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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Stroke. 2019 Aug 7;50(9):2547–2554. doi: 10.1161/STROKEAHA.119.026212

N-acetyl-seryl-aspartyl-lysyl-proline augments thrombolysis of tissue plasminogen activator in aged rats after stroke

Chao Li 1, Li Zhang 1, Chunyang Wang 1, Hua Teng 1, Baoyan Fan 1, Michael Chopp 1,2, Zheng Gang Zhang 1
PMCID: PMC6710137  NIHMSID: NIHMS1534936  PMID: 31387512

Abstract

Background and Purpose:

Stroke is a leading cause of disability worldwide, mainly affecting the elderly. However, preclinical studies in aged ischemic animals are limited. N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a naturally occurring tetrapeptide with vascular-protective properties. The present study investigated the effect of AcSDKP on tissue plasminogen activator(tPA) induced thrombolysis in aged rats after ischemic stroke.

Methods:

Aged male rats (18 months) were subjected to embolic middle cerebral artery occlusion (MCAO). Rats subjected to 4h of MCAO were randomized into the following groups: 1) AcSDKP; 2) tPA; 3) AcSDKP in combination with tPA; and 4) saline. Neurological deficits, cerebral microvascular patency and integrity and infarction were examined at 1 day and 7 days after MCAO. In vitro experiments were performed to examine the effect of AcSDKP on aged cerebral endothelial cell permeability.

Results:

Compared with saline, AcSDKP or tPA as monotherapy did not have any therapeutic effects, whereas AcSDKP in combination with tPA significantly reduced cerebral tissue infarction and improved neurological outcome without increasing cerebral hemorrhage. Concurrently, the combination treatment significantly augmented microvascular perfusion and reduced thrombosis and blood brain barrier (BBB) leakage. In vitro, compared with cerebral endothelial cells from ischemic adult rats, the endothelial cells from ischemic aged rats exhibited significantly increased leakage. AcSDKP suppressed tPA-induced aged endothelial cell leakage and reduced expression of ICAM-1 and NF-κB.

Conclusions:

The present study provides evidence for the therapeutic efficacy of AcSDKP in combination tPA for the treatment of embolic stroke in aged rats at 4h after stroke onset. AcSDKP likely acts on cerebral endothelial cells to enhance the benefits of tPA by increasing tissue perfusion and augmenting the integrity of the BBB.

Keywords: Stroke, Aged rats, tPA, AcSDKP

Graphical Abstract

graphic file with name nihms-1534936-f0001.jpg

Introduction

Stroke is a leading cause of disability worldwide, mainly affecting elderly1, 2. Although the therapeutic efficacy of mechanical thrombectomy has been demonstrated in treating patients with acute ischemic stroke induced by large arterial occlusion 3, tissue plasminogen activator (tPA) remains as the only Food and Drug Administration approved agent to treat acute ischemic stroke within 4.5h of stroke onset. Due to the narrow therapeutic window and high incidence of cerebral hemorrhage, fewer than 10% of patients with acute ischemic stroke receive tPA treatment 4-6. Elderly patients with acute stroke exhibit increased blood brain barrier (BBB) leakage and are less responsive to tPA treatment compared to adult patients1. Thus, elderly patients are often excluded from the tPA treatment1. Development of new adjunctive therapies with tPA particularly for the aged population is highly clinically relevant. Unfortunately, the majority of experimental studies on acute stroke employ young adult animals, which may have contributed to the unsuccessful translation of preclinically validated neuroprotective drugs to patients with acute stroke 1, 5-9.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is endogenously released by bone marrow from its precursor thymosin-β4 (Tβ4) that contains Ac-SDKP in N-terminus and naturally presents in plasma and in circulating mononuclear cells of humans and rodents10-12. AcSDKP has a wide range of vascular protective effects including anti-inflammatory, anti-fibrotic, and pro-angiogenic properties that go beyond its originally recognized role in hematopoiesis regulation13-15. We previously demonstrated that in young adult rats, adjuvant administration of AcSDKP effectively reduces tPA potentiated neurovascular damage, and subsequently extends the therapeutic window of tPA after embolic stroke16. To extend our findings, the present study examined that the therapeutic effects of AcSDKP in combination with tPA on acute ischemic stroke in aged rats. Our study is in accordance with the Stroke Therapy Academic Industrial Roundtable (STAIR) guidelines, which call for including aged animals in experimental stroke to improve modeling of the clinical situation17.

Materials and Methods

The data that support the findings of this study are available from the corresponding author on reasonable request.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital. The treatments were not blinded, however, all outcome measurements including neurological function were performed by observers blinded to the experimental conditions.

Animal model:

Male Wistar rats at the age of 18 months were subjected to embolic middle cerebral artery occlusion (MCAO)18. Briefly, the right common carotid arteries (CCA), the right external carotid artery (ECA) and the internal carotid artery (ICA) were exposed via a midline incision. A modified PE-50 catheter was placed from the ECA into the ICA until its tip positioned at the origin of the MCA. A fibrin-rich clot obtained from a donor rat was placed at the origin of the right MCA via the catheter. The catheter was withdrawn immediately after clot injection. Neurological evaluations were carried out 1h after MCAO prior to the treatment by means of a five-point Zea-Longa scale (0 = normal, 1 = failure to extend left forepaw fully, 2 = circling to the left, 3 = falling to the left, and 4 = loss of consciousness)18. Rats with a score ≥1 were enrolled in the experimental groups.

Experimental protocols:

Previous studies have demonstrated that in aged ischemic rats, a dose of tPA at 10 mg/kg, but not at 5mg/kg, causes a high early mortality, and that tPA at either dose given 4h after embolic MCAO in the rat is ineffective19, 20. Thus, to examine whether AcSDKP enhances the therapeutic effect of tPA, a time point of 4h after stroke was selected and tPA was administered at a dose of 5 mg/kg. Stroke and/or tPA induces a second phase of BBB leakage that occurs between 24 to 48h after stroke21, 22, accordingly, AcSDKP was infused for 3 days after stroke. Rats subjected to 4h of MCAO were randomly assigned to the following groups:1) AcSDKP alone; 2) tPA alone; 3) the combination of AcSDKP and tPA; and 4) saline. AcSDKP at a dose of 0.8 mg/kg/day was infused subcutaneously (SC) using an osmotic pump (Alzet model 1003D, Alzet) for 3 days starting 4h after MCAO. tPA (Genentech) at a dose of 5mg/kg was given intravenously (IV) as a 10% bolus and the remainder 90% was infused continuously over a 30 min interval using an infusion pump starting at 4h after MCAO. Ischemic rats receiving the same volume of saline for the subcutaneous infusion were used as control. To examine the effect of AcSDKP combined with tPA on neurological outcome and infarct volume, the rats were sacrificed 7 days after MCAO. To examine the effect of the combination treatment on cerebral hemorrhage and vascular perfusion and integrity, the rats were sacrificed 24h after MCAO.

Neurological outcomes:

Sensorimotor deficits were examined using the foot-fault test and the modified neurological severity score (mNSS) evaluation performed at 1d, 3d and 7d after MCAO. For the foot-fault test, rats were tested for placement dysfunctions of forelimbs 23. Data are presented as a percentage of left foot-faults. mNSS is a composite of motor, sensory, reflex, and balance tests, which was graded on a scale of 0 (normal) to 18 (maximal deficit), as previously described24.

Measurements of infarct volume and hemorrhage:

Infarct volume was measured on seven hematoxylin and eosin (H&E) stained brain coronal sections, as previously described18. Gross hemorrhage, defined by visible accumulation of blood on brain tissue was evaluated on H&E stained coronal sections for each rat, and data are presented as the percentage of animals per group with visible hemorrhage on any coronal sections18.

Measurements of microvascular patency:

To examine patency of cerebral microvessels, fluorescein isothiocyanate (FITC)-dextran (2×106 molecular weight, Sigma-Aldrich, 1 ml of 50 mg/ml) was administered IV to the rats 24 h after MCAO, as previously reported 25. FITC-dextran perfused cerebral vessels on three brain coronal sections (100 μm) from each rat, which encompassed the center of ischemic lesion at Bregma 0.2 to −0.8 mm, were imaged and analyzed with MicroComputer Imaging Device (MCID) system (Imaging Research Inc). Ten fields of view (4.4 mm2) of fluorescence images within the territory supplied by the right MCA and contralateral homologous regions were acquired 26. Data are presented as the numbers of FITC pixels divided by the contralateral FITC pixels and expressed as a percentage.

Immunohistochemistry:

Double immunofluorescent staining was performed on brain coronal sections, according to our published protocols26, 27. The following primary antibodies were used: antibodies against thrombocyte for platelets (1:2000, Inter-Cell Technologies), myeloperoxidase for leukocytes (MPO, 1:500, ab208670, Abcam), fibrinogen for fibrin (1:1000, ab34269, Abcam), intercellular adhesion molecule-1 for inflammation (ICAM-1, 1:1000, ab53013, Abcam), endothelial barrier antigen for cerebral vessels (EBA, 1:1000, 836805, Sternberger Monoclonals Inc). For the quantification of thrombosis and inflammatory response, numbers of vessels immunoreacted with platelets, leukocytes, fibrin, and ICAM-1 were counted throughout the ischemic territory. For the measurement of cerebral vascular leakage, numbers of vessels with extravascular fibrin deposition were counted.

Cerebral endothelial integrity assay:

Primary cerebral endothelial cells were isolated from cerebral microvessels of young adult and aged rats without MCAO (normal group) or were subjected to 24h MCAO (ischemic group), as previously described28. Briefly, cerebral cortex and sub-cortex were cut into small pieces and homogenized. Use of centrifuged and collagenase/dispase (Roche), endothelial cells from microvessels were isolated and cultured in endothelial growth medium (Sigma-Aldrich). Passage 2–4 endothelial cells were employed in the present study.

To examine the direct effects of AcSDKP and tPA on cerebral endothelial cell integrity, an in vitro endothelial monolayer assay was employed. Briefly, cultured endothelial cells for 7 days at a density of 5×104/well were seeded as monolayer onto the inner chamber of trans-well insert (Corning)29. AcSDKP (1nM) and/or tPA (10μg/ml) were added into the culture medium for 6h. The same volume of culture medium was used as control. Fluorescent-conjugated dextran (0.5mg/ml, 70kDa, D1830, Thermo Fischer Scientific) was then added on the top of the trans-wells for 60 minutes. Fluorescent signals in the bottom medium were then measured using a plate reader at excitation and emission wave lengths of 595 and 615 nm, respectively. Trans-endothelial permeability was calculated as % signals = (OD experimental-OD vehicle)/OD vehicle x100. All data were obtained from three individual experiments.

A Trans-Endothelial Electrical Resistance (TEER) assay was employed to measure the integrity of the tight junctions in cell culture model of cerebral endothelial monolayer30. The TEER value was measured with a Millicell ERS system equipped with chopstick electrodes (Millipore), according to published protocol 31. The values were standardized by the area of the culture inserts and presented as Ω·cm2 by subtracting the resistance of blank. All data were obtained from three individual experiments.

Western blot:

Proteins were extracted from cultured endothelial cells. The following primary antibodies were used: rabbit monoclonal anti-ICAM-1 (1:1000, ab53013, Abcam), rabbit polyclonal anti-ZO1 (1:1000, ab96587, Abcam), and rabbit monoclonal anti-phosphorylated NF-κB p65 at Ser536 (pp65, 1:1000, 3033S, Cell Signaling Technology). β-actin (1:5000, ab8226, Abcam) was used as the loading control. Western blots were performed at least three times in individual experiments.

Statistical analysis:

The Cochran-Mantel-Haenszel (CMH) test was used to analyze the baseline Zea-Longa score acquired 1h after MCAO. One-way ANOVA was used to compare multiple-group values (i.e., measurements of lesion volume, immunohistological data, and vascular perfusion data). If the main effect of treatment group was significant at p<0.05, then pair-wise comparisons between treatment groups were tested. Adjustments for multiple comparisons were made using Hochberg’s method. Fisher Exact test was used to test the gross hemorrhagic rates and mortality rates among the groups. Data are presented as the mean ± SD.

Results

AcSDKP in combination with tPA reduces infarct volume and improves neurological function:

Analysis of neurological deficits measured prior to the treatment showed that all rats at 1h after MCAO exhibited Zea-Longa score ≥ 1; therefore, no rats were excluded from the study. There were no significant differences of neurological deficits among experimental groups: 1.9±0.1 for saline, 2.0±0.1 for AcSDKP alone, 1.8±0.1 for tPA alone, and 1.9±0.1 for combination of AcSDKP and tPA. However, administration of AcSDKP in combination with tPA 4h after MCAO significantly (p<0.05) improved neurological outcome at days 1, 3 and 7 compared with the treatment of saline, and monotherapy with AcSDKP or tPA, respectively (Fig.1). No significant improvements of neurological function were detected in ischemic rats treated with AcSDKP or tPA monotherapy compared with the saline treatment. Histopathological analysis of ischemic brains 7 days after MCAO revealed that monotherapy with AcSDKP or tPA 4h after MCAO did not significantly reduce infarct volume compared with the saline treatment; however, the combination treatment significantly (p<0.05) reduced infarct volume compared with saline, AcSDKP alone, and tPA alone (Fig.1). There were no significant differences in mortality among the experimental groups, 36% (4 out of 11, saline), 33% (3 out of 9, AcSDKP alone), 45% (5 out of 11, tPA alone), and 25% (2 out of 8, of AcSDKP and tPA). Moreover, the gross hemorrhage measured 24h after MCAO revealed that the incidence of gross hemorrhage was not significantly different among experimental groups, 25% (2 out of 8 for saline), 13% (1 out of 8, for AcSDKP), 38% (3 out of 8, tPA), and 13% (1 out of 8, AcSDKP and tPA).

Figure 1.

Figure 1.

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AcSDKP in combination with tPA reduces infarct volume and improves neurological function. Panels A and B show the neurological outcome measured using mNSS and foot-fault test at 1d, 3d, and 7d after MCAO, respectively. Panel C shows infarct volume measured 7 days after MCAO. N=7 for saline and N=6/group for AcSDKP, tPA, and AcSDKP in combination with tPA. Values are mean ± SD.

AcSDKP in combination with tPA reduces microvascular thrombosis and enhances vascular patency and integrity:

To examine the effect of AcSDKP and tPA on microvascular patency and integrity, ischemic rats were sacrificed 24h after MCAO. Immunofluorescent staining analysis showed that AcSDKP combined with tPA significantly reduced the number of microvessels containing fibrin, platelets, and leukocytes compared with saline, tPA alone and AcSDKP alone, whereas monotherapy with tPA did not significantly reduce the intravascular fibrin, platelet, leukocyte accumulation (Fig.2). To further examine microvascular patency, FITC-dextran was administered intravenously to ischemic rats 24 hours after MCAO, and FITC-dextran perfused cerebral blood vessels were measured. Ischemic rats treated with AcSDKP in combination with tPA exhibited significantly increased FITC–dextran perfused vessels in the ipsilateral territory supplied by the MCA; however, monotherapy with AcSDKP or tPA did not significantly augment FITC-dextran perfused vessels (Fig.2). Moreover, adjuvant treatment with AcSDKP and tPA significantly reduced number of microvessels with extravascular fibrin deposition, an indicator of BBB leakage 32, whereas tPA or AcSDKP alone did not significantly decrease extravascular fibrin deposition (Fig.2).

Figure 2.

Figure 2.

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AcSDKP in combination with tPA reduces thrombosis and BBB leakage and augments plasma perfusion. Panels A, B and C show representative images of double immunofluorescent staining of EBA (red) with thrombocyte (A, green), MPO (B, green), and fibrin (C, green) as well as their corresponding quantitative data obtained at 24h after MCAO. The representative images were acquired from peri-ischemic lesion of the cortex, while the quantitative data were based on measurements from the ipsilateral cortex and striatum. Panel D shows representative coronal section images of cerebral vessels perfused by FITC-dextran and their quantitative data at 24h after MCAO. N=5/group. Values are mean ± SD.

Upregulation of ICAM-1 by stroke induces microvasculature damage 33. Double immunofluorescent staining analysis showed that ICAM-1 immunoreactivity was primarily localized to EBA positive vessels within the ischemic regions, which was further augmented by tPA monotherapy (Fig.3). However, the combination treatment with AcSDKP and tPA significantly reduced the number of ICAM-1 immunoreacted vessels compared with saline, tPA alone and AcSDKP alone (Fig. 3).

Figure 3.

Figure 3.

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AcSDKP in combination with tPA reduces ICAM-1. Representative images of double immunofluorescent staining of EBA (red) and ICAM-1 (green) immunoreactive cerebral vessels and their quantitative data from ischemic aged rats treated with saline, tPA alone, AcSDKP alone, and the combination of AcSDKP and tPA, respectively. The representative images were acquired from peri-ischemic lesion of the cortex, while the quantitative data were based on measurements from the ipsilateral cortex and striatum 24 after MCAO. N=5/group. Values are mean ± SD.

AcSDKP suppresses tPA-induced cerebral endothelial barrier leakage:

Aforementioned in vivo results suggest that AcSDKP acts on cerebral microvessels. We thus performed in vitro experiments to directly investigate the effect of AcSDKP on primary cerebral endothelial cells isolated from young adult and aged rats. In vitro trans-cerebral endothelial permeability assay showed that there was no significant difference of cerebral endothelial integrity measured by means of FITC signals and TEER between non-ischemic young adult and aged rats (Suppl. Fig. I). However, cerebral endothelial cells isolated from ischemic adult and aged rats exhibited significant increases of trans-endothelial permeability with more robust leakage in aged rats (Fig.4, Suppl. Fig.I), suggesting that the aged cerebral endothelial cells are more susceptible to vascular damage after stroke. We then treated the ischemic aged endothelial cells with AcSDKP or tPA and found that addition of tPA, but not AcSDKP, significantly increased trans-endothelial permeability compared with the non-treatment (Fig.5). In the presence of AcSDKP, tPA did not augment leakage (Fig.5). Western blot analysis showed that there were no significant differences of protein levels of ICAM-1, ZO1, and phosphorylated p65 (pp65) between non-ischemic adult and aged endothelial cells (Suppl. Fig. I). Stroke significantly increased levels of ICAM-1 and pp65 and decreased ZO1, while ischemic aged endothelial cells further significantly altered these proteins compared with adult ones (Fig.4). Treatment of ischemic aged endothelial cells with tPA, but not AcSDKP, significantly augmented ICAM-1 and pp65 and decreased ZO1 compared with saline treatment, whereas AcSDKP abolished tPA-altered proteins (Fig.5). These in vitro data indicate that AcSDKP reduces stroke- and tPA-induced endothelial barrier damage in aged cerebral endothelial cells.

Figure 4.

Figure 4.

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Primary cerebral endothelial cells from ischemic aged rats are more permeable than the endothelial cells from non-ischemic aged rats. Panels A and B show schematics of cerebral endothelial cell permeability and TEER assays, respectively, and corresponding quantitative data of cerebral endothelial cells harvested from non-ischemic and ischemic aged rats. Panel C shows representative Western blot images and their quantitative data of listed proteins in the cerebral endothelial cells (please also see the representative Western blot image in Suppl. Fig. I). Beta actin was used as an internal control. All data were acquired from three individual experiments. Values are mean ± SD.

Figure 5.

Figure 5.

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The effect of tPA and AcSDKP on ischemic aged cerebral endothelial cells. Panels A and B show the effect of tPA alone or in combination with AcSDKP on permeability of cerebral endothelial cells harvested from ischemic aged rats, which were measured by FITC-dextran signals and TEER method, respectively. Panel C shows representative Western blots of ICAM-1, ZO1 and pp65 and their quantitative data. Values are mean ± SD.

Discussion:

The present study demonstrated that AcSDKP in combination with tPA administered 4h after embolic stroke significantly reduced infarct volume and improved neurological function without increasing cerebral hemorrhage in male aged rats. The adjunctive therapy substantially reduced microvascular thrombosis and improved vascular perfusion and integrity. In vitro data showed that AcSDKP directly attenuated tPA induced leakage of cerebral endothelial cells harvested from aged ischemic rats. Together, our in vivo and vitro data suggest that augmentation of microvascular perfusion and reduction of microvascular thrombosis and BBB leakage by AcSDKP likely contribute to the therapeutic effect of the combination on acute stroke.

Aged patients with acute stroke have high mortality and poor clinical outcome, and advanced age is the most important predictor of intracerebral hemorrhage in patients receiving thrombolytic therapy34,35,36. However, preclinical studies in aged ischemic animals are limited 37. Compared with ischemic young adult rats, ischemic aged rats exhibit worse spontaneously functional recovery 38. Using aged rats, we previously demonstrated that a potent proteasome inhibitor, VELCADE, in combination with tPA administered 2h after embolic stroke robustly reduces infarction and improves neurological function 39.The present study provides evidence that the adjunctive therapy of AcSDKP with tPA administered 4h after embolic MCAO augments tPA thrombolysis. AcSDKP is endogenously released by bone marrow from its precursor thymosin-β4 in human and rodents, and AcSDKP crosses the BBB under physiological and ischemic conditions 10-12, 16. AcSDKP reduces inflammation and maintains the hemostatic balance of fibrinolytic and procoagulant factors10-12, 40, 41. In young adult rats, we previously demonstrated that the effect of AcSDKP on augmentation of tPA fibrinolytic effect is specific, because blockage of AcSDKP with a neutralizing antibody abolishes the therapeutic effect of AcSDKP combined tPA on acute stroke16. The model of embolic MCAO used in the present study represents acute ischemic stroke with large artery occlusion42. Endovascular thrombectomy is now a standard of care for treatment of acute ischemic stroke with large vessel occlusion43-49. However, recanalization of upstream large arteries may not always restore downstream cerebral microvascular perfusion. Given the fact that recanalization of the occluded large artery by the endovascular thrombectomy often leads to incomplete cerebral tissue reperfusion43-47, our data suggest that AcSDKP has translational potential as an adjuvant agent to tPA thrombolytic therapy and to endovascular thrombolysis for patients with acute stroke.

The present study indicates that AcSDKP primarily acts to augment microvascular perfusion and integrity by suppressing stroke- and tPA-induced secondary thrombosis and BBB leakage. These data are consistent with clinical findings that patients with ischemic stroke including elderly patients, who undergo endovascular thrombectomy and exhibit brain tissue reperfusion, have good clinical outcome44, 47, 50, 51. We and others have demonstrated extravascular deposition of fibrin as an indicator of BBB leakage52, 53. However, in addition to a marker, parenchymal deposition of fibrin directly induces neuronal damage54. Thus, reduction of brain deposition of fibrin by AcSDKP may also contribute to reduction of neurovascular damage. Cerebral endothelial cells are highly sensitive to ischemic insult by rapidly converting homoeostasis into a prothrombotic and proinflammatory state involving activations of ICAM-1 and NF-κB55, 56. The present study showed that cerebral endothelial cells from ischemic aged rats exhibited severe leakage associated with activation of pro-inflammatory ICAM-1 and NF-κB and reduction of tight junction protein ZO1 compared with the endothelial cells derived from adult ischemic rats. Under non-ischemic condition endothelial permeability between adult and aged rats was relatively comparable. Furthermore, treatment of cerebral endothelial cells derived from ischemic aged rats with tPA further exacerbated endothelial cell leakage, whereas AcSDKP suppressed tPA-induced endothelial leakage. These novel in vitro data complement our in vivo findings. We speculate that AcSDKP converts dysfunctional endothelial cells to functional ones by reducing stroke- and/or tPA-induced genes that mediate formation of the secondary thrombosis and BBB impairment. This consequently promotes recanalization of the occluded the MCA and amplifies downstream microvascular patency and integrity. AcSDKP crosses the BBB16 and in vitro study shows that Tβ4, the precursor of AcSDKP, protects against excitotoxicity-induced neuronal death57, 58. Thus, in addition to acting on cerebral endothelial cells, AcSDKP may directly reduce ischemic neuronal damage, which may contribute to its observed therapeutic effect in the present study.

In conclusion, we present the results of AcSDKP in combination with tPA treatment of acute embolic stroke in the aged rat. This adjunctive therapy is found to be effective in reducing infarction and improving neurological outcome even when the treatment was administered 4h after embolic MCAO.

Supplementary Material

Supplemental Material figure 1

Acknowledgments

The authors wish to thank Feng Jie Wang, Min Wei, Julie Landschoot-Ward for technical assistance.

Sources of Funding

This work was supported by National Institutes of Health grants, RO1NS079612(ZGZ), RO1NS111801(ZGZ), and RO1NS102744(LZ).

Footnotes

Disclosures

None.

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