- Email: [email protected]
Whole body hypothermia extends tissue plasminogen activator treatment window in the rat model of embolic stroke
Life Sciences 256 (2020) 117450
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Whole body hypothermia extends tissue plasminogen activator treatment window in the rat model of embolic stroke Mahsa Hassanipoura,b, Mohammadreza Zarisfic, Vahid Ehsanib,c, Mohammad Allahtavakolia,b,
T ⁎
a
Physiology-Pharmacology Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Physiology and Pharmacology, Rafsanjan University of Medical Sciences, Rafsanjan, Iran c Student Research Committee, Rafsanjan University of Medical Sciences, Rafsanjan, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hypothermia Cerebral ischemia Tissue plasminogen activator Matrix metalloproteinase-9
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, wholebody 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.
1. Introduction Stroke is one of the main causes of morbidity and serious long-term disability throughout the world [1]. Tissue plasminogen activator (tPA) was approved in 1996 by FDA for treatment of ischemic stroke and can be administered 3–4.5 h after occluding at least one of the brain arteries by a clot [2–4]. When fibrinolytic therapy with drug tPA is given beyond 4.5 h post stroke, some deleterious effects including hemorrhagic transformation and reperfusion injury leads to severe outcomes in stroke patients [5–7]. Some studies have shown the neurotoxic and deleterious effects of delayed thrombolysis [6]. Most of the delayed tPA-induced adverse effects are related to a reperfusion-associated injury, dysfuction of matrix metalloproteinase's (MMP) activity, break of the blood brain barrier (BBB) and damages to microvessels [6,8]. By these limitations, finding new combination approaches for extending the therapeutic time
⁎
window of tPA is of great importance. One neuroprotective strategy against stroke is induction of mild (33 to 36 °C) to moderate (28 to 32 °C) hypothermia [9–12]. We have recently shown that transient locally brain cooling reduces the reperfusion injury of delayed tPA and enhances its therapeutic time window in a rat stroke model [13]. Local hypothermia needs surgical procedure and is hard to translate in stroke patients whereas whole-body hypothermia induction can be easily performed in patients in clinic [10,14]. However, translation of cooling strategy to the clinic for stroke management is still in its early stages. Many obstacles remained, including onset time, duration and optimal depth of hypothermia, speed of rewarming and management of side effects related to therapeutic hypothermia [15]. Hypothermia exerts protection via different molecular pathways such as reducing free radical formation, decreasing cerebral metabolism, suppressing the glutamate release, reducing neuroinflammatory
Corresponding author at: School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. E-mail address: [email protected] (M. Allahtavakoli).
https://doi.org/10.1016/j.lfs.2020.117450 Received 27 October 2019; Received in revised form 13 February 2020; Accepted 19 February 2020 Available online 20 February 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
ventral of neck skin. The 50-μl Hamilton lock syringe was linked to a modified PE-50 tube containing 20 mm clot, after the ligation of the distal portion of the external carotid artery, a catheter was inserted into internal carotid and the tube was pushed 17–19 mm inside until it reached the MCA. The tube was extracted after injection of the embolus. The incision was sutured and rat was turned back to the cage [22]. Maximum duration of the surgery did not reach > 30 min and the surgeon was blinded throughout the operation.
responses and interrupting apoptosis or necrosis [14,16–18]. It has been reported that very mild hypothermia diminishes brain injury and BBB breakdown when followed by tPA therapy after ischemia in mice [19]. Some detrimental effects of tPA beyond 4.5 h is the result of its ability to activate MMP-9 [20,21]. We have previously shown that local hypothermia declined MMP-9 level and this might be a mechanism for the protective effect of hypothermia as MMP-9 increases the BBB permeability and edema in stroke [13]. The aim of this study was therefore to evaluate whether transient and mild whole-body hypothermia could show neuroprotective properties and extend the time window of tPA when applied at 5.5 h after induction of embolic stroke in the male rat. This type of transient and mild whole body hypothermia is more relevant to the clinical application if it would be neuroprotective and widen time window of thrombolysis therapy.
2.4. Conduction of hypothermia After anesthesia, core body temperature maintained constant near 37 °C during the procedure by setting a heating pad (Harvard apparatus, UK) in control and tPA groups. Whole-body hypothermia was induced 5 h after stroke by sprinkling ethanol (95%) on the skin surface under anesthesia. A Small fan was used to accelerate this process. Body temperature fell gradually below 34 °C about 40 min after cooling and then maintained about 33 °C through the whole experiments by sprinkling ethanol periodically. In all groups, actual core body temperature was monitored by a rectal probe [23,24].
2. Material and methods 2.1. Animals and treatments A total number of 32 male Wistar rats weighing 200 to 250 g were maintained on a 12-h light-dark cycle with free access to food and water. Rats were handled in accordance with the Guide for Care and Use of Laboratory Animals. Ethic committee of Rafsanjan University of Medical Sciences, Rafsanjan, Iran, approved all procedures. The registration code in Rafsanjan University of Medical Sciences was 9/20/ 2628. After induction of stroke, animals were randomly divided into 4 experimental groups (N = 8 in each group) as following: Control (saline 0.1 ml/100 g; i.v.), tPA (3 mg/kg; i.v.), hypothermia and a combination of hypothermia+tPA. Hypothermia was induced at 5 h after stroke. Infarct volume, BBB permeability, brain edema and serum level of MMP-9 were determined 48 h after stroke. Behavioral outcomes were evaluated 24 and 48 h post-stroke. Saline or tPA was intravenously injected 5.5 h after stroke. Body temperature was monitored in all animals for 90 min using a rectal thermometer. Regional cerebral blood flow (rCBF) was monitored with Laser-Doppler flowmetry (LDF) for 90 min after the time of hypothermia induction. Three animals that did not exhibit adequate response to thrombolysis were excluded from the study and were replaced. The dose of tPA (Actilyse, BoehringerIngelheim, Germany) was determined according to the previously published papers [13].
2.5. Infarct volume and brain edema evaluation Animals were euthanized at 48 h after stroke injury. For staining the brains, they were gently removed from the skull, then sliced into equal 2-mm-thick coronal sections and stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma) for 30 min at 37 °C. Finally, brain sections were immersed in 10% of formalin overnight. For measurement of total infarct area, the infarct zone of all sections which is calculated by the use of Image J software (NIH Image, version 1.61), were added and multiplied by the thickness (2 mm) of the brain slice. As the brain edema increases the infarct volume, the corrected infarct volume was determined with this formula: corrected infarct area = measured infarct area × (1-[(ipsilateral hemisphere area-contralateral hemisphere area)/contralateral hemisphere]). The formula [1-(DW/WW)] × 100 was used to calculating the percent of water content (%WC) of each hemisphere. In this eq. (WW) is referred to the wet weight of each hemisphere and (DW) is referred to the dry weight of each hemisphere that baked at 100 °C for 48 h and weighted again. By deduction of %WC of the normal hemisphere from the %WC of infarcted hemisphere, the percent of edema in each brain was calculated [13,25].
2.2. Surgical preparation
2.6. Evaluation of BBB integrity
Rats were anesthetized by 1.5% halothane and 79% N2 mixture. Normothermic (37 ± 5 °C) body temperature was preserved during the surgery. In order to prepare the required clots, blood was taken from the right common carotid artery of a donor rat. After artery catheterization, the blood was transferred into a 20 cm of PE-50 tube then it maintained at 37 °C for 2 h and next placed in 4 °C temperature for 22 h before usage. The clot was then transferred into 0.3 mm outer diameter PE-50 tube and was injected into right middle cerebral artery (MCA) as previously reported [22]. The approach for rCBF monitoring was initiated with scalp opening and gentle separation of right temporalis muscle from its origin. Then using a cooled-dental drill thinning of right skull bone was performed at 1.7 mm posterior and 5 mm mediolateral to the bregma and the probe was placed on the skull bone for rCBF measurement. CBF was measured from the onset of hypothermia induction for 90 min.
For determining the permeability of the BBB, 4 ml/kg of 2% Evans blue (EB; Sigma, Germany) solution was injected intravenously via the femoral vein. One hour later rats were anesthetized and warm saline (37 °C) was given through the cardiac ventricle to clear the Evans blue stain from the whole body. Clearance was achieved when the drainage of colorless fluid infusion from the atrium was observed. After sacrificing the animals, brains were removed and divided equally to left and right hemisphere and each one was weighted and preserved in formamide (1 ml/100 mg) at 60 °C for 24 h. The density of dye extracted from each brain was measured by using spectrophotometry (UV7500, Spectro Lab, England) at 620 nm and data was presented as absorbance per gram of tissue [13]. 2.7. Behavioral tests For evaluation of sensorimotor function, the adhesive removal test was used as previously reported [22]. In brief, animals were trained during three days before the stroke and were tested again at 24 and 48 h post-ischemia. The time to remove each sticky tape from forelimb was recorded. The grasping ability or forelimb strength was assessed by hanging wire test. Each rat was suspended from a stretched wire 60 cm above a foam pillow and the duration between suspending and falling
2.3. Induction of embolic stroke model For induction of embolic stroke model, the prepared clot was injected via MCA as previously described. In brief, for exposing the right common carotid artery, internal carotid artery, and external carotid artery, we approach through a 1.5 cm longitudinal incision on the 2
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
tPA injection not only prevented the hyper-perfusion after thrombolysis, but caused a significant reduction in rCBFcompared with control group from about 15 min to 35 min after tPA injection (P < 0.05). While in combined therapy animals blood flow gradually was returned to the level of untreated rats at about 50 min after tPA administration, hypothermia alone preserved it until the end of experiments compared with control or tPA + hypothermia groups (Fig. 2, P < 0.05).
was recorded. 2.8. MMP-9 ELISA assay For evaluation of MMP-9 levels, the enzyme-linked immunosorbent assay (ELISA) method, which utilizes the quantitative sandwich enzyme immunoassay technique was used. After sacrificing rats, blood samples were collected and then maintained in room temperature for 2 h to form a clot and then were centrifuged for 20 min at 1000g. The obtained serum produced by centrifuging, was extracted and assessed immediately or was aliquoted and stored at ≤−20 °C. By adding 180 μl of Calibrator Diluent RD5-10 to 20 μl of sample, the samples were 10fold diluted. The assay was continued by pipetting samples, standards and control into wells, then the immobilized antibody trapped any existing rat MMP-9. Unbound substances were excluded from the wells by washing and enzyme-linked polyclonal antibody specific for rat MMP-9 was added. Mixing the wells with a substrate solution caused enzyme reaction which turned the color to blue. This blue product changed to yellow when the stop solution was added. When more amount of MMP-9 is present and bound by the immobilized antibody, the intensity of the measured color is higher. At last the standard curve was used for reading off the sample values. All these procedures were done according to company instruction of ELISA kit (R&D Systems, Inc.)
3.2. Whole-body hypothermia diminished infarct volume Infarct size of the injured hemisphere in the control, tPA, hypothermia and tPA + hypothermia groups were 34.25 ± 6.27%, 37.9437 ± 5.19%, 14.12 ± 1.6%, and 6.62 ± 0.8%, respectively. Whole body cooling at 5 h after induction of stroke significantly reduced infarct volume about 58% and 62% compared with the control or tPA groups, respectively (P < 0.01). Induction of hypothermia 30 min before the injection of tPA significantly reduced infarct size about 80% and 82% compared with the control or tPA groups, respectively (P < 0.001). Furthermore, combination therapy significantly reduced infarct volume compared with hypothermia alone about 50% (P < 0.05; Fig. 3).
2.9. Statistical analysis
3.3. Whole-body hypothermia hindered BBB disruption and brain edema
Infarct volume, BBB integrity and brain edema data were analyzed using the one-way ANOVA and Tukey's test. The data from temperature, Laser Doppler and behavioral tests were compared by repeated measure ANOVA. Data are presented as mean ± SEM and a value of P < 0.05 was regarded to be significant.
While late tPA therapy caused a severe brain edema, hypothermia combined with tPA at 5 h after stroke significantly reduced brain edema compared with tPA alone (P < 0.05; Fig. 4). Hypothermia alone did not produce any change in brain edema compared with tPA treated or untreated animals. Although administration of tPA alone disturbed BBB integrity, when combined with whole-body hypothermia, it did not worsen BBB disruption (P < 0.05). In addition, compared with the tPA group, induction of whole-body hypothermia 5 h after the embolic stroke reduced the BBB leakage (P < 0.05; Fig. 5).
3. Results 3.1. Hypothermia decreased body temperature and prevented thrombolysisinduced hyperemia
3.4. Whole-body hypothermia decreased blood MMP-9 levels
While body temperature of tPA and control groups were maintained around normal levels, either in hypothermia or tPA + hypothermia groups the rectal temperature was gradually declined from about 25 to 60 min after hypothermia induction (P < 0.01; Fig. 1). As expected, tPA (3 mg/kg) alone was associated with a prompt increase in rCBF or reperfusion from 30 min after drug administration and afterwards (Fig. 2, P < 0.001). However, induction of hypothermia 30 min before
Compared with the tPA group, general hypothermia at 5 h after stroke reduced the level of MMP-9 (P < 0.05). Furthermore, the combination of whole-body hypothermia and tPA reduced levels of MMP-9 compared with control and tPA groups (P < 0.05, P < 0.01 respectively; Fig. 6). Fig. 1. Effects of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on body temperature. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Body temperature was monitored by rectal thermometer. Data are expressed as mean ± S.E.M. *P < 0.5, **P < 0.01 compared with the control and tPA and #P < 0.05 compared with all groups (N = 8).
3
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
Fig. 2. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on MCA territory blood flow of ipsilateral hemisphere after ischemic brain injury. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke, respectively. Blood flow was measured 5 h after embolic stroke and followed for 90 min. Data are presented as mean ± S.E.M. ***P < 0.001 tPA vs. all groups, #P < 0.05 vs. tPA+ hypothermia and control groups.
Fig. 5. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on BBB leakage. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. BBB leakage was measured 48 h after embolic stroke. Data are expressed as mean ± S.E.M. *P < 0.05 vs. tPA group.
Fig. 3. Effects of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on infarct volume. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Infarct volume was measured at 48 h after embolic stroke in TTC-stained brain sections. Data are expressed as mean ± S.E.M. **P < 0.01 and ***P < 0.001 vs. control or tPA groups, #P < 0.05 vs. whole-body hypothermia.
Fig. 6. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on MMP-9 level. tPA and hypothermia were applied 5.5 and 5 h after stroke, respectively. MMP-9 level was measured at 48 h after embolic stroke. Data are represented as mean ± S.E.M. *P < 0.05 and **P < 0.01 vs. control and tPA groups. Fig. 4. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on brain edema. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Brain edema was measured 48 h after embolic stroke. Data are expressed as mean ± S.E.M. *P < 0. 05 vs. tPA group.
grasping ability or forelimb strength of animal 24 h following stroke compared with tPA, control or hypothermia alone treated animals (P < 0.01). Combination of hypothermia and tPA increased forelimb strength of rats 48 h after stroke compared with tPA alone or control rats (P < 0.001). Interestingly, combination therapy was more effective in grasping ability recovery compared with hypothermia alone (P < 0.05; Fig. 7A). Whole-body cooling did not show significant improvement in sensorimotor function at 24 h after stroke. However, when combined with tPA, hypothermia significantly reduced latency to
3.5. Whole-body hypothermia improved behavioral performance in adhesive removal test and hanging wire test Whole-body hypothermia in combination with tPA increased 4
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
patients after recanalization improves neurological performance and lowers the risk of cerebral edema, hemorrhagic transformation [10]. Tang and colleages have also shown that mild hypothermia in a permanent middle cerebral artery occlusion in mice reduced the side effects of tPA such as brain hemorrhage and BBB disruption, suggesting that combination therapy with mild hypothermia and tPA appeared safe [24]. However, to the best of our knowledge, the effect of wholebody cooling on the efficacy of tPA after ischemic stroke has not been reported. The novelty of our current study is that short duration wholebody hypothermia just before thrombolysis may extend time window of tPA and also has the potential to be transferred to patients in clinics safely. It has been investigated that therapeutic hypothermia protects the brain barrier from ischemic damage via attenuation the inflammatory mediators and apoptosis [27]. Another study reported that very mild hypothermia is associated with infarct volume and BBB breakdown reduction following tPA treatment [19]. Several studies investigated the feasibility and safety of combining endovascular hypothermia combined with intravenous thrombolysis after stroke [9,28]. We have recently reported that ischemic postconditioning or mild local brain hypothermia resulted in gradual reperfusion following delayed tPA, reduction of infarct volume and brain edema as well as BBB leakage [13,26]. Hypothermia showed neuroprotective function in multiple experimental models, in combination with tPA or without tPA or in combination with various drugs, at different time points and temperatures after stroke and the important obstacle is the side effects of this method and finding efficacious approaches to encounter the hypothermia adverse effects which is important to be addressed in future studies [10,12,13,24]. In this study, we therefore induced mild and transient hypothermia (about 32–34 °C and only 1 h) in order to limit the side effects of strategy. 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. This sudden enhancement continued for 1 h after thrombolytic therapy. It has been shown that hypothermia shortens the period of reactive hyperemia in the initial ischemia-reperfusion stage [31]. Application of hypothermia reduces the side effects of tPA treatment and reperfusion in stroke [16]. Mild hypothermia-induced neuroprotection against ischemia-reperfusion injury is related to inhibition of elevated dynamin related protein 1 activation and cell apoptosis [32]. Our study revealed that controlling tissue reperfusion with mild hypothermia can decrease deleterious effects of tPA and provides a strategy for extending therapeutic time window of tPA. Many studies demonstrated the consequences of delayed tPA administration in brain edema, BBB disruption, basement membrane and tight junction proteins degradation after stroke [16,24,33]. Delayed use of tPA leads to hyperperfusion and accumulation of free radicals in the brain parenchyma [8]. Our data showed that tPA at 5.5 h after stroke was associated with a higher brain edema and BBB disruption. When hypothermia was used 30 min before the beginning of tPA, it could hamper the brain edema and BBB leakage. In parallel with our results, it has been reported that hypothermia reduces brain hemorrhage, BBB disruption and combination therapy of mild hypothermia and tPA appears to be safe [24]. Matrix metalloproteinases (MMPs) regulate several biologic processes such as inflammation, neurite growth, angiogenesis and bone remodeling [34]. Both the expression and activity of MMPs especially
Fig. 7. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on forelimb strength of animals which was measured by hanging test during 60 s (panel A); and on sensorimotor function which was measured by adhesive removal test during 120 s (panel B). tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Data are presented as mean ± S.E.M. Panel A: **P < 0.01 vs. all groups at 24 h; ***P < 0.001 vs. tPA or control and #P < 0.01 vs. hypothermia at 48 h after stroke. Panel B: **P < 0.01 vs. all groups at 24 h; ***P < 0.001 vs. tPA or control, #P < 0.05 vs. hypothermia and $P < 0.05 vs. control and tPA at 48 h after stroke.
detach sticky labels from the disabled contralateral forepaw at 24 h after stroke (P < 0.01). At 48 h after ischemia, the combination of tPA and body hypothermia (P < 0.001) as well as hypothermia alone (P < 0.05) significantly decreased latency to detach sticky labels from disabled contralateral forepaw (left) compared to tPA or control groups. In addition, the combination of body cooling and tPA treatment was more effective in sensorimotor improvement than cooling alone (P < 0.05; Fig. 7B). 4. Discussion In this study, the effectiveness of whole-body hypothermia alone or in combination with tPA on embolic stroke model in rats was investigated. Application of whole-body cooling at 5 h after stroke and 30 min before thrombolytic therapy led to the reduction of hyperemia, infarct volume, brain edema, BBB disruption, serum level of MMP-9 and the improvement of neurological performance. In our previous studies, we showed that short-term and local brain hypothermia mitigated the reperfusion injury following delayed tPA and also extended its therapeutic time window up to 6 h [13]. We have also shown in another study that brain ischemic postconditioning diminishes the hyperemic response of delayed tPA and extends its therapeutic time window up to 6 h in a rat model of embolic stroke [26]. However, translation of this strategy to bedside requires further efforts and construction of some devices because the application of ischemic conditioning is an invasive method in animals studies. However, in this study, we administered whole-body hypothermia in order to evaluate a possible method that can be applied in clinic. It has been reported that induction of hypothermia in stroke 5
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
MMP-9 increases under different neurologic conditions and CNS disorders such as excitotoxic or neuroinflammatory processes and ischemic stroke [35]. Overexpression of MMP-9 is associated with excitotoxicity and BBB disruption which leads to cerebral edema, neuronal damage, hemorrhagic transformation and apoptosis [20]. It has been indicated that the adverse effects of tPA is mediated via MMPs [36]. Evidence strongly suggests that MMP-9 plays a crucial role in tPAmediated neurotoxic effects [37]. In current study, we observed that administration of tPA at 5.5 h after stroke led to MMP-9 serum level enhancement and this might be a mechanism for the development of BBB leakage and brain edema. The reduction of MMP-9 activity by hypothermia has been reported in both MCAO [18] model and clinical researches [11]. Our results suggest that whole-body mild hypothermia prevents the elevation of MMP-9 following delayed tPA treatment. In an in vitro study, it has been shown that clot lysis by tPA might be affected by temperature and the use of hypothermia as a neuroprotective strategy may negatively impact the therapeutic benefit of thrombolytic agents [38]. However, findings of our current in vivo experiment were different from the above study. In this study, by measuring rCBF from the onset of hypothermia, tPA effectively increased reperfusion at about 30 min after administration.
management of patients with acute ischemic stroke, Stroke 44 (3) (2013) 870–947. [8] M. Allahtavakoli, F. Amin, A. Esmaeeli-Nadimi, A. Shamsizadeh, M. KazemiArababadi, D. Kennedy, Ascorbic acid reduces the adverse effects of delayed administration of tissue plasminogen activator in a rat stroke model, Basic & clinical pharmacology & toxicology 117 (5) (2015) 335–339. [9] T.M. Hemmen, R. Raman, K.Z. Guluma, B.C. Meyer, J.A. Gomes, S. Cruz-Flores, C.A. Wijman, K.S. Rapp, J.C. Grotta, P.D. Lyden, Intravenous thrombolysis plus hypothermia for acute treatment of ischemic stroke (ICTuS-L), Stroke 41 (10) (2010) 2265–2270. [10] J.M. Hong, J.S. Lee, H.-J. Song, H.S. Jeong, H.A. Choi, K. Lee, Therapeutic hypothermia after recanalization in patients with acute ischemic stroke, Stroke 45 (1) (2014) 134–140. [11] S. Horstmann, P. Kalb, J. Koziol, H. Gardner, S. Wagner, Profiles of matrix metalloproteinases, their inhibitors, and laminin in stroke patients, Stroke 34 (9) (2003) 2165–2170. [12] J.-H. Kim, M. Seo, H. Soo Han, J. Park, K. Suk, The neurovascular protection afforded by delayed local hypothermia after transient middle cerebral artery occlusion, Curr. Neurovasc. Res. 10 (2) (2013) 134–143. [13] M. Zarisfi, F. Allahtavakoli, M. Hassanipour, M. Khaksari, H. Rezazadeh, M. Allahtavakoli, M.M. Taghavi, Transient brain hypothermia reduces the reperfusion injury of delayed tissue plasminogen activator and extends its therapeutic time window in a focal embolic stroke model, Brain Res. Bull. 134 (2017) 85–90. [14] T.-C. Wu, J.C. Grotta, Hypothermia for acute ischaemic stroke, The Lancet Neurology 12 (3) (2013) 275–284. [15] F. Frank, G. Broessner, Is there still a role for hypothermia in neurocritical care? Curr. Opin. Crit. Care 23 (2) (2017) 115–121. [16] B. Kallmünzer, S. Schwab, R. Kollmar, Mild hypothermia of 34 C reduces side effects of rt-PA treatment after thromboembolic stroke in rats, Experimental & translational stroke medicine 4 (1) (2012) 3. [17] P.D. Lyden, D. Krieger, M. Yenari, W.D. Dietrich, Therapeutic hypothermia for acute stroke, Int. J. Stroke 1 (1) (2006) 9–19. [18] S. Wagner, S. Nagel, B. Kluge, S. Schwab, S. Heiland, J. Koziol, H. Gardner, W. Hacke, Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia, Brain Res. 984 (1) (2003) 63–75. [19] B.K. Cechmanek, U.I. Tuor, D. Rushforth, P.A. Barber, Very mild hypothermia (35° C) postischemia reduces infarct volume and blood/brain barrier breakdown following tPA treatment in the mouse, Therapeutic Hypothermia and Temperature Management 5 (4) (2015) 203–208. [20] M. Chaturvedi, L. Kaczmarek, Mmp-9 inhibition: a therapeutic strategy in ischemic stroke, Mol. Neurobiol. 49 (1) (2014) 563–573. [21] J.C. Copin, D.J. Bengualid, R.F. Da Silva, O. Kargiotis, K. Schaller, Y. Gasche, Recombinant tissue plasminogen activator induces blood–brain barrier breakdown by a matrix metalloproteinase-9-independent pathway after transient focal cerebral ischemia in mouse, Eur. J. Neurosci. 34 (7) (2011) 1085–1092. [22] M. Allahtavakoli, B. Jarrott, Sigma-1 receptor ligand PRE-084 reduced infarct volume, neurological deficits, pro-inflammatory cytokines and enhanced anti-inflammatory cytokines after embolic stroke in rats, Brain Res. Bull. 85 (3) (2011) 219–224. [23] C.M. Maier, M.L. Cheng, J.E. Lee, M.A. Yenari, G.K. Steinberg, Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia, Stroke 29 (10) (1998) 2171–2180. [24] X.N. Tang, L. Liu, M.A. Koike, M.A. Yenari, Mild hypothermia reduces tissue plasminogen activator-related hemorrhage and blood brain barrier disruption after experimental stroke, Therapeutic hypothermia and temperature management 3 (2) (2013) 74–83. [25] M.A. Yenari, L. Xu, X.N. Tang, Y. Qiao, R.G. Giffard, Microglia potentiate damage to blood–brain barrier constituents, Stroke 37 (4) (2006) 1087–1093. [26] A. Esmaeeli-Nadimi, D. Kennedy, M. Allahtavakoli, Opening the window: ischemic postconditioning reduces the hyperemic response of delayed tissue plasminogen activator and extends its therapeutic time window in an embolic stroke model, Eur. J. Pharmacol. 764 (2015) 55–62. [27] M. Mathur, U. Bayraktutan, Therapeutic hypothermia protects in vitro brain barrier from ischaemic damage through attenuation of inflammatory cytokine release and apoptosis, Stroke Research & Therapy 2 (1) (2017). [28] Z. Han, X. Liu, Y. Luo, X. Ji, Therapeutic hypothermia for stroke: where to go? Exp. Neurol. 272 (2015) 67–77. [29] A. Chevilley, F. Lesept, S. Lenoir, C. Ali, J. Parcq, D. Vivien, Impacts of tissue-type plasminogen activator (tPA) on neuronal survival, Front. Cell. Neurosci. 9 (2015) 415. [30] Y. Zhang, Y. Wang, Z. Zuo, Z. Wang, J. Roy, Q. Hou, E. Tong, A. Hoffmann, E. Sperberg, J. Bredno, Effects of tissue plasminogen activator timing on blood–brain barrier permeability and hemorrhagic transformation in rats with transient ischemic stroke, J. Neurol. Sci. 347 (1) (2014) 148–154. [31] T. Kunimatsu, A. Yamashita, H. Kitahama, T. Misaki, T. Yamamoto, Measurement of cerebral reactive hyperemia at the initial post-ischemia reperfusion stage under normothermia and moderate hypothermia in rats, J. Oral Sci. 51 (4) (2009) 615–621. [32] Y. Tang, X. Liu, J. Zhao, X. Tan, B. Liu, G. Zhang, L. Sun, D. Han, H. Chen, M. Wang, Hypothermia-induced ischemic tolerance is associated with Drp1 inhibition in cerebral ischemia-reperfusion injury of mice, Brain Res. 1646 (2016) 73–83. [33] D.S. Liebeskind, Reperfusion for acute ischemic stroke: arterial revascularization and collateral therapeutics, Curr. Opin. Neurol. 23 (1) (2010) 36–45. [34] H. Nagase, R. Visse, G. Murphy, Structure and function of matrix metalloproteinases and TIMPs, Cardiovasc. Res. 69 (3) (2006) 562–573. [35] V.W. Yong, P.A. Forsyth, R. Bell, C.A. Krekoski, D.R. Edwards, Matrix metalloproteinases and diseases of the CNS, Trends Neurosci. 21 (2) (1998) 75–80.
5. Conclusion The results of our study showed that using whole-body mild hypothermia for only 1 h prevents the destructive effects of delayed tPA therapy and improves neurological function. Additive effect of the combination of hypothermia and thrombolytic therapy was observed when tPA was used with a delay of 5.5 h after embolic stroke. Wholebody hypothermia inhibited BBB disruption, brain edema and reduced infarct volume as well as MMP-9 when evaluated 48 h after cerebral ischemia. In addition, hyperemic response due to the delayed use of tPA was significantly attenuated when combined with mild hypothermia. Further studies are required to address the effect of such strategy on tPA administration in patients. Declaration of competing interest The authors declare that there is no conflict of interest associated with this work. Acknowledgements This paper is derived from Mr. Vahid Ehsani's M.Sc. thesis and was supported by grant no. 9/20/2628 from the Vice Chancellor for Research and Technology, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. References [1] A. Durukan, T. Tatlisumak, Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia, Pharmacol. Biochem. Behav. 87 (1) (2007) 179–197. [2] K.M. Chapman, A.R. Woolfenden, D. Graeb, D.C. Johnston, J. Beckman, M. Schulzer, P.A. Teal, Intravenous tissue plasminogen activator for acute ischemic stroke, Stroke 31 (12) (2000) 2920–2924. [3] P. Gurman, O. Miranda, A. Nathan, C. Washington, Y. Rosen, N. Elman, Recombinant tissue plasminogen activators (rtPA): a review, Clin. Pharmacol. Ther. 97 (3) (2015) 274–285. [4] A. Qureshi, R. Pande, S. Kim, R. Hanel, J. Kirmani, A. Yahia, Third generation thrombolytics for the treatment of ischemic stroke, Current opinion in investigational drugs (London, England: 2000) 3(12) (2002) 1729-1732. [5] C.R. Carpenter, S.M. Keim, W.K. Milne, W.J. Meurer, W.G. Barsan, B.E.i.E.M.I. Group, Thrombolytic therapy for acute ischemic stroke beyond three hours, The Journal of emergency medicine 40(1) (2011) 82–92. [6] I. dela Peña, C. Borlongan, G. Shen, W. Davis, Strategies to extend thrombolytic time window for ischemic stroke treatment: an unmet clinical need, Journal of stroke 19(1) (2017) 50. [7] E.C. Jauch, J.L. Saver, H.P. Adams, A. Bruno, B.M. Demaerschalk, P. Khatri, P.W. McMullan, A.I. Qureshi, K. Rosenfield, P.A. Scott, Guidelines for the early
6
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
tissue-type plasminogen activator, Neurobiol. Dis. 38 (3) (2010) 376–385. [38] J.M. Meunier, W.-T.W. Chang, B. Bluett, E. Wenker, C.J. Lindsell, G.J. Shaw, Temperature affects thrombolytic efficacy using rt-PA and eptifibatide, an in vitro study, Therapeutic hypothermia and temperature management 2 (3) (2012) 112–118.
[36] S.E. Lakhan, A. Kirchgessner, D. Tepper, A. Leonard, Matrix Metalloproteinases and Blood-brain Barrier Disruption in Acute Ischemic Stroke, Frontiers in Neurology 4, (2013). [37] R. Jin, G. Yang, G. Li, Molecular insights and therapeutic targets for blood–brain barrier disruption in ischemic stroke: critical role of matrix metalloproteinases and
7
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Whole body hypothermia extends tissue plasminogen activator treatment window in the rat model of embolic stroke Mahsa Hassanipoura,b, Mohammadreza Zarisfic, Vahid Ehsanib,c, Mohammad Allahtavakolia,b,
T ⁎
a
Physiology-Pharmacology Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Physiology and Pharmacology, Rafsanjan University of Medical Sciences, Rafsanjan, Iran c Student Research Committee, Rafsanjan University of Medical Sciences, Rafsanjan, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hypothermia Cerebral ischemia Tissue plasminogen activator Matrix metalloproteinase-9
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, wholebody 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.
1. Introduction Stroke is one of the main causes of morbidity and serious long-term disability throughout the world [1]. Tissue plasminogen activator (tPA) was approved in 1996 by FDA for treatment of ischemic stroke and can be administered 3–4.5 h after occluding at least one of the brain arteries by a clot [2–4]. When fibrinolytic therapy with drug tPA is given beyond 4.5 h post stroke, some deleterious effects including hemorrhagic transformation and reperfusion injury leads to severe outcomes in stroke patients [5–7]. Some studies have shown the neurotoxic and deleterious effects of delayed thrombolysis [6]. Most of the delayed tPA-induced adverse effects are related to a reperfusion-associated injury, dysfuction of matrix metalloproteinase's (MMP) activity, break of the blood brain barrier (BBB) and damages to microvessels [6,8]. By these limitations, finding new combination approaches for extending the therapeutic time
⁎
window of tPA is of great importance. One neuroprotective strategy against stroke is induction of mild (33 to 36 °C) to moderate (28 to 32 °C) hypothermia [9–12]. We have recently shown that transient locally brain cooling reduces the reperfusion injury of delayed tPA and enhances its therapeutic time window in a rat stroke model [13]. Local hypothermia needs surgical procedure and is hard to translate in stroke patients whereas whole-body hypothermia induction can be easily performed in patients in clinic [10,14]. However, translation of cooling strategy to the clinic for stroke management is still in its early stages. Many obstacles remained, including onset time, duration and optimal depth of hypothermia, speed of rewarming and management of side effects related to therapeutic hypothermia [15]. Hypothermia exerts protection via different molecular pathways such as reducing free radical formation, decreasing cerebral metabolism, suppressing the glutamate release, reducing neuroinflammatory
Corresponding author at: School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. E-mail address: [email protected] (M. Allahtavakoli).
https://doi.org/10.1016/j.lfs.2020.117450 Received 27 October 2019; Received in revised form 13 February 2020; Accepted 19 February 2020 Available online 20 February 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
ventral of neck skin. The 50-μl Hamilton lock syringe was linked to a modified PE-50 tube containing 20 mm clot, after the ligation of the distal portion of the external carotid artery, a catheter was inserted into internal carotid and the tube was pushed 17–19 mm inside until it reached the MCA. The tube was extracted after injection of the embolus. The incision was sutured and rat was turned back to the cage [22]. Maximum duration of the surgery did not reach > 30 min and the surgeon was blinded throughout the operation.
responses and interrupting apoptosis or necrosis [14,16–18]. It has been reported that very mild hypothermia diminishes brain injury and BBB breakdown when followed by tPA therapy after ischemia in mice [19]. Some detrimental effects of tPA beyond 4.5 h is the result of its ability to activate MMP-9 [20,21]. We have previously shown that local hypothermia declined MMP-9 level and this might be a mechanism for the protective effect of hypothermia as MMP-9 increases the BBB permeability and edema in stroke [13]. The aim of this study was therefore to evaluate whether transient and mild whole-body hypothermia could show neuroprotective properties and extend the time window of tPA when applied at 5.5 h after induction of embolic stroke in the male rat. This type of transient and mild whole body hypothermia is more relevant to the clinical application if it would be neuroprotective and widen time window of thrombolysis therapy.
2.4. Conduction of hypothermia After anesthesia, core body temperature maintained constant near 37 °C during the procedure by setting a heating pad (Harvard apparatus, UK) in control and tPA groups. Whole-body hypothermia was induced 5 h after stroke by sprinkling ethanol (95%) on the skin surface under anesthesia. A Small fan was used to accelerate this process. Body temperature fell gradually below 34 °C about 40 min after cooling and then maintained about 33 °C through the whole experiments by sprinkling ethanol periodically. In all groups, actual core body temperature was monitored by a rectal probe [23,24].
2. Material and methods 2.1. Animals and treatments A total number of 32 male Wistar rats weighing 200 to 250 g were maintained on a 12-h light-dark cycle with free access to food and water. Rats were handled in accordance with the Guide for Care and Use of Laboratory Animals. Ethic committee of Rafsanjan University of Medical Sciences, Rafsanjan, Iran, approved all procedures. The registration code in Rafsanjan University of Medical Sciences was 9/20/ 2628. After induction of stroke, animals were randomly divided into 4 experimental groups (N = 8 in each group) as following: Control (saline 0.1 ml/100 g; i.v.), tPA (3 mg/kg; i.v.), hypothermia and a combination of hypothermia+tPA. Hypothermia was induced at 5 h after stroke. Infarct volume, BBB permeability, brain edema and serum level of MMP-9 were determined 48 h after stroke. Behavioral outcomes were evaluated 24 and 48 h post-stroke. Saline or tPA was intravenously injected 5.5 h after stroke. Body temperature was monitored in all animals for 90 min using a rectal thermometer. Regional cerebral blood flow (rCBF) was monitored with Laser-Doppler flowmetry (LDF) for 90 min after the time of hypothermia induction. Three animals that did not exhibit adequate response to thrombolysis were excluded from the study and were replaced. The dose of tPA (Actilyse, BoehringerIngelheim, Germany) was determined according to the previously published papers [13].
2.5. Infarct volume and brain edema evaluation Animals were euthanized at 48 h after stroke injury. For staining the brains, they were gently removed from the skull, then sliced into equal 2-mm-thick coronal sections and stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma) for 30 min at 37 °C. Finally, brain sections were immersed in 10% of formalin overnight. For measurement of total infarct area, the infarct zone of all sections which is calculated by the use of Image J software (NIH Image, version 1.61), were added and multiplied by the thickness (2 mm) of the brain slice. As the brain edema increases the infarct volume, the corrected infarct volume was determined with this formula: corrected infarct area = measured infarct area × (1-[(ipsilateral hemisphere area-contralateral hemisphere area)/contralateral hemisphere]). The formula [1-(DW/WW)] × 100 was used to calculating the percent of water content (%WC) of each hemisphere. In this eq. (WW) is referred to the wet weight of each hemisphere and (DW) is referred to the dry weight of each hemisphere that baked at 100 °C for 48 h and weighted again. By deduction of %WC of the normal hemisphere from the %WC of infarcted hemisphere, the percent of edema in each brain was calculated [13,25].
2.2. Surgical preparation
2.6. Evaluation of BBB integrity
Rats were anesthetized by 1.5% halothane and 79% N2 mixture. Normothermic (37 ± 5 °C) body temperature was preserved during the surgery. In order to prepare the required clots, blood was taken from the right common carotid artery of a donor rat. After artery catheterization, the blood was transferred into a 20 cm of PE-50 tube then it maintained at 37 °C for 2 h and next placed in 4 °C temperature for 22 h before usage. The clot was then transferred into 0.3 mm outer diameter PE-50 tube and was injected into right middle cerebral artery (MCA) as previously reported [22]. The approach for rCBF monitoring was initiated with scalp opening and gentle separation of right temporalis muscle from its origin. Then using a cooled-dental drill thinning of right skull bone was performed at 1.7 mm posterior and 5 mm mediolateral to the bregma and the probe was placed on the skull bone for rCBF measurement. CBF was measured from the onset of hypothermia induction for 90 min.
For determining the permeability of the BBB, 4 ml/kg of 2% Evans blue (EB; Sigma, Germany) solution was injected intravenously via the femoral vein. One hour later rats were anesthetized and warm saline (37 °C) was given through the cardiac ventricle to clear the Evans blue stain from the whole body. Clearance was achieved when the drainage of colorless fluid infusion from the atrium was observed. After sacrificing the animals, brains were removed and divided equally to left and right hemisphere and each one was weighted and preserved in formamide (1 ml/100 mg) at 60 °C for 24 h. The density of dye extracted from each brain was measured by using spectrophotometry (UV7500, Spectro Lab, England) at 620 nm and data was presented as absorbance per gram of tissue [13]. 2.7. Behavioral tests For evaluation of sensorimotor function, the adhesive removal test was used as previously reported [22]. In brief, animals were trained during three days before the stroke and were tested again at 24 and 48 h post-ischemia. The time to remove each sticky tape from forelimb was recorded. The grasping ability or forelimb strength was assessed by hanging wire test. Each rat was suspended from a stretched wire 60 cm above a foam pillow and the duration between suspending and falling
2.3. Induction of embolic stroke model For induction of embolic stroke model, the prepared clot was injected via MCA as previously described. In brief, for exposing the right common carotid artery, internal carotid artery, and external carotid artery, we approach through a 1.5 cm longitudinal incision on the 2
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
tPA injection not only prevented the hyper-perfusion after thrombolysis, but caused a significant reduction in rCBFcompared with control group from about 15 min to 35 min after tPA injection (P < 0.05). While in combined therapy animals blood flow gradually was returned to the level of untreated rats at about 50 min after tPA administration, hypothermia alone preserved it until the end of experiments compared with control or tPA + hypothermia groups (Fig. 2, P < 0.05).
was recorded. 2.8. MMP-9 ELISA assay For evaluation of MMP-9 levels, the enzyme-linked immunosorbent assay (ELISA) method, which utilizes the quantitative sandwich enzyme immunoassay technique was used. After sacrificing rats, blood samples were collected and then maintained in room temperature for 2 h to form a clot and then were centrifuged for 20 min at 1000g. The obtained serum produced by centrifuging, was extracted and assessed immediately or was aliquoted and stored at ≤−20 °C. By adding 180 μl of Calibrator Diluent RD5-10 to 20 μl of sample, the samples were 10fold diluted. The assay was continued by pipetting samples, standards and control into wells, then the immobilized antibody trapped any existing rat MMP-9. Unbound substances were excluded from the wells by washing and enzyme-linked polyclonal antibody specific for rat MMP-9 was added. Mixing the wells with a substrate solution caused enzyme reaction which turned the color to blue. This blue product changed to yellow when the stop solution was added. When more amount of MMP-9 is present and bound by the immobilized antibody, the intensity of the measured color is higher. At last the standard curve was used for reading off the sample values. All these procedures were done according to company instruction of ELISA kit (R&D Systems, Inc.)
3.2. Whole-body hypothermia diminished infarct volume Infarct size of the injured hemisphere in the control, tPA, hypothermia and tPA + hypothermia groups were 34.25 ± 6.27%, 37.9437 ± 5.19%, 14.12 ± 1.6%, and 6.62 ± 0.8%, respectively. Whole body cooling at 5 h after induction of stroke significantly reduced infarct volume about 58% and 62% compared with the control or tPA groups, respectively (P < 0.01). Induction of hypothermia 30 min before the injection of tPA significantly reduced infarct size about 80% and 82% compared with the control or tPA groups, respectively (P < 0.001). Furthermore, combination therapy significantly reduced infarct volume compared with hypothermia alone about 50% (P < 0.05; Fig. 3).
2.9. Statistical analysis
3.3. Whole-body hypothermia hindered BBB disruption and brain edema
Infarct volume, BBB integrity and brain edema data were analyzed using the one-way ANOVA and Tukey's test. The data from temperature, Laser Doppler and behavioral tests were compared by repeated measure ANOVA. Data are presented as mean ± SEM and a value of P < 0.05 was regarded to be significant.
While late tPA therapy caused a severe brain edema, hypothermia combined with tPA at 5 h after stroke significantly reduced brain edema compared with tPA alone (P < 0.05; Fig. 4). Hypothermia alone did not produce any change in brain edema compared with tPA treated or untreated animals. Although administration of tPA alone disturbed BBB integrity, when combined with whole-body hypothermia, it did not worsen BBB disruption (P < 0.05). In addition, compared with the tPA group, induction of whole-body hypothermia 5 h after the embolic stroke reduced the BBB leakage (P < 0.05; Fig. 5).
3. Results 3.1. Hypothermia decreased body temperature and prevented thrombolysisinduced hyperemia
3.4. Whole-body hypothermia decreased blood MMP-9 levels
While body temperature of tPA and control groups were maintained around normal levels, either in hypothermia or tPA + hypothermia groups the rectal temperature was gradually declined from about 25 to 60 min after hypothermia induction (P < 0.01; Fig. 1). As expected, tPA (3 mg/kg) alone was associated with a prompt increase in rCBF or reperfusion from 30 min after drug administration and afterwards (Fig. 2, P < 0.001). However, induction of hypothermia 30 min before
Compared with the tPA group, general hypothermia at 5 h after stroke reduced the level of MMP-9 (P < 0.05). Furthermore, the combination of whole-body hypothermia and tPA reduced levels of MMP-9 compared with control and tPA groups (P < 0.05, P < 0.01 respectively; Fig. 6). Fig. 1. Effects of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on body temperature. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Body temperature was monitored by rectal thermometer. Data are expressed as mean ± S.E.M. *P < 0.5, **P < 0.01 compared with the control and tPA and #P < 0.05 compared with all groups (N = 8).
3
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
Fig. 2. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on MCA territory blood flow of ipsilateral hemisphere after ischemic brain injury. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke, respectively. Blood flow was measured 5 h after embolic stroke and followed for 90 min. Data are presented as mean ± S.E.M. ***P < 0.001 tPA vs. all groups, #P < 0.05 vs. tPA+ hypothermia and control groups.
Fig. 5. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on BBB leakage. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. BBB leakage was measured 48 h after embolic stroke. Data are expressed as mean ± S.E.M. *P < 0.05 vs. tPA group.
Fig. 3. Effects of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on infarct volume. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Infarct volume was measured at 48 h after embolic stroke in TTC-stained brain sections. Data are expressed as mean ± S.E.M. **P < 0.01 and ***P < 0.001 vs. control or tPA groups, #P < 0.05 vs. whole-body hypothermia.
Fig. 6. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on MMP-9 level. tPA and hypothermia were applied 5.5 and 5 h after stroke, respectively. MMP-9 level was measured at 48 h after embolic stroke. Data are represented as mean ± S.E.M. *P < 0.05 and **P < 0.01 vs. control and tPA groups. Fig. 4. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on brain edema. tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Brain edema was measured 48 h after embolic stroke. Data are expressed as mean ± S.E.M. *P < 0. 05 vs. tPA group.
grasping ability or forelimb strength of animal 24 h following stroke compared with tPA, control or hypothermia alone treated animals (P < 0.01). Combination of hypothermia and tPA increased forelimb strength of rats 48 h after stroke compared with tPA alone or control rats (P < 0.001). Interestingly, combination therapy was more effective in grasping ability recovery compared with hypothermia alone (P < 0.05; Fig. 7A). Whole-body cooling did not show significant improvement in sensorimotor function at 24 h after stroke. However, when combined with tPA, hypothermia significantly reduced latency to
3.5. Whole-body hypothermia improved behavioral performance in adhesive removal test and hanging wire test Whole-body hypothermia in combination with tPA increased 4
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
patients after recanalization improves neurological performance and lowers the risk of cerebral edema, hemorrhagic transformation [10]. Tang and colleages have also shown that mild hypothermia in a permanent middle cerebral artery occlusion in mice reduced the side effects of tPA such as brain hemorrhage and BBB disruption, suggesting that combination therapy with mild hypothermia and tPA appeared safe [24]. However, to the best of our knowledge, the effect of wholebody cooling on the efficacy of tPA after ischemic stroke has not been reported. The novelty of our current study is that short duration wholebody hypothermia just before thrombolysis may extend time window of tPA and also has the potential to be transferred to patients in clinics safely. It has been investigated that therapeutic hypothermia protects the brain barrier from ischemic damage via attenuation the inflammatory mediators and apoptosis [27]. Another study reported that very mild hypothermia is associated with infarct volume and BBB breakdown reduction following tPA treatment [19]. Several studies investigated the feasibility and safety of combining endovascular hypothermia combined with intravenous thrombolysis after stroke [9,28]. We have recently reported that ischemic postconditioning or mild local brain hypothermia resulted in gradual reperfusion following delayed tPA, reduction of infarct volume and brain edema as well as BBB leakage [13,26]. Hypothermia showed neuroprotective function in multiple experimental models, in combination with tPA or without tPA or in combination with various drugs, at different time points and temperatures after stroke and the important obstacle is the side effects of this method and finding efficacious approaches to encounter the hypothermia adverse effects which is important to be addressed in future studies [10,12,13,24]. In this study, we therefore induced mild and transient hypothermia (about 32–34 °C and only 1 h) in order to limit the side effects of strategy. 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. This sudden enhancement continued for 1 h after thrombolytic therapy. It has been shown that hypothermia shortens the period of reactive hyperemia in the initial ischemia-reperfusion stage [31]. Application of hypothermia reduces the side effects of tPA treatment and reperfusion in stroke [16]. Mild hypothermia-induced neuroprotection against ischemia-reperfusion injury is related to inhibition of elevated dynamin related protein 1 activation and cell apoptosis [32]. Our study revealed that controlling tissue reperfusion with mild hypothermia can decrease deleterious effects of tPA and provides a strategy for extending therapeutic time window of tPA. Many studies demonstrated the consequences of delayed tPA administration in brain edema, BBB disruption, basement membrane and tight junction proteins degradation after stroke [16,24,33]. Delayed use of tPA leads to hyperperfusion and accumulation of free radicals in the brain parenchyma [8]. Our data showed that tPA at 5.5 h after stroke was associated with a higher brain edema and BBB disruption. When hypothermia was used 30 min before the beginning of tPA, it could hamper the brain edema and BBB leakage. In parallel with our results, it has been reported that hypothermia reduces brain hemorrhage, BBB disruption and combination therapy of mild hypothermia and tPA appears to be safe [24]. Matrix metalloproteinases (MMPs) regulate several biologic processes such as inflammation, neurite growth, angiogenesis and bone remodeling [34]. Both the expression and activity of MMPs especially
Fig. 7. Effect of tPA (3 mg/kg i.v.) and whole-body hypothermia alone or in combination on forelimb strength of animals which was measured by hanging test during 60 s (panel A); and on sensorimotor function which was measured by adhesive removal test during 120 s (panel B). tPA administration and hypothermia induction were performed 5.5 and 5 h after stroke respectively. Data are presented as mean ± S.E.M. Panel A: **P < 0.01 vs. all groups at 24 h; ***P < 0.001 vs. tPA or control and #P < 0.01 vs. hypothermia at 48 h after stroke. Panel B: **P < 0.01 vs. all groups at 24 h; ***P < 0.001 vs. tPA or control, #P < 0.05 vs. hypothermia and $P < 0.05 vs. control and tPA at 48 h after stroke.
detach sticky labels from the disabled contralateral forepaw at 24 h after stroke (P < 0.01). At 48 h after ischemia, the combination of tPA and body hypothermia (P < 0.001) as well as hypothermia alone (P < 0.05) significantly decreased latency to detach sticky labels from disabled contralateral forepaw (left) compared to tPA or control groups. In addition, the combination of body cooling and tPA treatment was more effective in sensorimotor improvement than cooling alone (P < 0.05; Fig. 7B). 4. Discussion In this study, the effectiveness of whole-body hypothermia alone or in combination with tPA on embolic stroke model in rats was investigated. Application of whole-body cooling at 5 h after stroke and 30 min before thrombolytic therapy led to the reduction of hyperemia, infarct volume, brain edema, BBB disruption, serum level of MMP-9 and the improvement of neurological performance. In our previous studies, we showed that short-term and local brain hypothermia mitigated the reperfusion injury following delayed tPA and also extended its therapeutic time window up to 6 h [13]. We have also shown in another study that brain ischemic postconditioning diminishes the hyperemic response of delayed tPA and extends its therapeutic time window up to 6 h in a rat model of embolic stroke [26]. However, translation of this strategy to bedside requires further efforts and construction of some devices because the application of ischemic conditioning is an invasive method in animals studies. However, in this study, we administered whole-body hypothermia in order to evaluate a possible method that can be applied in clinic. It has been reported that induction of hypothermia in stroke 5
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
MMP-9 increases under different neurologic conditions and CNS disorders such as excitotoxic or neuroinflammatory processes and ischemic stroke [35]. Overexpression of MMP-9 is associated with excitotoxicity and BBB disruption which leads to cerebral edema, neuronal damage, hemorrhagic transformation and apoptosis [20]. It has been indicated that the adverse effects of tPA is mediated via MMPs [36]. Evidence strongly suggests that MMP-9 plays a crucial role in tPAmediated neurotoxic effects [37]. In current study, we observed that administration of tPA at 5.5 h after stroke led to MMP-9 serum level enhancement and this might be a mechanism for the development of BBB leakage and brain edema. The reduction of MMP-9 activity by hypothermia has been reported in both MCAO [18] model and clinical researches [11]. Our results suggest that whole-body mild hypothermia prevents the elevation of MMP-9 following delayed tPA treatment. In an in vitro study, it has been shown that clot lysis by tPA might be affected by temperature and the use of hypothermia as a neuroprotective strategy may negatively impact the therapeutic benefit of thrombolytic agents [38]. However, findings of our current in vivo experiment were different from the above study. In this study, by measuring rCBF from the onset of hypothermia, tPA effectively increased reperfusion at about 30 min after administration.
management of patients with acute ischemic stroke, Stroke 44 (3) (2013) 870–947. [8] M. Allahtavakoli, F. Amin, A. Esmaeeli-Nadimi, A. Shamsizadeh, M. KazemiArababadi, D. Kennedy, Ascorbic acid reduces the adverse effects of delayed administration of tissue plasminogen activator in a rat stroke model, Basic & clinical pharmacology & toxicology 117 (5) (2015) 335–339. [9] T.M. Hemmen, R. Raman, K.Z. Guluma, B.C. Meyer, J.A. Gomes, S. Cruz-Flores, C.A. Wijman, K.S. Rapp, J.C. Grotta, P.D. Lyden, Intravenous thrombolysis plus hypothermia for acute treatment of ischemic stroke (ICTuS-L), Stroke 41 (10) (2010) 2265–2270. [10] J.M. Hong, J.S. Lee, H.-J. Song, H.S. Jeong, H.A. Choi, K. Lee, Therapeutic hypothermia after recanalization in patients with acute ischemic stroke, Stroke 45 (1) (2014) 134–140. [11] S. Horstmann, P. Kalb, J. Koziol, H. Gardner, S. Wagner, Profiles of matrix metalloproteinases, their inhibitors, and laminin in stroke patients, Stroke 34 (9) (2003) 2165–2170. [12] J.-H. Kim, M. Seo, H. Soo Han, J. Park, K. Suk, The neurovascular protection afforded by delayed local hypothermia after transient middle cerebral artery occlusion, Curr. Neurovasc. Res. 10 (2) (2013) 134–143. [13] M. Zarisfi, F. Allahtavakoli, M. Hassanipour, M. Khaksari, H. Rezazadeh, M. Allahtavakoli, M.M. Taghavi, Transient brain hypothermia reduces the reperfusion injury of delayed tissue plasminogen activator and extends its therapeutic time window in a focal embolic stroke model, Brain Res. Bull. 134 (2017) 85–90. [14] T.-C. Wu, J.C. Grotta, Hypothermia for acute ischaemic stroke, The Lancet Neurology 12 (3) (2013) 275–284. [15] F. Frank, G. Broessner, Is there still a role for hypothermia in neurocritical care? Curr. Opin. Crit. Care 23 (2) (2017) 115–121. [16] B. Kallmünzer, S. Schwab, R. Kollmar, Mild hypothermia of 34 C reduces side effects of rt-PA treatment after thromboembolic stroke in rats, Experimental & translational stroke medicine 4 (1) (2012) 3. [17] P.D. Lyden, D. Krieger, M. Yenari, W.D. Dietrich, Therapeutic hypothermia for acute stroke, Int. J. Stroke 1 (1) (2006) 9–19. [18] S. Wagner, S. Nagel, B. Kluge, S. Schwab, S. Heiland, J. Koziol, H. Gardner, W. Hacke, Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia, Brain Res. 984 (1) (2003) 63–75. [19] B.K. Cechmanek, U.I. Tuor, D. Rushforth, P.A. Barber, Very mild hypothermia (35° C) postischemia reduces infarct volume and blood/brain barrier breakdown following tPA treatment in the mouse, Therapeutic Hypothermia and Temperature Management 5 (4) (2015) 203–208. [20] M. Chaturvedi, L. Kaczmarek, Mmp-9 inhibition: a therapeutic strategy in ischemic stroke, Mol. Neurobiol. 49 (1) (2014) 563–573. [21] J.C. Copin, D.J. Bengualid, R.F. Da Silva, O. Kargiotis, K. Schaller, Y. Gasche, Recombinant tissue plasminogen activator induces blood–brain barrier breakdown by a matrix metalloproteinase-9-independent pathway after transient focal cerebral ischemia in mouse, Eur. J. Neurosci. 34 (7) (2011) 1085–1092. [22] M. Allahtavakoli, B. Jarrott, Sigma-1 receptor ligand PRE-084 reduced infarct volume, neurological deficits, pro-inflammatory cytokines and enhanced anti-inflammatory cytokines after embolic stroke in rats, Brain Res. Bull. 85 (3) (2011) 219–224. [23] C.M. Maier, M.L. Cheng, J.E. Lee, M.A. Yenari, G.K. Steinberg, Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia, Stroke 29 (10) (1998) 2171–2180. [24] X.N. Tang, L. Liu, M.A. Koike, M.A. Yenari, Mild hypothermia reduces tissue plasminogen activator-related hemorrhage and blood brain barrier disruption after experimental stroke, Therapeutic hypothermia and temperature management 3 (2) (2013) 74–83. [25] M.A. Yenari, L. Xu, X.N. Tang, Y. Qiao, R.G. Giffard, Microglia potentiate damage to blood–brain barrier constituents, Stroke 37 (4) (2006) 1087–1093. [26] A. Esmaeeli-Nadimi, D. Kennedy, M. Allahtavakoli, Opening the window: ischemic postconditioning reduces the hyperemic response of delayed tissue plasminogen activator and extends its therapeutic time window in an embolic stroke model, Eur. J. Pharmacol. 764 (2015) 55–62. [27] M. Mathur, U. Bayraktutan, Therapeutic hypothermia protects in vitro brain barrier from ischaemic damage through attenuation of inflammatory cytokine release and apoptosis, Stroke Research & Therapy 2 (1) (2017). [28] Z. Han, X. Liu, Y. Luo, X. Ji, Therapeutic hypothermia for stroke: where to go? Exp. Neurol. 272 (2015) 67–77. [29] A. Chevilley, F. Lesept, S. Lenoir, C. Ali, J. Parcq, D. Vivien, Impacts of tissue-type plasminogen activator (tPA) on neuronal survival, Front. Cell. Neurosci. 9 (2015) 415. [30] Y. Zhang, Y. Wang, Z. Zuo, Z. Wang, J. Roy, Q. Hou, E. Tong, A. Hoffmann, E. Sperberg, J. Bredno, Effects of tissue plasminogen activator timing on blood–brain barrier permeability and hemorrhagic transformation in rats with transient ischemic stroke, J. Neurol. Sci. 347 (1) (2014) 148–154. [31] T. Kunimatsu, A. Yamashita, H. Kitahama, T. Misaki, T. Yamamoto, Measurement of cerebral reactive hyperemia at the initial post-ischemia reperfusion stage under normothermia and moderate hypothermia in rats, J. Oral Sci. 51 (4) (2009) 615–621. [32] Y. Tang, X. Liu, J. Zhao, X. Tan, B. Liu, G. Zhang, L. Sun, D. Han, H. Chen, M. Wang, Hypothermia-induced ischemic tolerance is associated with Drp1 inhibition in cerebral ischemia-reperfusion injury of mice, Brain Res. 1646 (2016) 73–83. [33] D.S. Liebeskind, Reperfusion for acute ischemic stroke: arterial revascularization and collateral therapeutics, Curr. Opin. Neurol. 23 (1) (2010) 36–45. [34] H. Nagase, R. Visse, G. Murphy, Structure and function of matrix metalloproteinases and TIMPs, Cardiovasc. Res. 69 (3) (2006) 562–573. [35] V.W. Yong, P.A. Forsyth, R. Bell, C.A. Krekoski, D.R. Edwards, Matrix metalloproteinases and diseases of the CNS, Trends Neurosci. 21 (2) (1998) 75–80.
5. Conclusion The results of our study showed that using whole-body mild hypothermia for only 1 h prevents the destructive effects of delayed tPA therapy and improves neurological function. Additive effect of the combination of hypothermia and thrombolytic therapy was observed when tPA was used with a delay of 5.5 h after embolic stroke. Wholebody hypothermia inhibited BBB disruption, brain edema and reduced infarct volume as well as MMP-9 when evaluated 48 h after cerebral ischemia. In addition, hyperemic response due to the delayed use of tPA was significantly attenuated when combined with mild hypothermia. Further studies are required to address the effect of such strategy on tPA administration in patients. Declaration of competing interest The authors declare that there is no conflict of interest associated with this work. Acknowledgements This paper is derived from Mr. Vahid Ehsani's M.Sc. thesis and was supported by grant no. 9/20/2628 from the Vice Chancellor for Research and Technology, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. References [1] A. Durukan, T. Tatlisumak, Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia, Pharmacol. Biochem. Behav. 87 (1) (2007) 179–197. [2] K.M. Chapman, A.R. Woolfenden, D. Graeb, D.C. Johnston, J. Beckman, M. Schulzer, P.A. Teal, Intravenous tissue plasminogen activator for acute ischemic stroke, Stroke 31 (12) (2000) 2920–2924. [3] P. Gurman, O. Miranda, A. Nathan, C. Washington, Y. Rosen, N. Elman, Recombinant tissue plasminogen activators (rtPA): a review, Clin. Pharmacol. Ther. 97 (3) (2015) 274–285. [4] A. Qureshi, R. Pande, S. Kim, R. Hanel, J. Kirmani, A. Yahia, Third generation thrombolytics for the treatment of ischemic stroke, Current opinion in investigational drugs (London, England: 2000) 3(12) (2002) 1729-1732. [5] C.R. Carpenter, S.M. Keim, W.K. Milne, W.J. Meurer, W.G. Barsan, B.E.i.E.M.I. Group, Thrombolytic therapy for acute ischemic stroke beyond three hours, The Journal of emergency medicine 40(1) (2011) 82–92. [6] I. dela Peña, C. Borlongan, G. Shen, W. Davis, Strategies to extend thrombolytic time window for ischemic stroke treatment: an unmet clinical need, Journal of stroke 19(1) (2017) 50. [7] E.C. Jauch, J.L. Saver, H.P. Adams, A. Bruno, B.M. Demaerschalk, P. Khatri, P.W. McMullan, A.I. Qureshi, K. Rosenfield, P.A. Scott, Guidelines for the early
6
Life Sciences 256 (2020) 117450
M. Hassanipour, et al.
tissue-type plasminogen activator, Neurobiol. Dis. 38 (3) (2010) 376–385. [38] J.M. Meunier, W.-T.W. Chang, B. Bluett, E. Wenker, C.J. Lindsell, G.J. Shaw, Temperature affects thrombolytic efficacy using rt-PA and eptifibatide, an in vitro study, Therapeutic hypothermia and temperature management 2 (3) (2012) 112–118.
[36] S.E. Lakhan, A. Kirchgessner, D. Tepper, A. Leonard, Matrix Metalloproteinases and Blood-brain Barrier Disruption in Acute Ischemic Stroke, Frontiers in Neurology 4, (2013). [37] R. Jin, G. Yang, G. Li, Molecular insights and therapeutic targets for blood–brain barrier disruption in ischemic stroke: critical role of matrix metalloproteinases and
7