Tissue inhibitor of metalloproteinases-3 mediates the death of immature oligodendrocytes via TNF-α/TACE in focal cerebral ischemia in mice

The study was approved by the University of New Mexico Animal Care Committee and conformed to the National Institutes of Health Guide for the Care and Use of Animals in research. Eighty-nine male Timp-3 KO (n = 44) and WT (n = 45) mice weighing 20-30 g (age 10-12 weeks) were employed in this study. Fifty-nine animals were subjected to 90 min MCAO using the intraluminal thread method followed by 24 or 72 h reperfusion. MCAO was performed as previously described [15, 17]. Mice underwent reperfusion via the circle of Willis. Thirty sham-operated animals were anesthetized and had a suture introduced into the common carotid artery and immediately removed.

Glial fibrillary acidic protein (GFAP) (1:400; Sigma), Iba-1 (1:200; Wako Pure Chemical Industries), GST-π (1:600, Abcam), GalC (1:300, Millipore), Fas (1:400; Santa Cruz Biotechnology), Fas ligand (1:400; Santa Cruz Biotechnology), MMP-2 (1:250; Chemicon), MMP-3 (1:1500; Chemicon), MMP-9 (1:1000; Chemicon), caspase-3 (1:1000, Cell Signaling), TIMP-3 (1:1000; Chemicon), TNF-α (15 μg/ml; R&D Systems), TNF Receptor I (1:1000; Abcam), von Willebrand Factor (vWF) (10 μg/ml; Chemicon), myeloperoxidase (MPO; 1:250; Santa Cruz Biotechnology), and several markers for oligodendroglia; RIP (1:200,000; Chemicon), CNPase (15 μg/ml; Chemicon), and CC-1 (APC; 10 μg/ml; Calbiochem).

Brain tissues fixed with 2% paraformaldehyde, 0.1 M sodium periodate, 0.075 M lysine in 100 mM phosphate buffer at pH 7.3 (PLP) were used for immunohistochemical analysis. For immunofluorescence, 10 μm PLP-fixed brain sections were treated with acetone and blocked with 5% normal serum. Primary antibodies were incubated for two nights at 4°C. Slides were incubated for 90 min at room temperature with secondary antibodies conjugated with FITC or Cy-3 (Jackson). 4'-6-diamidino-2-phenylindole (DAPI; Molecular Probes) was used to label cell nuclei.

For brightfield immunolabeling, 10 μm PLP-fixed sections were rehydrated through alcohol series and incubated in phosphate buffered saline and 0.1% Tween-20 pH 7.3 (PBT) prior to blocking with 5% normal serum in PBT. Slides were incubated with primary antibodies for 48 h at 4°C followed by 1 h incubation with biotinylated secondary antibodies, Vectastain Elite ABC reagents (Vector Laboratories) and reacted with 3,3'-diaminobenzidine (DAB) was used for visualization of immunolabeling. Cresyl violet acetate (CVA, Sigma-Aldrich) was used for counterstaining.

Immunohistochemistry negative controls were incubated without the primary antibody or with normal (non-immune) IgGs and did not exhibit specific immunolabelling. All immunohistochemically stained slides were viewed on an Olympus BX-51 bright field and fluorescence microscope (Olympus America Inc.) equipped with an Optronics digital camera and Picture Frame image capture software (Optronics). Dual immunofluorescence slides were also imaged using high resolution confocal microscopy to verify co-localization of antigens (Zeiss LSM 510, Carl Zeiss Microimaging). Immunofluorescence was quantified using ImageJ software (NIH software).

The cellular composition of the white matter is comprised of > 90% OLs [18] consisting of OL progenitors, immature, and mature OLs. Antibodies used to identify mature OLs, including CC-1, RIP, and CNPase, either labeled too few OL cell bodies or obscured the cell bodies in the processes. To measure white matter damage in mouse focal ischemia, we first assessed loss of OL-like cells in white matter [2] using multiple-antibody immunostaining [19]. Labeling cells with a Nissl substance stain, such as cresyl violet acetate (CVA), all cells are clearly observable and an assessment of OL density can be determined stereologically. To distinguish OLs from astrocytes and microglia, we stained slides with GFAP for astrocytes and Iba-1 for microglia, using the DAB method. All slides were counterstained with CVA. By eliminating DAB-stained astrocytes and microglia, small CVA-positive cell bodies that were negative for GFAP and Iba-1 immunoreactivity were identified as OL-like cells and quantified using unbiased stereology with StereoInvestigator (Version 6, MBF Bioscience) software controlling a motorized stage equipped Olympus BX-51 microscope [19]. Employing the optical fractionator function of StereoInvestigator, we estimated OL-like cell number in the medial corpus callosum (CC) and external capsule (EC) regions of white matter over a total distance of 3.0 mm rostral and caudal to bregma. Tissue sections were blinded to the investigator as to animal identity and reperfusion time. A subset of slides was stained with myeloperoxidase or von Willebrand factor to identify neutrophils and endothelial cells. These could be distinguished from the OL-like cells based on morphology (data not shown).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was done to identify the extent of DNA fragmentation. Using the NeuroTACS II kit (Trevigen) brain sections were treated following the procedure specified by the manufacturer. For dual immunofluorescence, the brain sections were then exposed to the primary antibodies and streptavidin fluorescein and Cy-3 secondary antibodies. For positive controls the nuclease provided with the kit was added to the labeling reaction mix and induced TUNEL positivity in all cells. As negative controls, slides were prepared in a labeling reaction mix without the TdT enzyme resulting in no TUNEL positivity (data not shown).

MMP-3 enzymatic activity was assessed using a peptide-based FRET assay. In the intact FRET peptide, Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH₂, obtained from Bachem (Torrance, CA) the fluorescence of Mca (7-methoxycoumarin-4-acetyl) is quenched by Dnp (2,4-dinitrophenyl). Upon cleavage into two separate fragments by the MMP-3 present in the sample, the fluorescence of Mca was recovered, and monitored at λ_ex = 328 nm and λ_em= 393 nm using a spectrofluorimeter (Perkin-Elmer LS 50B).

Brain tissue was homogenized in lysis buffer containing 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij-35, 0.02% NaN₃, and 1% Triton X-100, and centrifuged at 12,000 × g for 5 min at 4°C. Equal amount of total protein (6-10 μg) was mixed with assay buffer containing 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10 mM CaCl₂, 0.01% Brij-35, and fluorogenic substrate (1 μM final concentration) to a final volume of 200 μl. Fluorescence was monitored for 15 min. The relationship between fluorescence units and nM of product produced was determined from the fluorescence values obtained when all the substrate was hydrolyzed.

TACE activity was measured using the fluorometric SensoLyte 520 TACE (α-Secretase) Activity Assay Kit (AnaSpec) according to the manufacturer's instructions. SensoLyte™ 520 TACE Activity Assay Kit uses a 5-FAM (fluorophore) and QXL™ 520 (quencher) labeled FRET peptide substrate for continuous measurement of enzyme activity. In the intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL™ 520. Upon cleavage of the FRET peptide by the active enzyme, the fluorescence of 5-FAM is recovered, and can be continuously monitored at λ_ex = 490 nm/520 nm. TACE activity was measured in the brain tissue homogenized as described before for MMP-3 activity. Equal amount of total protein was mixed with assay buffer (AnaSpec) and TACE substrate to a final volume of 100 μl. Fluorescence was monitored for 60 min with data recorded every 5 min. Fluorescence was expressed as relative fluorescence units (RFU). Some brain samples from KO and WT mice had 10 μl of 10 μM TACE inhibitor, TAPI-0, added as a control.

Statistical comparisons among groups were done using ANOVA with post-hoc analysis for multiple t- tests (Prism 4.0, GraphPad Software Inc.). All data are presented as mean ± standard error of the mean (S.E.M.). Statistical significance was set at p < 0.05.

Two regions of ischemic white matter were analyzed: the corpus callosum (CC) and the external capsule (EC). No OL-like cell loss was seen in ischemic CCs in both KO and WT mice at 24 and 72 h (data not shown) or in ischemic EC at 24 h (Figure 1A, top two panels). Representative regions from the nonischemic and the ischemic EC at 72 h showed the normal appearing OL-like cells in the nonischemic sides and the reactive astrocytosis and/or microglia/macrophages in the ischemic ECs in KO and WT (Figure 1A. bottom two panels). The ischemic EC in KO showed no observable loss of OL-like cells, while the ischemic EC in WT had marked loss of OL-like cells (Figure 1A, bottom two panels).

Next, the loss of OL-like cells in ischemic white matter was quantified by unbiased stereology. At 24 h, KO and WT mice showed similar cell counts in the ischemic and the nonischemic hemispheres (Figure 1B). By 72 h, there was a significant loss of OL-like cells in the ischemic EC of WT compared to KO mice (p < 0.01). Furthermore, there was a significant loss of cells in the ischemic compared to the nonischemic hemisphere in WT only (p < 0.05). In the CC, no significant reduction of OL-like cells was detected in either WT or KO ischemic CC at 72 h (data not shown). Next, we investigated apoptotic cell death in ischemic EC at 72 h of reperfusion by TUNEL assay. More TUNEL-positive cells were detected in ischemic WT hemisphere including the EC region compared with KO (Figure 1C). The difference in the distribution of TUNEL-positive cells between the ischemic KO and WT hemispheres is consistent with the patterns of the neuronal degeneration revealed by Fluoro-Jade staining (Figure 1D). This result suggested that ischemia-reperfusion injury induced a delayed cell loss in white matter in which TIMP-3 may be involved.

We then studied the expression of TIMP-3 in astrocytes and OLs in WT mice. At 24 h, very little TIMP-3 was seen in the white matter. We observed a new population of GFAP-positive astrocytes closely surrounding the core infarct areas at 72 h (data not shown), which has been reported by others [20]. Maximal TIMP-3 expression occurred at 72 h in GFAP-positive astrocytes in ischemic EC (Figure 2A). Most of the astrocytes were co-labeled with TIMP-3 and co-localization of TIMP-3 with CC1, an OL marker, was also seen by 72 h (Figure 2B). This temporal and spatial relationship of TIMP-3 expression and the loss of OL-like cells in ischemic EC indicated the involvement of TIMP-3 in the white matter damage in focal cerebral ischemia.

To further investigate the role of TIMP-3 in OL death, we examined the expression of myelin basic protein (MBP) and glutathione S-transferase π (GST-π, or GST-pi) [21], markers of mature OL, in ischemic ECs of KO and WT mice at 72 h using Western blot analysis (Figure 3A). Unexpectedly, we found no significant changes of MBP level either in ischemic ECs between KO and WT mice or between ischemic ECs and sham ECs. However, we observed a significant decrease of GST-π level in WT ischemic EC compared with sham WT mice. Next, we tested the expression of galactocerebroside (GalC), a marker of immature OL [22, 23]. We found that a significant increase in GalC was seen in ischemic ECs of WT and KO by 72 h reperfusion after ischemia compared with sham WT (p < 0.001) and sham KO (p < 0.05), respectively. Most importantly, TIMP-3 KO mouse shows significantly higher levels of GalC expression in white matter than WT mouse (p < 0.01). These findings suggested that TIMP-3-mediated delayed OL death mainly occurs in immature OLs in white matter after ischemic stroke with reperfusion injury.

To confirm the observation that TIMP-3 contributed to delayed immature OL death in ischemic white matter, we next performed immunohistochemistry of GST-π and GalC on brain tissue from KO and WT mice with 90 min-MCAO and 72 h reperfusion (Figure 3B). Double-immunostaining of GST-π and GalC demonstrated stronger immunohistochemical signals of GST-π and GalC in ischemic KO EC than WT EC. Furthermore, we detected apoptotic death using TUNEL assay in OLs. We found TUNEL-positive cells in ischemic EC at 72 h, which mostly co-localized with GalC-positive OLs (Figure 3C).

To study the possible mechanism of TIMP-3-mediated cell death, we used immunohistochemistry to determine the expression of caspase-3, since apoptosis has been implied in immature and mature OL death [1, 10, 24‐26]. In the ischemic EC of the WT, greater caspase-3 immunoreactivity was detected at 24 h of reperfusion than in the KO. There was loss of DAPI stained nuclei (blue) in the WT EC as compared with the KO EC at 72 h reperfusion (Figure 4A).

Western blots were done with an antibody that recognizes both pro- and cleaved-caspase-3. The activated caspase-3 often appears as multiple cleaved bands migrating variably in the 17-20 kDa range [27]. There was no detectable cleaved (active) caspase-3 (17/19 kDa) in white matter of KO and WT mice at 24 h after stroke. Western blots exhibited strong immunoreactive bands at 32 kDa corresponding to the pro- (inactive) caspase-3 (Figure 4B). A significant increase of pro-caspase-3 was demonstrated in ischemic white matter of WT mice (p < 0.05), indicating that caspase-3 was markedly induced in WT mice at 24 h reperfusion after stroke. By 72 h there were no differences in pro-caspase-3 expression in the white matter of KO or WT mice (Figure 4C). More importantly, a statistically increased level of cleaved caspase-3 (17/19 kDa) was seen in ischemic WT white matter (p < 0.05), implicating an apoptotic mechanism in OL loss (Figure 4C).

Since TIMP-3 regulates the activities of MMPs that are important in processes involved in growth, cell death, and tissue repair [28], we next investigated the expression of MMP-2, -3 and -9 in the ischemic white matter of Timp-3 KO and WT utilizing immunohistochemistry (Figure 5A). Co-labeling of the MMPs and GFAP showed more MMP-2 immunoreactivity in the KO white matter than in the WT at 72 h. Increased expression of MMP-2 in Timp-3 KO mice had been observed previously in other disease models and attributed to chronic damage in heart and lung [29, 30]. The increased MMP-2 expression was seen in GFAP-positive cells and around blood vessels. On the other hand, WT mice had more immunostaining for MMP-3 and -9 in EC than seen in the KO at 72 h. MMP-3 and-9 were seen in astrocytes around blood vessels (Figure 5A), while MMP-3 was also seen in OL cells and microglia/macrophages in the core infarcted white matter where very few astrocytes were detected (Figure 5A, B and 5C).

The observation of higher expression of MMP-3 in glial cells in WT white matter was unexpected since TIMP-3 is supposed to inhibit MMP-3 on the cell surface [28]. We quantified the MMP-3 protein in white matter by Western blot and measured its activity with a fluorogenic activity assay. We found increased MMP-3 (both pro- and active forms) in the ischemic hemisphere of the WT compared to the KO (Figure 5D). The fluorogenic assay showed a statistically significant increase in the activation of MMP-3 in the WT only (Figure 5E).

Death of OLs can occur through death receptors of the TNF superfamily, including Fas/FasL and TNF-α/TNFR. Double immunostaining with Fas and FasL antibodies demonstrated greater Fas/FasL signal in the gray matter of KO mice at 24 h (Figure 6). Considering the neuronal degeneration occurs earlier and greater in WT gray matter than KO (Figure 1D), the greater Fas/FasL signal in gray matter in KO mice at 24 h suggests delayed cell death compared with WT mice. However, Fas/FasL signal was not detected in white matter in KO or WT mice at both 24 and 72 h reperfusion after focal ischemia (Figure 6). Since TNF-α and activated microglia and macrophages are associated with white matter damage, we then investigated the TNF-α expression in microglia/macrophages. TNF-α was increased in white matter of Timp-3 WT mice after 72 h reperfusion, and it was co-localized with Iba-1-positive microglia/macrophages (Figure 7A and 7B). Furthermore, we observed greater Iba-1-positive microglia/macrophages that expressed TNF-α in WT white matter at 72 h. For quantification of Iba-1, we evaluated fields of the ischemic EC as well as cortical and striatal tissue immediately bordering the EC in each section, quantifying the amount of fluorescent intensity of Iba-1 as assessed in five brains per group at each time point. We found significantly greater fluorescence of Iba-1 in ischemic WT EC compared to KO at both 24 and 72 h after ischemia (Figure 7C). These results suggest that TIMP-3 facilitates the inflammatory response to ischemia-reperfusion injury.

Since TIMP-3 inhibits TACE, which both generates soluble TNF-α (17 kDa) from the 21 kDa cell surface form and cleaves the TNF receptors, we measured TACE activity in white matter from Timp-3 KO and WT mice at 72 h reperfusion. Ischemia with 72 h reperfusion resulted in a significant increase in activation of TACE in white matter of WT mouse compared with KO (Figure 7D). The double staining of TACE and Iba-1 or CC1 showed the expression of TACE in Iba-1-positive microglia/macrophages and OLs in WT white matter (Figure 7E).

The excess presence of TNF-α in white matter could trigger cell apoptosis by ligation with the TNFR-1. We determined the presence of this receptor and the cell type expressing it by immunohistochemistry. We found that the TNFR-1 co-localized with the OL marker, CC1 (Figure 7F). Western blot showed no difference of TNFR-1 expression between KO and WT mice (Figure 7F).