. Thoracic aorta microtome sections were deparaffinised and rehydrated in graded ethanols series as described in previous section. Afterwards the sections were antigen retrieved in Tris EGTA (TEG) buffer (pH 9) for 15 min using a pressure cooker and allowed to cool down in TEG buffer without lid for 15 min at room temperature (RT). Hereafter sections were rinsed in running tap water for 15 min followed by wash in distilled water (3-5 dips). The sections were rinsed 2 x 0.2MTrizma Saline Buffer (TBS) (pH 7.4) for 15 min. Hereafter the sections were washed in preheated 2X Standard Saline Citrate (SSC) solution (pH 7.31) solution for 1 to 2 minutes to avoid air bubbles on the tissue. Next, DNA was denatured in degassed 49% formamide solution diluted in 2XSSC (Cat #: 1.09684.1000, Merck Millipore, Germaney) for 2 hours at 60ºC. The sections were washed two times in preheated 2XSSC solution for 10 minutes at 60ºC followed by unmasking in preheated 2M HCl in 0.05M TBS solution for 30 minutes at 37 ºC. The sections were then neutralized for 10 minutes in 0.1M Sodium Borate Buffer (pH 8.5) and rinsed three times in 0.05M TBS for 15 minutes. Afterwards sections were blocked for endogen peroxidase activity in 0.033% hydrogenperoxide (Merck, Germaney) for 30 minutes and sections were washed in TBS for 5 to 10 minutes while shaking at 50 rpm at r.t. For permeabilisation of membrane the sections were washed in 0.05M TBS in 0.1% triton X-100 three times for 15 minutes. The excess TBS was removed the sections were pre-incubated in 200 μL 10% Fetal Bovine Serum (FBS) in TBS in incubation chamber for 30 minutes. After incubation the excess of FBS was removed and 200μL anti-rat BrdU (0.5 mg/mL; Cat #: OBT0030, BioLegend; 1:600 diluted in 10% FBS solution) was applied on the sections and incubated one hour at r.t. and followed by incubation overnight at 4 ºC. Next day, the sections were allowed to equilibrate at r.t. for 30 minutes. Afterwards the sections were rinsed three times in TBS in 0.1% triton X-100 for 15 minutes while shaking followed by incubation with 200 μL of secondary antibody Biotinylated anti-rat IgG (1.5 mg/mL,1:200, Cat #: BA-9400) for one hour at r.t. Then the sections were washed in 0.05M TBS in 0.1% triton X-100 and incubated with 200μL streptavidin conjugated Horse Radish Peroxidase (HRP) (1:200, GE Healthcare, Cat#: RPN1231V) was applied on the sections for one hour at r.t. Hereafter the sections were rinsed three times in TBS solution for 15 minutes. Cell proliferation were visualized using 0.009% 3,3’-diaminobenzidine (DAB) (50 mg; Sigma D5637) diluted in TBS with 30% H2O2 for 25 minutes. Then the sections were rinsed in TBS and the sections were counterstained with concentrated Mayer’s hematoxylin solution in 1 minute followed by washing in running tap water for 10 minutes. Then the sections were washed in distilled water for 2 min. Finally, the sections were dehydrated in 70% ethanol (rapidly 4-5 dips) followed by 3-4 dips in 96% ethanol, 10-15 dips in 96% ethanol, 3 x 50 dips in 99% ethanol and cleared in 3 x 5 min in xylene and excess of xylene was drained off before mounting with Depex (Merck, Germany). As negative controls; Isotype control (IgG2a; diluted 1:600 in 10% FBS solution) and blank control (10% FBS solution in TBS) without primary antibody were used. Perfused frozen ischemic brain tissue with infarct were used as positive control for BrdU staining.
In addition, staining for endogenous cell proliferation marker, Ki67 was stained as described above. Briefly, two months treated thoracic aortas were unmasked in TEG buffer and primary antibody were diluted 1:500 and 1:1250 diluted in 10% FBS (250 μg/ml, Cat #: 550609, BD Biosciences) and signal were amplified using biotinylated anti-mouse IgG (1:200, RPN1001V, GE Healthcare, UK) together with Streptavidin conjugated Horse Radish Peroxidase (HRP) (1:200, GE Healthcare, UK, Cat#: RPN1231V). As negative controls; Isotype mouse IgG1 κ diluted 1:200 or 1:500 (100 μg/ml, Cat#: X0931, Dako) were used and negative control 10% FBS in TBS without primary antibody were used in this study. Skeletal muscle tissue treated with incorporated BrdU was used as positive control for Ki67 staining.
TdT-mediated dUTP Nick-End Labelling (TUNEL) assay
To investigate the effects of treatment on apoptosis of SMCs and ECs TUNEL Andy FluorTM 594 Apoptosis Detection Kit (Cat#: A051, GeneCopoeia, Inc.) were used. The sections were deparaffinised as described before. Afterwards sections were washed 2 x 5 min in 1XPhosphate Saline Buffer (PBS) (Life technologies, 14200-067). The excess of PBS was removed and tissue were permeabilized by adding 50 µL 1X Proteinase K (50X diluted 1:50 in 1X PBS) for 30 min at 37°C followed by rinse in 2 x 5 min rinse in 1XPBS solution. For TUNEL reaction, samples were incubated with 45 μL TdT reaction buffer (prepared prior use) for 10 min. Hereafter the reaction buffer was removed and TdT reaction cocktail containing; TdT reaction buffer (Component A), TdT enzyme (Component B), Biotin-11-dUTP (component C) wad added on each tissue and the solution was spread over the surface of tissue and incubated in a humid chamber for 2 hours at 37ᴼC. After incubation, the samples were washed 3 x 5 min in 3% Bovine Serum Albumin (BSA) in PBS. Forty-five µL of Andy FlourTM 594-Streptavidin staining solution was added to each sample and incubated for 1 hour at r.t protected from light. Afterwards samples were washed 3 x 5 min in 3% BSA in 1XPBS and counterstained with 4',6-diamidino-2-phenylindole DAPI (diluted 1:1000 in 1XPBS) for nuclei visualization in 10 min followed by wash 2 x 5 min PBS. Finally, sections were mounted with Dako mounting medium and stored in dark place. Images were obtained using Fluorescence microscopy pE-300-White from CoolLED connected to Olympus BX51 microscope fitted with an Olympus DP26 digital camera connected to a PC setup with the CellSens software. Pictures were imported into ImageJ program and all TIFF files were converted to 8-bit. As negative controls without TdT enzyme and positive controls treated with Dnase enzyme was used in this study.
Mesoscale Multiplex analysis
The collected blood from XPro1595 (N=8), Etanercept (N=8) or saline (N=8) treated mice were centrifuged at 2.000g for 20 min and the resulting blood plasma frozen and stored at -80° C.
To assess changes of pro-inflammatory cytokines and chemokine and anti-inflammatory cytokines level (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α and CXCL1) in C57BL/6 mice (XPro1595, etanercept and saline treated mice for 2 months) we used a MSD Mouse Proinflammatory Panel 1 kit V-Plex (Cat. #: K15048D-1) (Mesoscale Discovery, Rockville, MD, USA) and a SECTOR Imager 6000 (Mesoscale Discovery) plate reader according to the manufacturer’s instructions. Calibrator solutions were prepared by diluting Pro-inflammatory Panel 1 (mouse) calibrator blend in diluent 41, and a 4-fold serial dilution steps and zero calibrator were made. Fifty μL/well of undiluted samples were added in duplicates per wall (96-well Pro-inflammatory Panel 1 plate) with vigorous shaking for 2 hours at RT. After 2 hours incubation, the plate was washed three times with 150 μL washing buffer per well and 25μL of 1X detection antibody solution (ten different detection antibody of diluted in Diluent 45) was added per well and the plate were allowed to incubate at RT with vigorous shaking for 2 hours. Finally, the plate was washed three times with washing buffer and 150 µL of 2X reading buffer T (diluted 2-fold with deionized water) was added per well and the data were analyzed using MSD Discovery Workbench Software analysis software.
Data analysis
In analysis of comparing more than two groups, statistical significance was tested by one-Way ANOVA followed by Tukey’s multiple comparison test. Analysis of media area, changes of expressions of anti-inflammatory cytokines and anti-inflammatory cytokines of saline, XPro1595 or etanercept treated mice was analyzed by using One-way ANOVA with Tukey’s multiple comparison test. In analysis, comparing only two group’s statistical significance was tested using an unpaired Student t-test. Statistical analyses will be performed using Prism 5.01 software for Macintosh, GraphPad Software, San Diego, CA, USA, http://www.graphpad.com. Data are presented as mean ± SEM with N indicating the number of mice used in this study. Statistical significance will be established for P < 0.05.