Establishment of a Reproducible Ischemic Stroke Model in Nestin-GFP Mice with High Survival Rates

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

Short-Vessel Occlusion Might Indicate Higher Possibility of Success in Reperfusion following Mechanical Thrombectomy in Acute Middle Cerebral Artery Occlusion

<b><i>Background:</i></b> The impact of the length of the occluded vessel in acute large-vessel occlusion on successful reperfusion by mechanical thrombectomy remains unclear. This study evaluated whether diameter and length of the occluded vessel in acute middle cerebral artery (MCA) occlusion might relate to successful reperfusion following mechanical thrombectomy. <b><i>Methods:</i></b> This retrospective study included patients with acute MCA occlusion who underwent intra-aortic injection of contrast medium to obtain maximum intensity projection (MIP) images acquired by flat-panel detector computed tomography (FD-CT) equipped with an angiographic system. All patients received mechanical thrombectomy and were divided into two groups: those with successful reperfusion (Thrombolysis in Cerebral Infarction [TICI] 2b/3) and those without. We compared the diameter and length of the occluded vessel between the groups. In the sub-analysis of patients with stent retriever use, ratio of length of occluded vessel to length of the active zone was compared. <b><i>Results:</i></b> We enrolled 29 patients (median age: 73, M1 occlusion: 51%, stent retriever use: 72%). Eighteen patients achieved TICI 2b/3 with significantly larger distal end diameter (1.7 [interquartile range: 1.5–1.9] vs. 1.2 [1.2–1.5] mm, <i>p</i> = 0.007) and shorter length (7.1 [4.9–9.7] vs. 12.3 [7.2–15.8] mm, <i>p</i> = 0.043) of the occluded vessel. Sub-analysis of 21 patients showed that the cut-off value for TICI 2b/3 reperfusion was 0.32 as the ratio between the occluded vessel and stent retriever active zone (receiver operating characteristic area under the curve: 0.90). <b><i>Conclusion:</i></b> In acute MCA occlusion, larger diameter of the distal end and shorter length of the occluded vessel on FD-CT MIP images might indicate a higher possibility of achieving TICI 2b/3 following mechanical thrombectomy.

Image processing-based mTICI grading after endovascular treatment for acute ischemic stroke

Introduction : The grade of reperfusion after endovascular treatment of ischemic stroke e.g. mechanical thrombectomy is determined based on the mTICI score. The mTICI score shows significant interrater variability; it is usually biased towards better reperfusion results if selfassessed by the operator. We therefore developed a semiautomated image processing technique for assessing and evaluating the degree of reperfusion independently, resulting in a more objective mTICI score. Methods: Fifty angiography datasets of patients who were treated with mechanical thrombectomy for middle cerebral artery (MCA) occlusion were selected from our database. Image datasets were standardized by adjustment of field of view and orientation. Based on pixel intensity features, the internal carotid artery (ICA) curve was detected automatically and used as a starting point for identifying the target downstream territory (TDT) of the MCA on the DSA series. Furthermore, a grid with predefined dimensions was used to divide the TDT into checkzones and be classified as perfused or unperfused. Results: The algorithm detected the TDT and classified each zone of the grid as perfused or unperfused. Lastly, the percentage of the perfused area in the TDT was calculated for each patient and compared to the grading of experienced clinical users. Conclusion: A semi-automatic image-processing workflow was developed to evaluate perfusion rate based on angiographic images. The approach can be used for the objective calculation of the mTICI score. The semi-automatic grading is currently feasible for MCA occlusion but can be extended for other brain territories. The work shows a starting point for a machine learning approach to achieve a fully automated system that can evaluate and give an accurate mTICI score to become a common AI-based grading standard in the coming near future.

Secondary Cerebellar Cortex Injury in Albino Male Rats after MCAO: A Histological and Biochemical Study

The present study focused on secondary injury following the middle cerebral artery (MCA) occlusion in rats not linked to the MCA’s feeding zone. This entity has been very rarely studied. Additionally, this study investigated the rates of expression of five fundamental angiogenic biomarkers called endoglin, vascular endothelial growth factors-A (VEGF-A), endothelin-1 (ET-1), 2granulocyte colony-stimulating factor (G-CSF), and angiopoietin-using the MCA occlusion (MCAO) model. The random allocation of twelve adult male albino rats was in two groups. As a sham control group, six rats were used. This group was subjected to a sham operation without MCAO. The MCAO group consisted of six rats that were subjected to MCAO operation. After three days, the rats were sacrificed. The cerebellar specimens were immediately processed for light microscopic examination. An angiogenic biomarkers multiplex assay from multiplex was used to assess endoglin levels, VEGF-A, ET-1, angiopoietin-2, and G-CSF in serum samples. Hematoxylin and eosin-stained sections showed that the cerebellar cortex of rats of the MCAO group was more affected than the sham control group. Furthermore, Nissl stain and immunohistochemical analysis revealed an apparent increase in the number of positive immunoreactive in the cerebellar cortex and an evident decrease in Nissl granules in Purkinje cells of the MCAO rats, in contrast to the control rats. In addition, there was a significant increase in angiogenic factors VEGF-A, ET-1, angiopoietin-2, and endoglin. Interestingly, there was an increase in the G-CSF but a non-significant in the MCAO rats compared to the control rats. Furthermore, there was a significant correlation between the angiopoietin-2 and ET-1, and between G-CSF and ET-1. VEGF-A also exhibited significant positive correlations with the G-CSF serum level parameter, Endoglin, and ET-1. Rats subjected to MCAO are a suitable model to study secondary injury away from MCA’s feeding zone. Additionally, valuable insights into the association and interaction between altered angiogenic factors and acute ischemic stroke induced by MCAO in rats.

Collateral status evaluation coupled with time window by dynamic axial computed tomographic angiography with a focus on the middle cerebral artery for mechanical thrombectomy

Background Dynamic axial computed tomographic angiography (dynax–CTA), covering a thin width, with a focus on the bilateral middle cerebral artery (MCA), can quickly visualize the internal carotid artery (ICA) or MCA occlusion. We aimed to investigate whether dynax–CTA appropriately evaluated the collateral status coupled with the upper limit of the onset-to-reperfusion (OtR) time to achieve a major neurological improvement (MNI) at a 24-h follow-up examination after mechanical thrombectomy (MT). Methods We included acute ischemic stroke patients admitted from 2018 to 2020 who underwent dynax–CTA on admission and emergent MT for ICA or MCA occlusion. We performed dynax–CTA using an 80-row CT scanner and acquired 25 volume scans, consisting of 40 images of 1-mm thickness and 4-cm width. We classified the collateral status as good, intermediate, and poor based on MCA branch opacification. We evaluated the collateral status and the upper OtR time limit to achieve MNI. Results Forty-eight patients met our inclusion criteria. Dynax–CTA findings demonstrated MCA and ICA occlusion in 30 and 18 patients, respectively. The collateral status was good, intermediate, and poor in four, 25, and 19 patients, respectively. The upper limits of the OtR time for MNI were 3.63, 8.08, and 8.67 h in patients with poor, intermediate, and intermediate or good collateral status, respectively. Conclusions Dynax–CTA appropriately evaluated the collateral status coupled with the upper limit of the OtR time before performing MT.

Genetic Deletion of mGlu3 Metabotropic Glutamate Receptors Amplifies Ischemic Brain Damage and Associated Neuroinflammation in Mice

Backgroud: Type-3 metabotropic glutamate (mGlu3) receptors are found in both neurons and glial cells and regulate synaptic transmission, astrocyte function, and microglial reactivity. Here we show that the genetic deletion of mGlu3 receptors amplifies ischemic brain damage and associated neuroinflammation in adult mice. An increased infarct size was observed in mGlu3−/− mice of both CD1 and C57Black strains 24 h following a permanent occlusion of the middle cerebral artery (MCA) as compared to their respective wild-type (mGlu3+/+ mice) counterparts. Increases in the expression of selected pro-inflammatory genes including those encoding interleukin-1β, type-2 cycloxygenase, tumor necrosis factor-α, CD86, and interleukin-6 were more prominent in the peri-infarct region of mGlu3−/− mice. In contrast, the expression of two genes associated with the anti-inflammatory phenotype of microglia (those encoding the mannose-1-phosphate receptor and the α-subunit of interleukin-4 receptor) and the gene encoding the neuroprotective factor, glial cell line-derived neurotrophic factor, was enhanced in the peri-infarct region of wild-type mice, but not mGlu3−/− mice, following MCA occlusion. In C57Black mice, the genetic deletion of mGlu3 receptors worsened the defect in the paw placement test as assessed in the contralateral forepaw at short times (4 h) following MCA occlusion. These findings suggest that mGlu3 receptors are protective against ischemic brain damage and support the way to the use of selective mGlu3 receptor agonists or positive allosteric modulators in experimental animal models of ischemic stroke.

A novel model of ischemia in rats with middle cerebral artery occlusion using a microcatheter and zirconia ball under fluoroscopy

The failure of neuroprotective treatment-related clinical trials may be partially caused by unestablished animal models. Existing animal models are less likely to provide occlusion confined to the middle cerebral artery (MCA), making transarterial intervention difficult. We aimed to develop a novel focal stroke model using a microcatheter and zirconium dioxide that is non-magnetic under fluoroscopic guidance, which can monitor MCA occlusion and can improve hemorrhagic complications. Using male Sprague Dawley rats (n = 10), a microcatheter was navigated from the caudal ventral artery to the left internal carotid artery using an X-ray fluoroscopy to establish local occlusion. All rat cerebral angiographies were successful. No rats had hemorrhagic complications. Eight (80%) rats underwent occlusion of the MCA bifurcation by zirconium dioxide. Accidentally, the left posterior cerebral artery was failure embolized in 2 rats (20%). The median operating time was 8 min. All rats of occlusion MCA revealed an incomplete hemiparesis on the right side with neurological deficit score ranging from 1 to 3 (median 1, interquartile range 1–3) at 24 h after the induction of ischemia. Moreover, 2% 2,3,5-triphenyl tetrazolium chloride staining showed that the median infarct volume (mm3) was 280 (interquartile range 267–333) 24 h after the left MCA bifurcation occlusion. We present a novel rat model for focal stroke using a microcatheter and zirconium dioxide which does not affect the MRI. The model is predictable which is well confined within the territory supplied by the MCA, and reproducibility of this model is 80%. Fluoroscopy was able to identify which the MCA occlusion and model success while creating the model. It permitted exclusion of animals with complications from the experiment.

The Long-Term Functional Effect of Thrombectomy on Patients with Middle Cerebral Artery Occlusion Who Exhibit Moderate to Severe Disability

Background and Objectives: Endovascular thrombectomy (EVT is an emerging gold standard treatment for acute cerebral infarction and may allow functional improvement after subacute cerebral infarction. However, the long-term functional benefits of EVT in patients with moderate to severe disability remain unclear. We investigated the effects of EVT on the activities of daily living (ADL), handicap, gait, and eating in patients with middle cerebral artery (MCA) occlusion who exhibited moderate to severe disability (score of 3–5 on the modified Rankin scale (mRS)) due to stroke, up to six months after onset. Materials and Methods: This retrospective longitudinal case–control study assessed 45 patients with MCA occlusion who exhibited moderate to severe disability (mRS score ≥ 3): 15 underwent EVT and 30 served as controls. Clinical assessments were conducted at two weeks (12–16 days), four weeks (26–30 days), and six months (180–210 days) after stroke onset. Functional assessments comprised the Korean version of the modified Barthel index (MBI), mRS, functional ambulation category (FAC), and dysphagia outcome severity scale (DOSS) to assess disability, handicap, gait, and eating. Results: The MBI, mRS, FAC, and DOSS scores all improved significantly (all p < 0.05) in the EVT group, compared to the controls. Conclusions: EVT has favorable effects on performing routine ADL, the handicap itself, walking, and eating. Therefore, EVT is recommended for patients with acute MCA occlusion, including those with severe disability at the initial assessment.

Middle Cerebral Artery Thrombectomy: Clinical Findings and Technical Pearls: 2-Dimensional Operative Video

Mechanical thrombectomy as a treatment for large vessel occlusion to achieve rapid revascularization is supported in the literature.1-3 The presenting symptoms will localize to functions of the ischemic area. The middle cerebral artery (MCA) supplies areas of the frontal, temporal, and parietal cortices, as well as the basal ganglia. Occlusion of the MCA will present with contralateral hemiplegia, sensory loss, and, if the dominant hemisphere is involved, language deficits. We present a right-hand-dominant 79-yr-old female with right MCA syndrome—her last known well time was 1.5 h prior to presentation. Her NIH (National Institutes of Health) Stroke Scale was 16, most notable for left hemiplegia. Although the patient presented early in the clinical time course, as part of our institution protocol, a computed tomography (CT) head, CT perfusion, and CT angiogram (CTA) were performed. CT head did not demonstrate acute hemorrhage, so she received intravenous tissue plasminogen activator. CTA demonstrated a right MCA occlusion and CT perfusion suggested a large area of salvageable tissue, so she was taken to the angiography suite for mechanical thrombectomy. Angiography of the right internal carotid artery (ICA) showed MCA occlusion (insular segment). A thrombectomy device was deployed over the area of occlusion and allowed to engage for 5 min. An aspiration catheter was advanced over the stentriever up against the clot. The stentriever device was withdrawn under continuous aspiration and follow-up angiography showed complete reperfusion. The patient demonstrated improvement and was eventually discharged to an inpatient rehabilitation center. Patient provided consent for photography per university protocol. Institutional review board (IRB) approval was not needed for the single-patient data included in this report.

A Novel Rat Model of Embolic Cerebral Ischemia Using a Microcatheter and Zirconia Ball Under Fluoroscopic Guidance

Background and Purpose: The failure of neuroprotective treatment-related clinical trials may be partially caused by unestablished animal models. Existing animal models are less likely to provide occlusion confined to the middle cerebral artery (MCA), making transarterial intervention difficult. We aimed to develop a novel focal stroke model using a microcatheter and zirconium dioxide that is non-magnetic under fluoroscopic guidance, which can monitor MCA occlusion and can improve hemorrhagic complications. Methods: Using male Sprague Dawley rats (n=8), a microcatheter was navigated from the caudal ventral artery to the left internal carotid artery using digital subtraction angiography to establish local occlusion. Results: All rat cerebral angiographies were successful. No rats had hemorrhagic complications. Six (75%) rats underwent occlusion of the MCA bifurcation by zirconium dioxide. Accidentally, the left posterior cerebral artery was failure embolized in 2 rats (25%) via the posterior communicating artery. The median operating time was 8.5 min. Moreover, 2% 2,3,5-triphenyl tetrazolium chloride staining showed that the median infarct volume (mm3) was 280 (interquartile range; 267-333) 24 hours after the left MCA bifurcation occlusion. Conclusions: We present a novel rat model for focal stroke using a microcatheter and zirconium dioxide which does not affect the MRI. The model is predictable and reproducible, which is well confined within the territory supplied by the MCA. Fluoroscopy was able to identify which the MCA occlusion and model success while creating the model. It permitted exclusion of animals with complications from the experiment.