So human testing needed. Will never occur since we have NO leadership and NO strategy anywhere in stroke. You, your children and grandchildren are screwed until we get survivors in charge.
Macrophage Infiltration Reduces Neurodegeneration and Improves Stroke Recovery after Delayed Recanalization in Rats
Jinwei Pang
,1,2 Nathanael Matei,3 Jianhua Peng,1,2 Wen Zheng,3 Jing Yu,3 Xu Luo,3 Richard Camara,3 Ligang Chen,1,2,4 Jiping Tang,3 John H. Zhang,3 and Yong Jiang1,2,41Department of Neurosurgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China
2Sichuan Clinical Research Center for Neurosurgery, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China
3Department of Anesthesiology, Neurosurgery and Neurology, School of Medicine, Loma Linda University, Loma Linda, CA 92354, USA
4Luzhou Key Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China
Abstract
Background.
Recent cerebrovascular recanalization therapy clinical trials have validated delayed recanalization in patients outside of the conventional window. However, a paucity of information on the pathophysiology of delayed recanalization and favorable outcomes remains. Since macrophages are extensively studied in tissue repair, we anticipate that they may play a critical role in delayed recanalization after ischemic stroke.
Methods.
In adult male Sprague-Dawley rats, two ischemic stroke groups were used: permanent middle cerebral artery occlusion (pMCAO) and delayed recanalization at 3 days following middle cerebral artery occlusion (rMCAO). To evaluate outcome, brain morphology, neurological function, macrophage infiltration, angiogenesis, and neurodegeneration were reported. Confirming the role of macrophages, after their depletion, we assessed angiogenesis and neurodegeneration after delayed recanalization.
Results.
No significant difference was observed in the rate of hemorrhage or animal mortality among pMCAO and rMCAO groups. Delayed recanalization increased angiogenesis, reduced infarct volumes and neurodegeneration, and improved neurological outcomes compared to nonrecanalized groups. In rMCAO groups, macrophage infiltration contributed to increased angiogenesis, which was characterized by increased vascular endothelial growth factor A and platelet-derived growth factor B. Confirming these links, macrophage depletion reduced angiogenesis, inflammation, neuronal survival in the peri-infarct region, and favorable outcome following delayed recanalization.
Conclusion.
If properly selected, delayed recanalization at day 3 postinfarct can significantly improve the neurological outcome after ischemic stroke. The sanguineous exposure of the infarct/peri-infarct to macrophages was essential for favorable outcomes after delayed recanalization at 3 days following ischemic stroke.
1. Introduction
Acute ischemic stroke (AIS) is a global health concern that often leads to lifelong disability or death of patients [1, 2]. Although vascular recanalization therapy is widely recognized to be effective for AIS patients with large vessel occlusions (LVO), it is limited in the clinical setting due to the required window of time between ischemia onset and intervention [1, 3–5]. Previously, AIS reperfusion therapy has been restricted to a 4.5 h window for rt-PA thrombolysis or 6 h for endovascular thrombectomy [6]. Despite these limitations, early arterial recanalization following AIS has been shown to improve cerebral blood perfusion, functional outcome, and survival. The conventional window for reperfusion has not been challenged beyond these time points mostly due to the risk of ischemia/reperfusion injury [7], hemorrhagic transformation risk [8], early arterial reocclusion [9], and other associated deleterious outcomes [10]. Most AIS patients fail to receive intervention due to delays that increase the door-to-needle time (DNT) or door-to-puncture time (DPT), e.g., failure in early diagnosis, onset of unidentified symptoms, and/or other associated limitations in current stroke protocols [11, 12]. The majority of AIS patients presenting with LVO do not receive recanalization therapy and experience unfavorable disability and mortality. New therapeutic strategies are thus urgently needed for AIS.
Following advances in brain imaging to identify salvageable tissue, thrombolysis therapies and endovascular thrombectomy have shown remarkable improvement in outcomes following delayed recanalization beyond 6 h in AIS patients with LVO [13]. Two landmark randomized controlled clinical trials (DAWN and DEFUSE 3) reported that selective delayed recanalization up to 24 hours after symptom onset via endovascular mechanical thrombectomy resulted in favorable outcomes [14, 15]. Moreover, endovascular thrombectomy for AIS patients beyond 24 hours from symptom onset has also been shown to be safe and effective [16]. In the EXTEND trial, evidence has shown thrombolysis with rt-PA to be effective and improve overall outcome when administered in a window of 4.5-9 hours after symptom onset [17, 18]. Along these lines, imaging-based intravenous thrombolysis with Tenecteplase is efficacious up to 24 hours after symptom onset in selected patients [19]. A study reanalyzed the data of the DEFUSE 3 trial and suggested that approximately 20% of patients with LVO who are not treated with thrombectomy had a salvageable penumbra for at least an additional 24 hours [20].
Notably, the long-term outcomes of patients who received recanalization beyond 6 hours were reported to be improved as well [16]. In a follow-up study of the REVASCAT trial, the difference in outcomes of patients in the endovascular therapy group versus standard therapy group continued to increase from 3 months to 1 year [21]. As most patients are assessed for possible treatment up to 24 hours from symptom onset, two questions remain: (1) can more accurate criteria be established to categorize patients evaluated within 24 h from symptom onset for possible treatment; (2) would patients with salvageable brain tissue beyond the 24 h window also benefit from “delayed recanalization”?
Basic science research has reported that delayed recanalization at 3 days after permanent middle cerebral artery occlusion (pMCAO) attenuated neuronal apoptosis and improved functional outcome in rats [22]. Paralleling these studies, others have reported that even recanalization at up to 14 days after middle cerebral artery occlusion improved rat sensorimotor function although no difference in infarct volume was observed compared to controls [23]. Taken together, delayed recanalization can potentially limit cell death and reduce neurological deficits associated with ischemia. Nevertheless, the cellular and molecular mechanisms surrounding delayed recanalization have not been fully characterized.
Following delayed recanalization and although not fully confirmed, many peripheral circulation factors and/or cells are likely necessary for vascular remodeling and cerebral repair of the infarct core and perilesion regions [24]. In support, peripheral circulation macrophage infiltration reduces unfavorable outcomes in transient MCAO models [25, 26]. Furthermore, evidence supports that sanguineous exposure to macrophages may promote tissue repair in ischemic tissue via accelerated angiogenesis [26, 27]. However, the role of peripheral circulation macrophages on delayed brain repair has not been evaluated. In this study, we aim to elucidate whether the underlying benefits of delayed recanalization on neurological recovery are mediated through macrophage-dependent angiogenesis in a rat MCAO model.
2. Methods
2.1. Laboratory Animals
Adult male Sprague-Dawley rats (260–280 g) were housed and treated in a humidity and temperature-controlled environment with regular light/dark cycle and free access to food and water. All animal procedures were approved by the Loma Linda University and the Southwest Medical University Institutional Animal Care and Use Committee (IACUC) and in accordance with Stroke Treatment Academic Industry Roundtable (STAIR) guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.2. Experiment Design, Excluding and Including
The current study consists of two parts. Based on previous studies [22], we performed recanalization 3 days after MCAO. Detailed group information and a schematic illustration of the experimental process are displayed in additional file 1: Fig. S1. Sample sizes were calculated according to a previously reported [28] (formula:( means the number of animals in each group, and means the number of groups)). A power of 0.8 and of 0.05 were used to choose the sample size.
First, rats at 3 d after MCAO were allocated randomly into either pMCAO or rMCAO groups. However, we found that some animals with poor neurological function could not tolerate recanalization surgery with resultant unfavorable outcomes, including death. MRI scanning revealed that poor neurological performance may be related to hematoma volume expansion. Therefore, animals with neurological scores less than 6 at day 3 after MCAO were excluded from the study. The remaining rats were allocated randomly into pMCAO or rMCAO groups. Detailed information about experimental and procedural design is listed in supplementary material (Additional file 1: Table S1).
2.3. Establishment of Middle Cerebral Artery Occlusion (MCAO) Model and Recanalization
Cerebral blood flow (CBF) monitoring was used to ensure MCAO model alignment with previous methods [23]. Briefly, rats were anesthetized with an intraperitoneal injection of ketamine/xylazine mixture (80/20 mg/kg) and subcutaneous injection of atropine (10 mg/kg). A scalp incision was made, and a burr hole was created over the MCA territory. CBF was measured via a laser Doppler flow probe (OxyFlo probe, MNP100XP, AdInstruments Inc., Colorado Springs, CO, USA) to obtain the baseline and post-MCAO CBF levels using specialized software (PowerLab PL3504 and LabChart Pro, AdInstruments Inc., Colorado Springs, CO, USA). After acquiring the CBF baseline, the MCAO surgery was initiated. An abrupt attenuation of CBF was the mark of successful vessel occlusion. CBF was continuously monitored during the MCAO surgery and recanalization processes.
All MCAO model surgical procedures were performed as previously described [23]. In permanent MCAO (pMCAO) groups, an incision in the neck was made, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. Next, a 4-0 monofilament suture with a silicon-coated tip was inserted into the middle cerebral artery (MCA) bifurcation from the ECA stump. The occlusion suture was permanently tied, and the excess thread was cut off. Next, the neck wound and scalp incision were sutured. All rats were separately placed into 37°C recovery chambers for anesthesia recovery. In delayed recanalization animal (rMCAO) groups, the scalp incision and neck wounds were reopened, and the occlusion suture was removed at 3 d following MCAO. Sham animals underwent all surgical procedures without occlusion.
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