Effects of tissue plasminogen activator timing on blood–brain barrier permeability and hemorrhagic transformation in rats with transient ischemic stroke

doi:10.1016/j.jns.2014.09.036

Journal of the Neurological Sciences

Volume 347, Issues 1–2, 15 December 2014, Pages 148-154

https://doi.org/10.1016/j.jns.2014.09.036 Get rights and content

Highlights

•
We studied a cerebral ischemia/reperfusion model in spontaneously hypertensive rats.
•
We measured a dynamic model of blood–brain barrier permeability (BBBP) using imaging.
•
We administered tPA at different timepoint: immediately and 4 h after reperfusion.
•
Different BBBP evolution and rates of hemorrhagic transformation (HT) were observed.
•
Late tPA administration was associated with increased BBBP, mortality, and risk of HT.

Abstract

The goal of our study was to determine if the timing of the tissue plasminogen activator (tPA) administration influenced its effect on blood–brain barrier (BBB) permeability and the subsequent risk of hemorrhagic transformation.

Thirty spontaneously hypertensive male rats were subjected to a 90-minute unilateral middle cerebral artery occlusion. Six rats did not receive tPA treatment (vehicle control: Group 0), intravenous tPA was administered immediately after reperfusion (Group 1) or 4 h after reperfusion (Group 2). Dynamic contrast enhancement (DCE) and gradient-echo (GRE) MR sequences were used to assess the dynamic evolution of BBB permeability and hemorrhagic transformation changes at the following time points: during occlusion, and 3 h, 6 h, and 24 h post reperfusion.

In all groups, BBB permeability values in the ischemic tissue were low during occlusion. In Group 0, BBB permeability values increased at 3 h after reperfusion (p = 0.007, compared with the values during occlusion), and further at 6 h after reperfusion (p = 0.004, compared with those at 3 h post reperfusion). At 24 h post reperfusion, the values decreased to a level relative to but still higher than those during occlusion (p = 0.025, compared with the values during occlusion). At 3 h after reperfusion, BBB permeability values in the ischemic tissue increased, but to a greater extent in Group 1 than in Group 0 (p = 0.034) and Group 2 (p = 0.010). At 6 h after reperfusion, BBB permeability values in the ischemic tissue increased further in Group 2 than in Group 0 (p = 0.006) and Group 1 (p = 0.001), while Group 1 exhibited BBB permeability that were still abnormal but less than those observed at 3 h (p = 0.001). Group 2 tended to have a higher hemorrhage incidence (36.4%, 4/11) than Group 1 (10.0%, 1/10, p = 0.311) and Group 0 (0%), and hemorrhages occurred around 6 h after reperfusion when BBB permeability values were the highest. Mortality was higher in Group 2 (63.6%, 7/11) than in Group 0 (0%) and Group 1 (10.0%, 1/10, p = 0.024).

The findings suggest that the timing of tPA administration is of importance for its impact on BBB permeability and subsequent risk of hemorrhagic transformation.

Graphical abstract

Introduction

Hemorrhagic transformation (HT) is a feared complication of acute ischemic stroke. It can arise as the result of an ischemic damage to the blood–brain barrier (BBB) with subsequent vascular leakage [7], [26]. It is often triggered by reperfusion [32].

Tissue plasminogen activator (tPA) has been shown to be a successful thrombolytic drug in acute ischemic stroke patients [11], [12] but significantly increases the risk of symptomatic HT [11], [33], [35]. In the National Institute of Neurological Disorders and Stroke (NINDS) tPA trial [1], the percentage of tPA-treated patients who developed significant HT following an ischemic stroke was 6.4% as compared with just 0.6% in the placebo group. One of the mechanisms by which tPA causes HT is its impact on the BBB permeability [[20], [27], [32]].

Reperfusion injury resulting from the thrombolytic effect of tPA involves reactive oxygen species and oxidative stress, which degrade protein and lipid components vital to the BBB function [14]. Independently of reperfusion, tPA activates matrix metalloproteinases (MMPs), which in turn alter the basal lamina of vascular endothelium, weaken vessels, and favors leakage and rupture [2], [5], [6], [13], [16], [24], [27], [29], [31], [32].

The BBB permeability changes evolve in a dynamic process after reperfusion. In a 2-hour temporary middle cerebral artery occlusion (MCAO) model, Belayev et al. explored the time course and regional pattern of blood–brain barrier (BBB) opening after reperfusion, the quantitation of Evans' blue extravasation indicated some degree of BBB disruption occurring at 3–4 h after MCAO, maximal disruption at 5 h, and delayed BBB disruption at 48–50 h [3]. In a study with 3-h MCAO, the water content accumulated with time in the ipsilateral hemisphere within 12 h post MCAO (Slivka et al., 1995) [36]. In our study, we wanted to focus on the direct effect of tPA on the BBB permeability and on the risk of hemorrhagic transformation. We thus assessed the impact of tPA injected at various timepoints, while maintaining the duration of the occlusion constant.

Section snippets

Study design

This animal study was approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Spontaneously hypertensive (SHR) male rats (Charles River Laboratories, Inc., Wilmington, MA.), body weight 260–280 g, 11–13 weeks old, were subjected to 90 min of transient focal cerebral ischemia. The animals were then

Study population

36 male SHR rats were subjected to a 90-minute fMCAO or 3VO, respectively. Of the 36 rats, 1 rat died right after surgery because of subarachnoid hemorrhage; 5 rats were excluded because of the inclusion/exclusion criteria; 3 rats died after reperfusion. For the remained 27 rats, 13 rats received 3VO (3 in Group 0, 5 in Group 1, 5 in Group 2), 14 rats received fMCAO (3 in Group 0, 5 in Group 1, 6 in Group 2). At 24 h post reperfusion, there were 6 rats left in Group 0, 9 rats left in Group 1(4

Discussion

The present study demonstrated that BBB permeability increased more when tPA was administered 4 h after reperfusion as compared to when it was administered at the time of reperfusion. In addition, hemorrhagic transformation and mortality were more frequent when tPA was administered late. Hemorrhagic transformation typically occurred when BBB permeability was the highest. The degree of reperfusion, luxury perfusion, and the type of surgery were not confounding factors as they did not differ

Conclusion

In conclusion, this study demonstrates that tPA administered at different timepoints has different effects in terms of BBB permeability and subsequent risk of hemorrhagic transformation. This may be related to the fact that a BBB already affected by ischemia may be more vulnerable to the effect of tPA, resulting in even higher BBB permeability and a higher risk of hemorrhagic transformation.

Conflict of interest

On behalf of all the authors, the corresponding author states that there is no conflict of interest.

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Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCβ phosphorylation of occludin
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Citation Excerpt :
Furthermore, the high systemic concentrations of recombinant tPA achieved during therapeutic thrombolysis can further increase permeability in an occludin phosphorylation–dependent manner. The extent to which thrombolytic tPA exacerbates barrier permeability and increases the risk of ICH is likely related to its penetration across the BBB, as the effects of thrombolytic tPA on the BBB seem to increase with time after stroke as spontaneous barrier dysfunction is intensifying.49 Importantly, our data show that PKCβ inhibitor treatment given after stroke was effective in preventing delayed tPA-induced ICH, suggesting the possibility of repurposing previously developed PKCβ inhibitors for stroke therapy.
The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cβ (PKCβ) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA^−/− mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCβ-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCβ, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCβ after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCβ phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCβ inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.
Whole body hypothermia extends tissue plasminogen activator treatment window in the rat model of embolic stroke
2020, Life Sciences
Citation Excerpt :
Although in patients with ischemic stroke tPA is the most effective treatment, however its neurotoxic effects have been identified through mechanisms such as proteolytic activity, influence on NMDAR (N-methyl-d-aspartate receptor), targeting plasminogen, affecting extracellular matrix components, inflammatory mediators and different proteases [29]. The effects of tPA timing on BBB leaking and hemorrhagic transforming in transient ischemic stroke model in rats revealed that late tPA administration was related to BBB permeability, mortality, and risk of hemorrhage [30]. The recorded data of rCBF in current study showed that the injection of tPA 5.5 h after induction of stroke enhanced rCBF.
Late treatment with tissue plasminogen activator (tPA) leads to reperfusion injury and poor outcome in ischemic stroke. We have recently shown the beneficial effects of local brain hypothermia after late thrombolysis. Herein, we investigated whether transient whole-body hypothermia was neuroprotective and could prevent the side effects of late tPA therapy at 5.5 h after embolic stroke. After induction of stroke, male rats were randomly assigned into four groups: Control, Hypothermia, tPA and Hypothermia+tPA. Hypothermia started at 5 h after embolic stroke and continued for 1 h. Thirty min after hypothermia, tPA was administrated. Infarct volume, brain edema, blood-brain barrier (BBB) and matrix metalloproteinase-9 (MMP-9) were assessed 48 h and neurological functions were assessed 24 and 48 hour post-stroke. Compared with the control or tPA groups, whole-body hypothermia decreased infarct volume (P < 0.01), BBB disruption (P < 0.05) and MMP-9 level (P < 0.05). However, compared with hypothermia alone a combination of hypothermia and tPA was more effective in reducing infarct volume. While hypothermia alone did not show any effect, its combination with tPA reduced brain edema (P < 0.05). Hypothermia alone or when combined with tPA decreased MMP-9 compared with control or tPA groups (P < 0.01). Although delayed tPA therapy exacerbated BBB integrity, general cooling hampered its leakage after late thrombolysis (P < 0.05). Moreover, only combination therapy significantly improved sensorimotor function as well as forelimb muscle strength at 24 or 48 h after stroke (P < 0.01). Transient whole-body hypothermia in combination with delayed thrombolysis therapy shows more neuroprotection and extends therapeutic time window of tPA up to 5.5 h.
Neutrophils in tPA-induced hemorrhagic transformations: Main culprit, accomplice or innocent bystander?
2019, Pharmacology and Therapeutics
Citation Excerpt :
A dynamic MRI study showed that after ischemia, the vascular dysfunction depended on the timing of tPA administration. Hemorrhages were increasingly numerous as the administration of tPA was delayed, owing to a greater impact on BBB permeability (Zhang et al., 2014). A contributing role for neutrophils was proposed given their unique influence on the neurogliovascular unit during cerebral ischemia, and their ability to interact with tPA (del Zoppo, 2009; Roever & Levine, 2015) (Fig. 1).
The risk of intracerebral hemorrhage still greatly limits the use of tPA in stroke patients. Research is ongoing in order to identify the pathophysiological mechanisms at play, detect predictive biomarkers and discover new pharmacological targets to develop preventive or curative treatments. Going through experimental and clinical studies, this review focuses on the role of neutrophils as key predictive biomarkers for thrombolysis-induced hemorrhages and as pharmacological targets to limit their occurrence. To date, there are no established pharmacological modulators of neutrophils for ischemic stroke and its hemorrhagic complications. Several strategies are under evaluation, including lipid-lowering drugs, free radical scavengers, or minocycline, as well as non-pharmacological interventions such as physical exercise.
Safety of Recanalization Therapy in Patients with Acute Ischemic Stroke Under Anticoagulation: A Systematic Review and Meta-Analysis
2018, Journal of Stroke and Cerebrovascular Diseases
Citation Excerpt :
In addition, several proteolytic enzymes such as matrix metalloproteinase, the trigger for the disruption of blood brain barrier (BBB), upregulated by recombinant tissue plasminogen activator (rt-PA) treatment.28,29 Moreover, during acute cerebral ischemia the disruption of BBB will lead to hemorrhagic transformation.30 However, mechanical thrombectomy pose a great advantage in the upregulation of proteolytic enzymes and direct impairment of hemostasis condition, thus reducing the risk of sICH.
Intravenous thrombolysis treatment (IVT) and endovascular therapy (EVT) have been proved as fist-line beneficial option for eligible patients who have acute ischemic stroke (AIS) with major safety concern of symptomatic intracranial hemorrhage (sICH). Unfortunately, the emergency management of patients with AIS taking vitamin K antagonists and with international normalized ratio higher than 1.7 or taking new oral anticoagulants (NOACs) represents a great challenge. We aim to comprehensively determine the safety of EVT in patients under prior-stroke anticoagulants and IVT in patients under NOAC use.
Clinical researches published in the Embase, PubMed, and Cochrane Library electronic databases up to December 2017 were identified for analysis. Subgroup and sensitivity analyses were also conducted to evaluate the robustness of the conclusions.
Overall, 9 studies involving 3885 patients met the inclusion criteria. The rate of sICH (risk ratio [RR] = .94, 95% CI = .61-1.47, P = .799), mortality (P = .495), and recanalization (P = .655) after EVT did not differ between patients under and those who were not under anticoagulants, although patients under anticoagulants were less likely to achieve good functional outcome (P < .001) than those who were not. Moreover, prior NOAC therapy was not significantly associated with increasing sICH in patients with AIS after IVT (RR = .79, 95% CI = .41-1.53, P = .492).
Patients under anticoagulation appear to be safe after EVT with relatively lower rate of good outcome; furthermore, prior NOAC therapy was not associated with an increasing sICH rate after IVT. This offered a practical information to select appropriate therapeutic strategies for patients under anticoagulation, although the level of evidence seems to be quite shaky.
Safety of Endovascular Intervention for Stroke on Therapeutic Anticoagulation: Multicenter Cohort Study and Meta-Analysis
2017, Journal of Stroke and Cerebrovascular Diseases
Citation Excerpt :
As all of the major randomized trials of tPA for acute ischemic stroke excluded patients on therapeutic anticoagulation, there are few high-quality data on the relative risks and benefits of thrombolytic therapy in patients on anticoagulation. Mechanistically, post-thrombolysis ICH may occur due to 3 major mechanisms: (1) direct impairment of hemostasis due to the thrombolytic and coagulopathic effect of tPA, (2) recanalization of an occluded artery leading to reperfusion injury, and (3) direct disruption of blood–brain barrier integrity by tPA.1 In any scenario, the presence of therapeutic anticoagulation might further increase the risk of intracranial bleeding; however, the degree of increased risk might vary substantially.
Intravenous (IV) tissue plasminogen activator (tPA) is contraindicated in therapeutically anti-coagulated patients. Such patients may be considered for endovascular intervention. However, there are limited data on its safety.
We performed a multicenter retrospective study of patients undergoing endovascular intervention for acute ischemic stroke while on therapeutic anticoagulation. We compared the observed rate of National Institute of Neurological Disorders and Stroke defined symptomatic intracerebral hemorrhage (sICH) with risk-adjusted historical control rates of sICH after IV tPA using weighted averages of the hemorrhage after thrombolysis (HAT) and Multicenter Stroke Survey (MSS) prediction scores. We also performed a metaanalysis of studies assessing risk of sICH with endovascular intervention in patients on anticoagulation.
Of 94 cases, mean age was 73 years and median National Institutes of Health Stroke Scale was 19. Anticoagulation consisted of warfarin (n = 51), dabigatran (n = 6), rivaroxaban (n = 13), apixaban (n = 1), IV heparin (n = 19), low molecular weight heparin (n = 3), and combined warfarin and IV heparin (n = 3). sICH was seen in 7 patients (7%, 95% confidence interval 4-15), all on warfarin. Predicted sICH rates for the cohort based on HAT and MSS scoring were 12% and 7%, respectively. Meta-analysis of 6 studies showed no significant difference in sICH between patients undergoing endovascular intervention on anticoagulation and comparator groups.
Endovascular intervention in subjects on therapeutic anticoagulation appears reasonably safe, with a sICH rate similar to patients not on anticoagulation receiving IV tPA.
Microglia at the blood brain barrier in health and disease
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Effects of tissue plasminogen activator timing on blood–brain barrier permeability and hemorrhagic transformation in rats with transient ischemic stroke

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Study design

Study population

Discussion

Conclusion

Conflict of interest

Brain Res

Thromb Res

Brain Res

Exp Neurol

Curr Opin Pharmacol

Lancet

Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group

N Engl J Med

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood–brain barrier and white matter components after cerebral ischemia

J Neurosci

Relative cerebral blood volume maps corrected for contrast agent extravasation significantly correlate with glioma tumor grade, whereas uncorrected maps do not

AJNR Am J Neuroradiol

Matrix metalloproteinases are associated with increased blood–brain barrier opening in vascular cognitive impairment

Stroke

Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke

J Neurol Neurosurg Psychiatry

Delayed rt-PA treatment in a rat embolic stroke model: diagnosis and prognosis of ischemic injury and hemorrhagic transformation with magnetic resonance imaging

J Cereb Blood Flow Metab

Patlak plots of Gd-DTPA MRI data yield blood–brain transfer constants concordant with those of 14C-sucrose in areas of blood–brain opening

Magn Reson Med

A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke

Stroke

Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS)

JAMA

Thrombolysis in acute ischemic stroke: controlled trials and clinical experience

Neurology

Hemorrhagic transformation and microvascular integrity during focal cerebral ischemia/reperfusion

J Cereb Blood Flow Metab

Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood–brain barrier dysfunction

J Neurochem