Genetic ablation of soluble tumor necrosis factor with preservation of membrane tumor necrosis factor is associated with neuroprotection after focal cerebral ischemia

Mice

Homozygous mTNF^Δ/Δ19 breeders, on a C57BL/6 background, were obtained from the Department of Biochemistry, University of Lusanne, kindly provided by Dr. Tacchini-Cottier and established as heterozygous mTNF^Δ/wt breeding colonies at the animal facility of the Biomedical Laboratory, University of Southern Denmark (BL-SDU). All experiments were performed blinded on age-matched (8–12 weeks old) homozygous mTNF^Δ/Δ male mice and mTNF^wt/wt littermates. TNF knockout (TNF^−/−) mice ⁴⁴ were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Age-matched C57BL/6 WT mice were purchased from Taconic A/S (Ry, Denmark). Animals were housed in ventilated cages with 1–3 cage-mates at a 12 h light/dark cycle, under controlled temperature and humidity, and with free access to food and water. Mice were cared for in accordance with the protocols and guidelines approved by the Danish Animal Health Care Committee; experiments are reported in accordance with the ARRIVE guidelines, and all efforts were made to minimize pain and distress (J. No. 2011/561-1950 and 2013-15-2934-00924).

Body composition measurements

Body composition was examined immediately before surgery on fully sedated mTNF^Δ/Δ (n = 18) and mTNF^wt/wt (n = 15) mice using non-invasive Dual-Energy X-ray Absorptiometry (DXA) (PIXImus 2, Version 1.44, GE Lunar, Madison, WI, USA). This method provides accurate assessments of total tissue mass (g), bone mineral density (BMD, g/cm²), bone mineral content (BMC, g), bone area (cm²), fat mass (g), % fat tissue and lean tissue mass (g). ⁴⁵

Behavioral assessments

Permanent focal cerebral ischemia

The distal part of the left middle cerebral artery (MCA) was permanently occluded under surgical anaesthesia consisting of a mixture of Hypnorm® (fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml, VetaPharma Ltd, Leeds, UK), Stesolid® (Diazepanum 5 mg/ml, Actavis, Gentofte, Denmark) and distilled H₂O in a 1:1:2 ratio, as previously described.^35,37 The surgery was conducted on a 37 ℃ heating pad to maintain a stable body temperature. An incision was made from the lateral part of the orbit to the external auditory meatus. The underlying superior pole of the parotid gland and the upper part of the temporal muscle were pushed in the distal direction after partial resection. A craniotomy was performed directly above the distal part of the MCA using a 0.8 mm high-speed microdrill. The bone was removed and the dura carefully opened. The distal part of the MCA was coagulated using bipolar forceps coupled to an electrosurgical unit (ICC 50, Erbe, Tübingen, Germany) ensuring a restricted cortical infarct. Post-operative treatment consisted of supplying the mice with physiological saline (0.9% NaCl) and Temgesic® (Buprenorphinum 0.3 mg/ml, RB Pharmaceuticals, North Chesterfield, VA, USA) three times during the first 24 h. The mice were allowed to survive for one or five days (n = 7–18 mice/group). Mortality was < 2% and only related to anaesthesia.

Sham mice were subjected to the same procedure, except the bipolar forceps were applied in the superficial brain parenchyma next to the MCA, without coagulating the MCA (n = 5/group).

Tissue processing and infarct volume analysis

Mice with 3 h (n = 5/group), one or five days post-surgical survival, as well as naive control (n = 5/group) mice, were anesthetized with pentobarbital (200 mg/ml)/lidocain hydrochloride (20 mg/ml) (Glostrup Apotek, Glostrup, Denmark) prior to euthanasia. The brains were removed, fresh-frozen in CO₂ snow and coronally cryo-sectioned into 30 μm thick sections in six parallel series, three series were placed on microscope slides and three series in eppendorf tubes. Every sixth series was stained with Toludine Blue (TB) for visualization of the infarct area. ⁴⁹ The infarct was estimated on TB-stained serial sections from mice with one and five days survival utilizing the computer-assisted stereological test system CAST^© 2000 (Olympus, Ballerup, Denmark) and applying Cavalieri's principle for volume estimation. ⁵⁰ The rostrocaudal distribution of the infarct was analyzed as previously described ⁵¹ using the anterior commissure as an anatomical landmark. In addition, to correct for edema, the volume of the contralateral and the non-ischemic ipsilateral cortex and the volume of injury spanning from 1080 μm anterior to 1080 μm posterior to the anterior commissure was compared in mTNF^Δ/Δ and mTNF^wt/wt mice at one and five days after pMCAO using an indirect method of infarct volume estimation.^37,52

Immunohistochemistry

Naive mice (n = 6 mice/group) were anesthetized with pentobarbital (200 mg/ml)/lidocain hydrochloride (20 mg/ml) and transcardially perfused with 4% paraformaldehyde (PFA) through the left ventricle. Brains were quickly removed and left in 4% PFA overnight, changed to 1% PFA and finally 0.1% PFA before they were cut horizontally on a vibratome (VT100S, Leica Microsystems A/S, Ballerup, Denmark) into six 60 μm-thick parallel series of free floating sections and cryoprotected in de Olmo's solution. ⁵³ One series from each animal was blocked for endogenous peroxidase activity using methanol and peroxide in tris-buffered saline (TBS), then incubated with 10% fetal bovine serum in 0.05 M TBS + 0.1 % Triton-X100 (TBS-T) for 30 min. Sections were incubated with anti-Iba1 primary antibody (rabbit, 1:600, Wako, Neuss, Germany) in TBS-T for two days at 4 ℃, followed by biotinylated anti-rabbit secondary antibody (1:200, GE Healthcare, Buckinghamshire, UK) in TBS-T for 1 h at RT. Sections were rinsed with TBS-T, incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:200, GE Healthcare) and developed using diaminobenzidine and hydrogen peroxide diluted in TBS. Finally, sections were transferred to gelatin-coated glass slides, counter-stained with TB, dehydrated and coverslipped using Depex.

Neocortical volume estimation and quantification of Iba1⁺ cells

The volume of the neocortex was estimated on one series of horizontally cut vibratome sections from naive control mice using Cavalieri's principle for volume estimation. ³⁷ The neocortex was delineated using a 2 × magnification and included the following regions: anterior part of the agranular insular cortex, orbital cortex, cingulate cortex, frontal cortex, pre-limbic cortex, granular insular cortex, retrosplenial agranular cortex, anterior parts of retrosplenial granular cortex, ectorhinal cortex, parietal cortex, temporal cortex and all areas of the occipital cortex. ⁵⁴ Immunolabeled Iba1⁺ cells were counted at a 60 × magnification in the right hemisphere of 10–14 sections from each mouse by the use of CAST1 (Visiopharm, Hørsholm, Denmark). The number of Iba1⁺ positive cells was recorded by systematically counting cells using a counting frame of 1012.6 μm² and a X-Y step size of 350 μm resulting in an area (a(step)) of 122,500 μm² to ensure that a representative sample was counted. ⁵⁴ The number of microglial cells per area unit was calculated as previously described ⁵⁴ and expressed as Iba1⁺ cells/mm². The person performing the cell counting was blinded to the genotype.

Total RNA isolation and real-time RT-PCR

Total RNA was extracted from one series of brain tissue sections with TRIZOL® according to manufacturer's instructions (Invitrogen, Grand Island, NY, USA). To ensure complete elimination of genomic DNA, RNA was further purified with RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA) in combination with DNA digestion using RNase-free DNase (Qiagen). Reverse transcription was performed with Omniscript (Qiagen), according to manufacturer's protocols. cDNA equal to 50 ng of initial total RNA was used as a template in each PCR reaction. Real-time PCR was performed in the Rotor-Gene 3000 Real-Time Cycler (Corbett Life Science, Sydney, Australia) with QuantiTect SYBR Green PCR MasterMix (Qiagen). Relative expression was calculated by comparison with a standard curve, after normalization to β-actin gene expression. Primers for gene amplification are listed in Supporting Table 1.

Protein extraction and Western Blotting

One series of brain tissue sections, parallel to those used for real-time RT-PCR, was homogenized in 300 μl of RIPA buffer supplemented with Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitor cocktail 1 (Sigma, St. Louis, MO, USA). The total protein concentration was quantified utilizing the Lowry assay. Proteins were resolved by SDS-PAGE on 8–11% gels transferred to nitrocellulose or polyvinylidene fluoride membranes and blocked in 5% non-fat milk in TBS-T. Membranes were probed overnight with anti-TNFR1 (mouse, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MHCII (mouse, 1:1000, Dako, Carpinteria, CA, USA), and anti-β-tubulin (mouse, 1:5000, Sigma), followed by HRP-conjugated secondary antibodies (GE Healthcare/Amersham, Brøndby, Denmark). Proteins were visualized with Super Signal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) and bands quantified with Quantity One software (Biorad, Hercules, CA; USA). Data were normalized to β-tubulin.

Multiplex analysis

Cytokine and chemokine protein concentrations were determined by multiplex technology in brain samples (25 μl/sample), parallel to those used for Western Blotting and real-time RT-PCR, from mTNF^Δ/Δ and mTNF^wt/wt mice using a MSD Mouse Pro-inflammatory V-Plex Plus Kit (Mesoscale Discovery, Rockville, MD, USA) and a SECTOR Imager 6000 (Mesoscale Discovery) Plate Reader according to the manufacturer's instructions. Samples were diluted two-fold in Diluent 41 prior to measurement. Data were analyzed using MSD Discovery Workbench software. Groups consisted of naive mTNF^Δ/Δ and mTNF^wt/wt mice, and mice with 3 h, one day or five days survival following focal cerebral ischemia (n = 5–6/group).

Flow cytometry

One day after pMCAO, mice (n = 5–6 mice/group) were anaesthetized with an overdose of pentobarbital (200 mg/ml)/lidocaine hydrochloride (20 mg/ml) and transcardially perfused with phosphate-buffered saline (PBS). Ipsilateral cortices were quickly removed and placed in RPMI medium containing 10% foetal calf serum. Single-cell suspensions were obtained as previously described,^37,38 and non-specific binding was blocked at 4 ℃ with anti-CD16/32 FcR. Cells were then incubated with the following antibodies: rat anti-mouse CD45-PerCP-Cy5.5 (BD Biosciences, clone 30-F11), rat anti-mouse CD11b-PE (BD Biosciences, clone M1/70), rat anti-mouse Ly6G/Ly6C-PE-Cy7 (Gr1) (Biolegend, Copenhagen, Denmark, clone RB6-8C5) and hamster anti-mouse CD3-APC (BD Biosciences, clone 145-2C11). Isotype controls used were hamster IgG1κ (BD Biosciences, clone A19-3) and rat IgG2b (BD Biosciences, clone A95-1 or Biolegend, clone RTK4530). Prior to fixation, suspensions were stained for live/dead cells for 30 min at 4 ℃ using the Fixable Viability Dye eFluor 506 (eBioscience, Aarhus, Denmark) diluted in PBS. Positive staining for microglia [CD45^dimCD11b⁺], macrophages [CD45^highCD11b⁺ Gr1⁻], granulocytes [CD45^highCD11b⁺Gr1⁺] and T cells [CD45^highCD3⁺] was determined based on fluorescence levels of the respective isotype controls and fluorescence minus one (FMO) controls, as previously described. ⁴⁷ Samples (n = 5–6/group) were run on a FACSVerse flow cytometer (BD Biosciences) equipped with FACSuite software for data analysis. ⁴⁷

Statistics

Real-time PCR and multiplex cytokine measurements were analyzed with parametric one-way ANOVA followed by Tukey's or Bonferroni's test for multiple comparisons. For single comparisons, Student's t-test was applied. Correlation analysis was performed using Pearson's correlation analysis. P values ≤ 0.05 were considered statistically significant. Data are presented as mean ± SEM.

No phenotypical abnormalities in naive mTNF^Δ/Δ mice

Since TNF has been implicated in the regulation of spatial memory and locomotor activity in physiological conditions,^15,16 we investigated whether absence of solTNF caused any phenotypical abnormalities in naive mTNF^Δ/Δ mice. Radiographic assessment of total body mass, bone mineral density, bone mineral content, bone area, % fat tissue, and % lean mass using the DXA scanner showed no differences between mTNF^Δ/Δ and mTNF^wt/wt mice (Supporting Table 2). In the Y-maze test for spontaneous alternation, which measures the willingness of rodents to explore new environments and involves areas of the brain such as hippocampus, septum, basal forebrain and prefrontal cortex, we found the total number of Y-maze entries (Figure 1(a)) and the spontaneous alternation percentage (Figure 1(b)) to be similar between mTNF^Δ/Δ and mTNF^wt/wt mice. In the open field test for spontaneous locomotion and anxiety-related behavior, we found no differences in all parameters evaluated including number of rearings (Figure 1(c)), total distance traveled (Figure 1(d)), and center/perimeter ratio (Figure 1(e)). Finally, the rotarod test showed comparable motor coordination (Figure 1(f)), and the grip strength test comparable proprioception and neuromuscular function (Figure 1(g)) between mTNF^Δ/Δ and mTNF^wt/wt mice. OPEN IN VIEWER

On the other hand, complete ablation of TNF in TNF^−/− mice did result in behavioral abnormalities. In the Y-maze, TNF^−/− mice showed a significant increase in the number of entries and total distance travelled (Supplementary Figure 1(a) and (b)), and a decrease in spontaneous alternation percentage (Supplementary Figure 1(c)) compared to WT mice. In the open field test, the number of rearings were comparable between TNF^−/− and WT mice (Supplementary Figure 1(d)), whereas total distance travelled (Supplementary Figure 1(e)) and center/perimeter ratio (Supplementary Figure 1(f)) were significantly increased in TNF^−/− mice. No differences were found in the rotarod and the grip strength tests, indicating that TNF^−/− mice have normal proprioception and neuromuscular function (Supplementary Figure 1(h)). Collectively, these data show that while complete ablation of TNF leads to impairment of basal cognitive and motor function, absence of solTNF alone is not sufficient to alter these behaviors.

Extensive evidence has shown that TNF is primarily produced by microglia following focal cerebral ischemia in mice;^35–38,55 therefore, we investigated whether genetic ablation of solTNF affected the total number of microglia in the neocortex. We found no difference in the number of Iba1⁺ microglial cells between the two genotypes (Figure 1(h) and (i)). Similarly, no obvious morphological differences were observed in cell shape or branching (Figure 1(h)). Total neocortex volume was also measured, and no differences were found (Figure 1(j)). These data indicate that absence of solTNF does not result in microglial alterations in the cortex and suggests that mTNF signaling is necessary and sufficient to maintain normal microglia homeostasis under naive conditions.

Absence of soluble TNF reduces infarct volume and improves asymmetry and neuromuscular function after pMCAO

To examine the effect of solTNF ablation on ischemic injury, we compared infarct volumes between mTNF^Δ/Δ and mTNF^wt/wt mice at one and five days after pMCAO (Figure 2(a) and (c)). The rostrocaudal distribution of the infarct showed that especially the sensory cortex, and in case of large infarcts also the motor cortex, is affected in this model (Figure 2(b), (d) and (e)). We found the volume of the injury to be significantly smaller in mTNF^Δ/Δ mice one day after pMCAO (Figure 2(c)). Also at five days after pMCAO, the injury volume was still significantly reduced in mTNF^Δ/Δ mice compared to mTNF^wt/wt littermates (Figure 2(c)). The similar rostrocaudal distribution of the infarcts both at day 1 and 5 in mTNF^Δ/Δ and mTNF^wt/wt mice showed that ablation of solTNF had no effect on the regional distribution of the MCA territory in mTNF^Δ/Δ mice (Figure 2(d) and (e)). When correcting for edema formation in the ischemic brain, the infarct was found to constitute 33% ± 6% of the ipsilateral cortex in mTNF^Δ/Δ mice, whereas the infarct was found to constitute 43% ± 4% of the ipsilateral cortex in mTNF^wt/wt mice one day after pMCAO. At five days, the infarct was found to constitute 14% ± 6% of the ipsilateral cortex in mTNF^Δ/Δ mice and 33% ± 7% in mTNF^wt/wt mice. OPEN IN VIEWER

To assess whether the decreased infarcts after pMCAO in mTNF^Δ/Δ mice translated into improved locomotor and/or sensory-motor function, we assessed mouse behavior two, three and five days after pMCAO. In the open field test two days after pMCAO, we found no difference between mTNF^Δ/Δ and mTNF^wt/wt mice in total distance traveled (Figure 2(f)), center/perimeter ratio (Figure 2(g)), and number of center and wall rearings (Figure 2(h)). Rung walk analysis on day 2 showed that mTNF^wt/wt mice displayed a clear asymmetry with significantly more missteps of their right front limbs compared to the left front limbs (Figure 3(a)). This correlated with the large cortical infarct measured in mTNF^wt/wt mice. In contrast, mTNF^Δ/Δ mice did not show significant asymmetry of their front limbs (Figure 3(a)), which may be due to the smaller cortical infarcts. The reduced infarct volume in mTNF^Δ/Δ did not result in improved motor coordination, as mTNF^Δ/Δ and mTNF^wt/wt mice performed similarly on the rotarod at both three and five days after pMCAO (Figure 3(b)). When analyzing proprioception and neuromuscular function using the grip strength test on the front paws, we observed significant asymmetry with loss of grip strength in the contralateral side (right, R) in mTNF^wt/wt mice at both three and five days post pMCAO, compared to baseline values. On the contrary, mTNF^Δ/Δ did not show loss of grip strength in the contralateral side as a consequence of the infarct and no significant asymmetry was measured between the front paws (Figure 3(c)). OPEN IN VIEWER

Absence of soluble TNF does not result in changes of pro-inflammatory gene expression after pMCAO

To investigate whether the functional improvement of mTNF^Δ/Δ mice was associated with a modulation of the inflammatory response, we first evaluated the expression of pro-inflammatory cytokines and chemokines using real-time RT-PCR 1 day after pMCAO, the time point where expression of several cytokines relevant for infarct development is known to peak. ²³ We found that IL-1β, IL-6, CXCL1, CXCL10 and CCL2 mRNA levels were significantly upregulated after stroke, but no significant difference was observed between mTNF^Δ/Δ and mTNF^wt/wt mice (Figure 4). TNF mRNA expression was also upregulated in sham-operated mice. In sham conditions, TNF mRNA upregulation was significantly higher in mTNF^wt/wt controls as compared to mTNF^Δ/Δ mice. OPEN IN VIEWER

Absence of soluble TNF reduces protein levels of pro-inflammtory cytokines after pMCAO

To further investigate possible changes in the inflammatory response, we assessed the expression of pro- and anti-inflammatory cytokines at 3 h and one and five days after pMCAO using multiplex technology. We found the pro-inflammatory molecules TNF, IL-1β, IL-6 and CXCL1 to be significantly decreased in mTNF^Δ/Δ mice compared to mTNF^wt/wt controls one day after pMCAO (Figure 5(a)). IL-5 was upregulated in mTNF^Δ/Δ mice one day after pMCAO, but no difference was found between mTNF^Δ/Δ and mTNF^wt/wt mice. In contrast, IL-2, IL-4 and IL-10, which have been associated with an anti-inflammatory function, did not change in the brain after pMCAO (Figure 5(a)). Correlation analysis (Supporting Table 3) showed a positive linear correlation between infarct volumes and IL-6 protein levels both at day 1 and 5. At day 1, IL-1β protein levels were also found to correlate with infarct volumes. In line with previous findings, ⁵⁶ TNF protein levels correlated with infarct volumes five days after pMCAO, and so did CXCL1 and IL-2 protein (Supporting Table 3). OPEN IN VIEWER

Since TNF has been shown to be neuroprotective through TNFR1,^37,57 we evaluated TNFR1 expression by Western Blot. We found significant upregulation in mTNF^Δ/Δ naive and sham mice compared to mTNF^wt/wt mice. Despite tendencies to increased TNFR1 protein levels in mTNF^Δ/Δ mice one day after pMCAO, no significant difference was observed between mTNF^Δ/Δ and mTNF^wt/wt mice (P = 0.08) (Figure 5(b)). These results suggest that genetic ablation of solTNF leads to suppression of the inflammatory response in the ischemic brain, contributing to an environment more favorable to functional recovery.

Absence of soluble TNF reduces macrophage infiltration into the ischemic cortex after pMCAO

Based on our previous findings of increased leukocyte infiltration in TNF^−/− mice one day after experimental stroke ³⁷ as well as altered microglial activation following anti-TNF treatment, ⁴⁷ we investigated changes in microglia and infiltrating leukocytes in the ipsilateral cortex of mTNF^Δ/Δ mice and mTNF^wt/wt controls one day after pMCAO (Figure 6). We found that the number and percentage of infiltrating CD45^highCD11b⁺Gr1⁻ macrophages were significantly decreased in mTNF^Δ/Δ mice, whereas the number and percentage of CD45^highCD11b⁺Gr1⁺ granulocytes and CD45^highCD3⁺ T cells were comparable between the two genotypes (Figure 6(a) to (c)). With respect to microglia, both number and percentage of CD45^dimCD11b⁺ microglia were comparable between mTNF^Δ/Δ mice and mTNF^wt/wt controls (Figure 6(d) and (e)), consistent with findings of comparable numbers of Iba1⁺ cells (Figure 1(i)). Finally, we evaluated by Western Blot MHCII expression as a marker of microglial activation (Figure 6(f)), and found a significant reduction in mTNF^Δ/Δ mice compared to mTNF^wt/wt one day after pMCAO (Figure 6(f)). These findings suggest that genetic ablation of solTNF, while preserving mTNF, reduces the inflammatory response following stroke possibly by dampening the infiltration of macrophages into the ischemic brain. OPEN IN VIEWER