Combination treatment with U0126 and rt-PA prevents adverse effects of the delayed rt-PA treatment after acute ischemic stroke

In acute ischemic stroke, the only FDA-approved drug; recombinant tissue plasminogen activator (rt-PA) is limited by restricted time-window due to an enhanced risk of hemorrhagic transformation which is thought to be caused by metalloproteinase (MMP). In experimental stroke inhibitors of the mitogen–activated protein kinase kinase extracellular signal–regulated kinase kinase (MEK) 1/2 pathways reduce the MMPs. This study evaluated whether a MEK1/2 inhibitor in combination with rt-PA can prevent the detrimental effects of delayed rt-PA therapy in stroke. Thromboembolic stroke was induced in C57 black/6J mice and the MEK1/2 inhibitor U0126 was administrated 3.5 h and rt-PA 4 h post stroke-onset. Treatment with rt-PA demonstrated enhanced MMP-9 protein levels and hemorrhagic transformation which was prevented when U0126 was given in conjunction with rt-PA. By blocking the MMP-9 with U0126 the safety of rt-PA administration was improved and demonstrates a promising adjuvant strategy to reduce the harmful effects of delayed rt-PA treatment in acute ischemic stroke.

Ischemic stroke remains a leading cause of death and disability worldwide and is an enormous economic, clinical and social burden. Current treatments for acute ischemic stroke are limited to thrombolytic therapies involving the use of endovascular thrombectomy, and the only effective FDA approved drug recombinant tissue plasminogen activator (rt-PA) 1 . Although rt-PA represents an important break-through for management of acute stroke, this treatment has several limitations, recanalization efficiency is far from optimal, and it can only be administered within 4.5 h after stroke onset because of the risk of hemorrhagic transformation 2,3 , cerebral edema and neurotoxicity 4 . Less than 10% of all stroke patients receive rt-PA, because of the narrow time-window 5 . The hemorrhagic transformation can lead to significant morbidity and mortality in stroke patients. It is thought that metalloproteinases (MMPs) are the key culprit in causing hemorrhagic transformation. Elevated plasma levels of MMP are correlated with the frequency of hemorrhagic transformation following stroke 6,7 . MMP precursors are expressed in brain blood vessels within several hours after ischemia, and these precursors become active enzymes if cleaved by rt-PA and its substrate plasmin, leading to breakdown of extracellular matrix and leakage in the vessel; this mechanism is thought to underlie hemorrhagic transformation. When a brain vessel is occluded, the reduction in blood flow triggers molecular regulatory "switches" that turn on a vascular injury response program that includes MMP expression. The mitogen-activated protein kinase kinase extracellular signal-regulated kinase kinase (MEK) 1/2 signaling pathway is an important "switch" in the blood vessel wall that leads to upregulation of MMP-9 as a response to ischemia. Studies have previously demonstrated that inhibitors targeting MEK1/2 reduces vascular MMP levels following experimental stroke 8,9 . By blocking the MMP-9 by adding a MEK1/2 inhibitor in combination with rt-PA, we propose that we can improve the safety of rt-PA administration by preventing the hemorrhagic transformation. Here, we investigated whether the combination therapy of the MEK/1/2 inhibitor U0126 and rt-PA can alleviate the detrimental effects of delayed rt-PA therapy in acute ischemic stroke.
U0126 and rt-PA in combination prevent activation of MMP-9. Zymography and western blot analysis were performed to investigate the expression of MMP-9 protein levels. We observed that MMP-9 protein levels were significantly decreased when U0126 was combined with rt-PA compared with rt-PA treatment alone. However, MMP-9 activity was not different between the groups (Fig. 3, Supplementary Fig. 1). Similar results were observed with the immunohistochemistry (Fig. 4). Using NIS basic research software, the ischemic area in each section was delineated as shown in Fig. 4a and the overall MMP-9 immunoreactivity was examined. The immunoreactivity in the control side of the brain was often organized in a radial pattern, corresponding to the distribution of the cortical neurons (Fig. 4b). Often a pearl-like distribution was observed ( Fig. 4b-I), suggesting staining of the neuronal processes. The distribution of the immunoreactive structures in the stroke areas was different in all groups (Fig. 4b). In addition to the radial distribution observed in the control side, a transverse distribution was found. This disorganization was found in all stroke sides, however, more or less pronounced. The amount of immunoreactive structures increased, with the largest difference observed in the group treated with rt-PA (Fig. 4c).
U0126 and rt-PA in combination prevent activation of pERK1/2. The pERK1/2 protein levels were significantly increased after ischemia (Fig. 5a). Interestingly, there was no effect of rt-PA treatment, nevertheless rt-PA in combination with U0126 and U0126 alone showed a tendency to prevent the enhanced protein www.nature.com/scientificreports/ level observed after ischemia (Fig. 5a), though no differences were observed. Similarly, there was an increase in pERK1/2 immunoreactivity after ischemia (Fig. 5b,c). Both combination treatment with rt-PA and U0126 and U0126 as a single treatment prevented this increase (Fig. 5c). Immunoreactivity of pERK1/2 was only found within the stroke core and in areas close to stroke in all groups. Consequently, only these areas are described below. All animals in the vehicle group contained immunoreactive cells in the core and the area close to the core. Also, immunoreactive vessels were found (Arrow heads; Figs. 5b). In the other groups, the number of positive cells decreased, with fewest immunoreactive cells in the group treated with the combination therapy rt-PA + U0126. In this group, no positive cells were found in the area close to the stroke area (Fig. 5b). In the groups treated with U0126, or rt-PA in combination with U0126, no immunoreactive vessels were found.

Discussion
This study evaluated the important question whether rt-PA-associated hemorrhage after delayed rt-PA treatment in an animal model of thromboembolic stroke can be prevented by blocking the MMP-9 through a specific MEK1/2 inhibitor. Our data demonstrate that by blocking the MMP-9 expression through the use of a MEK1/2 inhibitor the rt-PA induced hemorrhagic transformation was prevented, indicating that this may be a promising adjuvant strategy to alleviate the detrimental side effects of delayed rt-PA in acute ischemic stroke. However, there was a discrepancy between the effect on the cerebral infarct volume and hemorrhagic transformation. No effect on the infarct volume was observed even if the hemorrhagic transformation was prevented. In previous study where U0126 was administrated at both 6 h and 24 h after tMCAO a reduced infarct compared to vehicle was demonstrated over time with a significant difference at day 14 post-stroke 10 , but not at early time-points. The same dose was used in this study 30 mg/kg, however as one single dose, which may be one of the reasons why U0126 alone did not have any protective effect in the current study. Further explanations may be that the timepoint 24 h was too early to evaluate the infarct and that we still have ongoing processes and changes in infarct may occur much later. The activation of MMP-9 and enhanced expression of pERK1/2 are expressed in the vasculature. The MMP precursors are expressed in brain blood vessels after ischemia, and become active www.nature.com/scientificreports/ Representative zymography blots and scatter plots illustrate MMP-9 protein expression. The expression levels were normalized to control group which was set to 100%. All data are presented as median ± IQR, *P < 0.05 is considered statistically significant, n = 3-7. www.nature.com/scientificreports/ enzymes if cleaved by rt-PA, leading to vascular dysfunction, and hemorrhage resulting in neuronal damage. By preventing the hemorrhagic transformation the neuronal damage may become reversible which can be observed over time. Further long-term studies will be needed to assess the long-term outcome. The only effective drug therapy for treatment of acute ischemic stroke to date is thrombolysis with rt-PA 1 . The major complication of rt-PA is the hemorrhagic transformation which is associated with increased stroke morbidity and mortality. Recombinant tissue plasminogen activator increase the rate of hemorrhage by tenfold 11,12 and given that rt-PA is the only FDA approved treatment for acute ischemic stroke it is essential to prevent the detrimental effects of rt-PA and restore blood flow without complications. Elevated MMP plasma levels have been reported to correlate with the frequency of rt-PA induced hemorrhagic transformation following stroke in both animal models 6,7 and stroke patients 13 . It is demonstrated that rt-PA activates nuclear factor (NF)-КB signalling leading to activation of MMP-9, which in turn contribute to enhanced blood-brain barrier (BBB) permeability, vascular www.nature.com/scientificreports/ dysfunction and hemorrhage 14,15 . Despite the correlation between enhanced MMP-9 levels after rt-PA treatment and worsen outcomes of patients, there is no current therapy to prevent hemorrhagic complications. Our present study demonstrates that the specific MEK1/2 inhibitor U0126 prevent activation of MMP-9 in the acute phase of experimental stroke, which is in agreement with a previous study 16 . By adding U0126 in combination with rt-PA we prevent activation of MMP-9 expression leading to BBB leakage and hemorrhagic transformation. Despite tremendous attempts over the last decades in developing new treatments for ischemic stroke none of them demonstrated efficacy and safety in clinical trials 17,18 . Considering that stroke is a vascular disease, neuroprotection without restoration of tissue perfusion will have a low possibility of demonstrating efficiency in treating acute ischemic stroke 19 . In the present study, we use rt-PA to restore the cerebral blood flow and by adding U0126 as an adjuvant agent to block MMP-9 we are able to prevent the enhanced MMP-9 leading to BBB breakdown and hemorrhagic transformation which is one of the major complications of delayed rt-PA treatment.
In experimental ischemic stroke models broad spectrum MMP inhibitors have shown reduced infract and BBB permeability, however, clinical trials resulted in unsatisfactory outcome due to toxicity and adverse effects. It has been technically challenging to develop specific MMP-9 inhibitors, but recent generation of MMP inhibitors have overcame this obstacle and shown improved pharmacokinetics 20 . In this study we demonstrated that www.nature.com/scientificreports/ by indirectly inhibiting MMP-9 the hemorrhagic transformation was prevented. This suggest that targeting the MMP-9 activation may be a promising strategy to prevent the detrimental side effects of delayed rt-PA therapy. However, further studies need to be performed to investigate the use of the new promising MMP-9 inhibitors in acute ischemic stroke as well as functional and long-term outcome.

Materials and methods
Experimental design of the study. Thromboembolic stroke was induced in C57BL/6J mice (20-30 g) and all animals were randomly divided into 4 groups; (i) vehicle treated, (ii) treated with rt-PA, (iii) treated with rt-PA in combination with U0126 and (iv) treated with U0126. To induce thrombolysis, rt-PA (10 mg/ kg; Actilyse) was intravenously administrated 4 h after thrombin injection. It was injected as 10% bolus and 90% perfusion for 40 min. The MEK1/2 inhibitor U0126 (30 mg/kg; LC laboratories, Boston, MA, USA) or vehicle (dimethyl sulfoxide) was injected intraperitoneal at 30 min prior to rt-PA administration, in a random and blinded fashion. The dosage for U0126 was chosen based on previous studies 21 . After 24 h all mice were euthanized and brains removed and either fixed in 4% paraformaldehyde in phosphate buffered-saline (PBS) for immunohistochemistry and diaminobenzidine (DAB) staining or snap frozen in isopentane in order to collect tissue for western blot and zymography. The design of the study is illustrated in Fig. 6 Thromboembolic stroke. Thromboembolic stroke was induced by local injection of thrombin directly into the right middle cerebral artery (MCA) of mice as originally described by Orset et al. 22 . Male C57 black/6J mice (20-30 g) were anaesthetized using 5% isoflurane and thereafter maintained with 1-2% isoflurane during the surgical procedure. An electric temperature probe was inserted into the rectum of the mouse to record the temperature, and found to be maintained at 37 °C. A catheter was placed in the tail vein to allow the intravenous administration (200 µl) of vehicle or t-PA. The mice were placed in a stereotaxic device and the temporal muscle was retracted. A small craniotomy was performed, the dura was excised, and the MCA was exposed. The laser Doppler flow probe to measure cortical cerebral blood flow (CBF) was placed on the skull in the MCA territory. Finally, a micropipette filled with 1 µl of purified murine alpha-thrombin (1.5 UI) was introduced into MCA bifurcation lumen and injected carefully to induce the formation of a clot in situ. The pipette was removed 10 min after the injection at which time the clot had stabilized. CBF velocity was measured continuously by laser Doppler flowmetry allowing determination of spontaneous clot dissolution. A clot was defined as successful when the cerebral blood velocity decreased minimum 60% from baseline at the time of thrombin injection and remained stable during minimum 20 min.

Magnetic resonance imaging.
All experiments were performed on a Pharmascan 7T (Bruker, Germany) small animals' system. Animals were anaesthetized with 5% isoflurane and thereafter maintained with 1-2% isoflurane. During the scanning procedure body temperature was maintained using a water-heating system and breathing rate was monitored. The MR data set was acquired at 24 h after the onset of thromboembolic stroke. T2-weighted images to visualize infarction were acquired using MSME sequences (multi-spin multi-echo): echo time/repetition time: 51 ms/2500 ms with a 70 mm × 70 mm × 500 mm spatial resolution. Lesion size was measured 24 h post-surgery on T2-weighted images using ImageJ software. The measurements were performed by a blinded evaluator.

Intracerebral hemorrhage examination.
To evaluate the presence of hemorrhage after delayed rt-PA treatment, T2 * -weighted gradient echo images by MRI and diaminobenzidine staining was performed on 10 µm www.nature.com/scientificreports/ coronal cryosections. Diaminobenzidine staining is commonly used to define hemorrhagic areas in sections 23 and has previously been described 24 Hemorrhages were assessed by blinded histological evaluation on three sections from each animal. The incidence of hemorrhagic transformation was determined and counted microscopically by classifying them into grades 1-3; 1 = no hemorrhage, 2 = defined as more confluent petechiae within the damaged area, 3 = defined as blood clots in more than 30% of the damaged area.
Tissue collection for Zymography and western blot. Brains frozen in isopentane were used to collect tissue for zymography and western blot. Tissue samples were collected from the ischemic and contra-lateral side of each brain. All tissue samples were collected blinded. Tissues collected for all four groups were further used for protein extraction. Total protein concentration was determined using a BioRad Protein Assay kit (Hercules, CA, USA). Zymography and western blot analysis. Protein lysates from the 4 different groups and samples from the right hemisphere of the mice were compared. Quantification of band intensity was performed by using image J software (http:// rsb. info. nih. gov/ ij/). The optical density values were determined with repeated measurements and presented as percentage activity of the treated group compared with the control group (tissue from healthy mice), where the values for the control group were set to 100%. Each sample was replicated three times. For western blot the intensity of the bands for pERK1/2 and MMP-9 was normalized to β-actin which was used as loading control.

Zymography.
Immunohistochemistry. Immunohistochemistry was performed as previously described 10 . Brains were fixed in 4% paraformaldehyde and then sectioned into 12-μm thick slices in a cryostat. Brain sections were permeabilized in PBS containing 0.25% Triton X-100 (PBST) and blocked for 1 h in blocking solution containing PBS 0.25% Triton X-100, 1% BSA and 5% normal goat serum. The sections were incubated over night at 4 °C with the following primary antibodies: rabbit monoclonal MMP-9 (ab137867, Abcam, Cambridge, UK) diluted 1:100 or mouse dually phosphorylated ERK1/2 (#ab50011, Abcam, Cambridge, UK) diluted 1:200. The sections were subsequently incubated for 1 h at room temperature with FITC-conjugated donkey anti-mouse (1:100, Jackson ImmunoResearch) or FITC-conjugated goat anti-rabbit (1:100, Jackson ImmunoResearch) as secondary antibody. The sections were mounted with a mounting medium containing nucleus staining DAPI (Vecta-Shield, Vector Laboratories, Burlingame, CA, U.S.A.). The experiments were repeated to ensure reproducibility. Negative control was performed by omitting primary antibody. Brain sections were chosen where the infarct size was at largest, accordingly to the MRI measurements. Immunoreactivity of the brain sections was visualized with an epifluorescence microscope (Nikon 80i; Nikon, Tokyo, Japan) coupled to a Nikon DS-2 MV camera at the appropriate wavelength. All the images were taken at the same day for all the sections and images analysis were performed blinded. The image was taken on each of four sections for all the individual mice.

MMP-9.
Using NIS basic research software, the ischemic area in each section was delineated and the overall MMP9 immunoreactivity was examined. 100 µm towards the midline in relation to the ischemic area, an area measuring approximately 300 µm in width and spanning through all cortical layers was outlined. The corresponding area in the control side was defined and examined. The immunoreactivity here was judged as a baseline regarding the particular individual. A three-grade scale was used to examine the intensity, distribution and the structural features of MMP9 immunoreactivity in the ischemic area in relation to the base-line in the control side.
pERK1/2. The ischemic area was delineated and the overall pERK1/2 immunoreactivity was examined. As immunoreactivity was observed specifically within the stroke area and areas close to stroke, the calculations