Refined movement analysis in the staircase test reveals differential motor deficits in mouse models of stroke.

Accurate assessment of post-stroke deficits is crucial in translational research. Recent advances in machine learning offer precise quantification of rodent motor behavior post-stroke, yet detecting lesion-specific upper extremity deficits remains unclear. Employing proximal middle cerebral artery occlusion (MCAO) and cortical photothrombosis (PT) in mice, we assessed post-stroke impairments via the Staircase test. Lesion locations were identified using 7 T-MRI. Machine learning was applied to reconstruct forepaw kinematic trajectories and feature analysis was achieved with MouseReach, a new data-processing toolbox. Lesion reconstructions pinpointed ischemic centers in the striatum (MCAO) and sensorimotor cortex (PT). Pellet retrieval alterations were observed, but were unrelated to overall stroke volume. Instead, forepaw slips and relative reaching success correlated with increasing cortical lesion size in both models. Striatal lesion size after MCAO was associated with prolonged reach durations that occurred with delayed symptom onset. Further analysis on the impact of selective serotonin reuptake inhibitors in the PT model revealed no clear treatment effects but replicated strong effect sizes of slips for post-stroke deficit detection. In summary, refined movement analysis unveiled specific deficits in two widely-used mouse stroke models, emphasizing the value of deep behavioral profiling in preclinical stroke research to enhance model validity for clinical translation.

Small vessel disease burden and prognosis of recent subcortical ischaemic stroke differ by parent artery atherosclerosis.

BACKGROUND AND PURPOSE: Parent artery atherosclerosis is an important aetiology of recent subcortical ischaemic stroke (RSIS). However, comparisons of RSIS with different degrees of parent artery atherosclerosis are lacking. METHODS: Prospectively collected data from our multicentre cohort (all were tertiary centres) of the Stroke Imaging Package Study between 2015 and 2017 were retrospectively reviewed. The patients with RSIS defined as a single clinically relevant diffusion-weighted imaging positive lesion in the territory of lenticulostriate arteries were categorized into three subgroups: (1) normal middle cerebral artery (MCA) on magnetic resonance angiography and high-resolution magnetic resonance imaging (HR-MRI); (2) low-grade MCA atherosclerosis (normal or <50% stenosis on magnetic resonance angiography and with MCA plaques on HR-MRI); (3) steno-occlusive MCA atherosclerosis (stenosis ≥50% or occlusion). The primary outcome was 90-day functional dependence (modified Rankin Scale score >2). The clinical and imaging findings were compared between subgroups. RESULTS: A total of 239 patients (median age 60.0 [52.0-67.0] years, 72% male) were enrolled, including 140 with normal MCA, 64 with low-grade MCA atherosclerosis and 35 with steno-occlusive MCA atherosclerosis. Patients with steno-occlusive MCA atherosclerosis had the largest infarct volume. Low-grade MCA atherosclerosis was independently associated with cerebral microbleeding, more severe perivascular spaces in basal ganglia and higher total cerebral small vessel disease burden. Low-grade MCA atherosclerosis was an independent determinant of 90-day functional dependence (odds ratio 3.897; 95% confidence interval 1.309-11.604). CONCLUSIONS: Our study suggested RSIS with varying severity of parent artery atherosclerosis exhibits distinctive clinical and neuroimaging characteristics, with low-grade MCA atherosclerosis associating with higher cerebral small vessel disease burden and worse prognosis.

Cerebral Angiography and Neurobehavioral Patterns in a Non-human Primate Middle Cerebral Artery Occlusion Model.

BACKGROUND/AIM: Ischemic stroke is a major health concern globally and developing reliable animal models is crucial for understanding its pathophysiology. This study evaluated the relationship between cerebral angiographic findings and neurologic dysfunction in an acute non-human primate thromboembolic stroke model and determined the minimum clot length for suitable middle cerebral artery (MCA) occlusion. MATERIALS AND METHODS: A thromboembolic stroke model was developed by injecting autologous blood clots (length: 1, 2, 3, 4, 5, and 10 cm, n=1 to 3, 14 monkeys in total) into the internal carotid artery of male cynomolgus monkeys. Digital subtraction angiography (DSA) and neurologic deficit observation were performed pre-; immediately after (DSA only); and 1, 3, 6, and 24 h after embolization, and the relationship between clot length, neurologic deficits, and cerebral infarction was assessed. RESULTS: DSA confirmed MCA occlusion in all animals after the clot injection. Recanalization of the MCA was observed within 6 h post-embolization in animals with shorter clots (≤3 cm). Neurologic deficits were evident in animals with MCA occlusion and correlated with the clot length. Larger clots (≥5 cm) led to permanent MCA occlusion, significant neurologic deficits, and extensive cerebral infarction. Histopathological examination revealed ischemic damage in brain regions corresponding to the infarcted areas. CONCLUSION: Clot length is critical in determining the extent of neurologic dysfunction and cerebral infarction, with larger clots producing more severe outcomes. Furthermore, the minimum clot length required for model creation is 5 cm.

How i do it: individualized bypass strategy with tentative clamping method.

BACKGROUND: Cerebral Revascularization (CR) remained an indispensable arm in the neurosurgical arsenal, especially managing symptomatic hemisphere with misery perfusion (SHMP). METHOD: We described an a mid-aged gentleman diagnosed with progressive middle cerebral steno-occlusion following carotid endarterectomy by employing individualized arterial reconstruction with tentative clamping method (TCM) under supervision of intraoperative monitoring. An operative video was also accompanied to demonstrate further details. CONCLUSION: The optimal treatment strategy for SHMP should be tailored by individuals. The risk of postoperative adverse sequel can be minimized and improved neuro-cognitive status was accomplished with an aid of TCM for such prophylactic procedure. CLINICAL TRIAL REGISTRATION: NA.

Moyamoya Disease: A Rare Presentation and Literature Review.

Moyamoya disease is a rare vasculopathy involving the vessels of the central nervous system, predominantly the internal carotid arteries. A 21-year-old female patient from the Murshidabad district of West Bengal presented to us with an altered sensorium. She was referred to us from another hospital in Kolkata, where she was diagnosed and being treated as a case of ischemic stroke 2 weeks prior to the presentation. There was a significant past history of excruciating headaches, which would be more at night, and did not have any other obvious aggravating or relieving factors. On examination, the plantar was bilaterally extensor, and the patient was afebrile with no signs of meningeal irritation. Neuroimaging with noncontrast computed tomography (CT) head revealed a large right-sided middle cerebral artery (MCA) territory infarct along with acute hemorrhage in the left basal ganglia region. Upon a thorough perusal of the course of treatment at the previous hospital, it was ascertained that there was no usage of antiplatelet or anticoagulant agents there. A magnetic resonance (MR) angiogram of the intracranial blood vessels was therefore ordered, which was suggestive of moyamoya disease. Therefore, it was concluded that the spontaneous and near-simultaneous occurrence of infarct and hemorrhage was due to the vascular anomalies induced by moyamoya disease, making this case an uncommon presentation of a very rare disease.

Radiomics model based on dual-energy CT can determine the source of thrombus in strokes with middle cerebral artery occlusion.

PURPOSE: To develop thrombus radiomics models based on dual-energy CT (DECT) for predicting etiologic cause of stroke. METHODS: We retrospectively enrolled patients with occlusion of the middle cerebral artery who underwent computed tomography (NCCT) and DECT angiography (DECTA). 70 keV virtual monoenergetic images (simulate conventional 120kVp CTA images) and iodine overlay maps (IOM) were reconstructed for analysis. Five logistic regression radiomics models for predicting cardioembolism (CE) were built based on the features extracted from NCCT, CTA and IOM images. From these, the best one was selected to integrate with clinical information for further construction of the combined model. The performance of the different models was evaluated and compared using ROC curve analysis, clinical decision curves (DCA), calibration curves and Delong test. RESULTS: Among all the radiomic models, model NCCT+IOM performed the best, with AUC = 0.95 significantly higher than model NCCT, model CTA, model IOM and model NCCT+CTA in the training set (AUC = 0.88, 0.78, 0.90,0.87, respectively, P < 0.05), and AUC = 0.92 in the testing set, significantly higher than model CTA (AUC = 0.71, P < 0.05). Smoking and NIHSS score were independent predictors of CE (P < 0.05). The combined model performed similarly to the model NCCT+IOM, with no statistically significant difference in AUC either in the training or test sets. (0.96 vs. 0.95; 0.94 vs. 0.92, both P > 0.05). CONCLUSION: Radiomics models constructed based on NCCT and IOM images can effectively determine the source of thrombus in stroke without relying on clinical information.

Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial.

BACKGROUND: The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. METHODS: The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. RESULTS: Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. CONCLUSIONS: Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.

MCA Parallel Anatomic Scanning MR Imaging-Guided Recanalization of a Chronic Occluded MCA by Endovascular Treatment.

Basi-parallel anatomic scanning has been widely used for assessing the vascular morphology of vertebral basilar arteries. Previous studies have demonstrated its efficacy in evaluating the morphology of the MCA, which we refer to as MCA parallel anatomic scanning MR imaging (MCPAS). In this study, we present our experience with the application of MCPAS in patients with MCA occlusion. Endovascular treatment was performed on the patients with intact MCA morphology visible in on MCPAS, with no intracranial hemorrhage, occlusion, or other complications observed. No severe stenosis or re-occlusion was observed at the 12-month postoperative follow-up. In conclusion, MCPAS is an effective method for assessing the outer contour of an occlusive MCA. Endovascular treatment can be considered a safe and efficient option for patients who show a favorable MCA through MCPAS assessment.

Comparative Analysis of Mechanical Thrombectomy Outcomes of Middle Cerebral Artery M1, M2 Superior, and M2 Inferior Occlusion Strokes.

BACKGROUND: The M1 middle cerebral artery (MCA) commonly bifurcates into M2 superior and M2 inferior segments. However, MCA anatomy is highly variable rendering classification for mechanical thrombectomy trials difficult. This study explored safety and effectiveness of M2 MCA stroke thrombectomy stratified by M2 MCA anatomy. METHODS: Cases of large vessel occlusion strokes treated by mechanical thrombectomy between February 2016 and August 2022 were reviewed (N = 784). M1 (n = 431) and M2 (n = 118) MCA occlusions were assessed. Among M2 MCA occlusions, only prototypical MCA bifurcation anatomy cases were included (n = 99). Dominance was assessed based on angiography. Procedural and outcome data were compared between M1, M2 superior, and M2 inferior MCA occlusions. RESULTS: Baseline demographics and periprocedural criteria of M2 superior (n = 56) and M2 inferior (n = 43) occlusion mechanical thrombectomies were comparable. The occluded branch was dominant in 41/43 (95.3%) M2 inferior cases, but in only 37/56 (66.1%) M2 superior cases (P < 0.001). The 90-day favorable functional outcome (modified Rankin Scale score 0-2) and mortality (modified Rankin Scale score 6) rates were 60.0% and 8.9% in M2 superior, 42.9% and 32.6% in M2 inferior, and 44.1% and 26.0% in M1 (n = 431) cases. Compared with M2 superior cases, in M2 inferior cases, favorable outcome rates were lower (P = 0.094) and mortality rates were higher (P = 0.003) and resembled M1 rates (P = 0.750 and P = 0.355, respectively). CONCLUSIONS: In the setting of prototypical MCA bifurcation anatomy, thrombectomy of dominant M2 inferior occlusions had outcome rates similar to M1 occlusions. In contrast, M2 superior occlusions had significantly lower mortality rates and a trend toward better favorable functional outcome rates.

A novel NIR-II albumin-escaping probe for cerebral arteries and perfusion imaging in stroke mice model.

In order to guide the formulation of post-stroke treatment strategy in time, it is necessary to have real-time feedback on collateral circulation and revascularization. Currently used near-infrared II (NIR-II) probes have inherent binding with endogenous albumin, resulting in significant background signals and uncontrollable pharmacokinetics. Therefore, the albumin-escaping properties of the new probe, IR-808AC, was designed, which achieved timely excretion and low background signal, enabling the short-term repeatable injection for visualization of cerebral vessels and perfusion. We further achieved continuous observation of changes in collateral vessels and perfusion during the 7-d period in middle cerebral artery occlusion mice using IR-808AC in vivo. Furthermore, using IR-808AC, we confirmed that remote ischemic conditioning could promote collateral vessels and perfusion. Finally, we evaluated the revascularization after thrombolysis on time in embolic stroke mice using IR-808AC. Overall, our study introduces a novel methodology for safe, non-invasive, and repeatable assessment of collateral circulation and revascularization in real-time that is crucial for the optimization of treatment strategies.

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion.

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

[123I]CLINDE SPECT as a neuroinflammation imaging approach in a rat model of stroke.

Poststroke neuroinflammation exacerbates disease progression. [11C]PK11195-positron emission tomography (PET) imaging has been used to visualize neuroinflammation; however, its short half-life of 20 min limits its clinical use. [123I]CLINDE has a longer half-life (13h); therefore, [123I]CLINDE-single-photon emission computed tomography (SPECT) imaging is potentially more practical than [11C]PK11195-PET imaging in clinical settings. The objectives of this study were to 1) validate neuroinflammation imaging using [123I]CLINDE and 2) investigate the mechanisms underlying stroke in association with neuroinflammation using multimodal techniques, including magnetic resonance imaging (MRI), gas-PET, and histological analysis, in a rat model of ischemic stroke, that is, permanent middle cerebral artery occlusion (pMCAo). At 6 days post-pMCAo, [123I]CLINDE-SPECT considerably corresponded to the immunohistochemical images stained with the CD68 antibody (a marker for microglia/microphages), comparable to the level observed in [11C]PK11195-PET images. In addition, the [123I]CLINDE-SPECT images corresponded well with autoradiography images. Rats with severe infarcts, as defined by MRI, exhibited marked neuroinflammation in the peri-infarct area and less neuroinflammation in the ischemic core, accompanied by a substantial reduction in the cerebral metabolic rate of oxygen (CMRO2) in 15O-gas-PET. Rats with moderate-to-mild infarcts exhibited neuroinflammation in the ischemic core, where CMRO2 levels were mildly reduced. This study demonstrates that [123I]CLINDE-SPECT imaging is suitable for neuroinflammation imaging and that the distribution of neuroinflammation varies depending on the severity of infarction.

Ischemia-reperfusion injury in a salvaged penumbra: Longitudinal high-tesla perfusion magnetic resonance imaging in a rat model.

INTRODUCTION: Although ischemia-reperfusion (I/R) injury varies between cortical and subcortical regions, its effects on specific regions remain unclear. In this study, we used various magnetic resonance imaging (MRI) techniques to examine the spatiotemporal dynamics of I/R injury within the salvaged ischemic penumbra (IP) and reperfused ischemic core (IC) of a rodent model, with the aim of enhancing therapeutic strategies by elucidating these dynamics. MATERIALS AND METHODS: A total of 17 Sprague-Dawley rats were subjected to 1 h of transient middle cerebral artery occlusion with a suture model. MRI, including diffusion tensor imaging (DTI), T2-weighted imaging, perfusion-weighted imaging, and T1 mapping, was conducted at multiple time points for up to 5 days during the I/R phases. The spatiotemporal dynamics of blood-brain barrier (BBB) modifications were characterized through changes in T1 within the IP and IC regions and compared with mean diffusivity (MD), T2, and cerebral blood flow. RESULTS: During the I/R phases, the MD of the IC initially decreased, normalized after recanalization, decreased again at 24 h, and peaked on day 5. By contrast, the IP remained relatively stable. Both the IP and IC exhibited hyperperfusion, with the IP reaching its peak at 24 h, followed by resolution, whereas hyperperfusion was maintained in the IC until day 5. Despite hyperperfusion, the IP maintained an intact BBB, whereas the IC experienced persistent BBB leakage. At 24 h, the IC exhibited an increase in the T2 signal, corresponding to regions exhibiting BBB disruption at 5 days. CONCLUSIONS: Hyperperfusion and BBB impairment have distinct patterns in the IP and IC. Quantitative T1 mapping may serve as a supplementary tool for the early detection of malignant hyperemia accompanied by BBB leakage, aiding in precise interventions after recanalization. These findings underscore the value of MRI markers in monitoring ischemia-specific regions and customizing therapeutic strategies to improve patient outcomes.

Correlation between asymmetrical vein sign of SWI and long-term clinical outcomes in patients with middle cerebral artery ischemic stroke.

OBJECTIVE: To evaluate the association of asymmetrical cortical vein sign (ACVS) and asymmetrical medullary vein sign (AMVS) on susceptibility-weighted imaging (SWI) with 90-day poor outcomes in patients with unilateral middle cerebral artery acute ischemic stroke (AIS) after conservative drug treatment. METHODS: Clinical data for the participants included age, sex, smoking, alcohol, hypertension, diabetes, hyperlipidemia, coronary heart disease, NHISS-admission, and NHISS-discharge scores. Participants underwent magnetic resonance imaging (MRI) within 12 h of hospital admission, including conventional scan sequences and a SWI sequence. Poor prognosis was defined as a modified Rankin scale (mRS) ≥ 3 at 90 days. RESULTS: A total of 108 patients were included from January 2021 to March 2022. Twenty-seven (25%) patients had a poor outcome at 90 days. Univariate analysis indicated that diabetes, NHISS-admission, NHISS-discharge, DWI-ASPECTS, SWI-ASPECTS, FLAIR-ASPECTS, and AMVS + were associated with 90-day poor outcome. Multivariate regression analysis showed that AMVS + was associated with 90-day poor outcome from the three models (OR = 3.57, P = 0.006; OR = 3.74, P = 0.005; OR = 5.14, P = 0.0057). However, no significant association was found between ACVS + and 90-day poor outcome. CONCLUSIONS: AMVS might be a helpful neuroimaging predictor for poor outcome at 90 days compared to ACVS in drug-conserving treatment of patients with unilateral middle cerebral artery ischemic stroke.

Vascular syndrome predicts the development and course of epilepsy after perinatal stroke.

OBJECTIVE: Epilepsy develops in one third of the patients after perinatal stroke. It is still unclear which vascular syndrome of ischemic stroke carries higher risk of epilepsy. The aim of the current study was to evaluate the risk of epilepsy according to the vascular syndrome of perinatal stroke. METHODS: The study included 39 children with perinatal arterial ischemic stroke (13 with anterior or posterior trunk of the distal middle cerebral artery occlusion, 23 with proximal or distal M1 middle cerebral artery occlusion and three with lenticulostriate arteria infarction), and 44 children with presumed perinatal venous infarction. Magnetic resonance imaging obtained at the chronic stage was used to evaluate the vascular syndrome of stroke. RESULTS: The median follow-up time was 15.1 years (95% CI: 12.4-16.5 years), epilepsy developed in 19/83 (22.9%) patients. The cumulative probability to be without epilepsy at 15 years was 75.4% (95% CI: 65.8-86.4). The probability of having epilepsy was higher in the group of proximal or distal M1 artery occlusion compared to patients with periventricular venous infarction (HR 7.2, 95% CI: 2.5-26, p = .0007). Patients with periventricular venous infarction had significantly more often status epilepticus or spike-wave activation in sleep ≥85% of it compared to patients with anterior or posterior trunk of the distal middle cerebral artery occlusion (OR = 81; 95% CI: 1.3-5046, p = .029). SIGNIFICANCE: The emphasis of this study is placed on classifying the vascular syndrome of perinatal stroke and on the targeted follow-up of patients for epilepsy until young adulthood. The risk for having epilepsy after perinatal stroke is the highest in children with proximal or distal M1 middle cerebral artery occlusion. Patients with periventricular venous infarction have a more severe course of epilepsy.

Preparation of NIR-II Polymer Nanoprobe Through Twisted Intramolecular Charge Transfer and Förster Resonance Energy Transfer of NIR-I Dye.

Near-infrared-II (NIR-II) fluorescence imaging is pivotal in biomedical research. Organic probes exhibit high potential in clinical translation, due to advantages such as precise structure design, low toxicity, and post-modifications convenience. In related preparation, enhancement of NIR-II tail emission from NIR-I dyes is an efficient method. In particular, the promotion of twisted intramolecular charge transfer (TICT) of relevant NIR-I dyes is a convenient protocol. However, present TICT-type probes still show disadvantages in relatively low emission, large particle sizes, or limited choice of NIR-I dyes, etc. Herein, the synthesis of stable small-sized polymer NIR-II fluoroprobes (e.g., 7.2 nm), integrating TICT and Förster resonance energy transfer process to synergistically enhance the NIR-II emission is reported. Strong enhanced emissions can be obtained from various NIR-I dyes and lanthanide elements (e.g., twelvefold at 1250 nm from Nd-DTPA/IR-808 sample). The fluorophore provides high-resolution angiography, with high-contrast imaging on middle cerebral artery occlusion model mice for distinguishing occlusion. The fluorophore can be rapidly excreted from the kidney (urine ≈65% within 4 h) in normal mice and exhibits long-term renal retention on acute kidney injury mice, showing potential applications in the prognosis of kidney diseases. This development provides an effective strategy to design and synthesize effective NIR-II fluoroprobes.

Innovative statistical approaches: the use of neural networks reduces the sample size in the splenectomy-MCAO mouse model.

AIM: To compare the effectiveness of artificial neural network (ANN) and traditional statistical analysis on identical data sets within the splenectomy-middle carotid artery occlusion (MCAO) mouse model. METHODS: Mice were divided into the splenectomized (SPLX) and sham-operated (SPLX-sham) group. A splenectomy was conducted 14 days before middle carotid artery occlusion (MCAO). Magnetic resonance imaging (MRI), bioluminescent imaging, neurological scoring (NS), and histological analysis, were conducted at two, four, seven, and 28 days after MCAO. Frequentist statistical analyses and ANN analysis employing a multi-layer perceptron architecture were performed to assess the probability of discriminating between SPLX and SPLX-sham mice. RESULTS: Repeated measures ANOVA showed no significant differences in body weight (F (5, 45)=0.696, P=0.629), NS (F (2.024, 18.218)=1.032, P=0.377) and brain infarct size on MRI between the SPLX and SPLX-sham groups post-MCAO (F (2, 24)=0.267, P=0.768). ANN analysis was employed to predict SPLX and SPL-sham classes. The highest accuracy in predicting SPLX class was observed when the model was trained on a data set containing all variables (0.7736±0.0234). For SPL-sham class, the highest accuracy was achieved when it was trained on a data set excluding the variable combination MR contralateral/animal mass/NS (0.9284±0.0366). CONCLUSION: This study validated the neuroprotective impact of splenectomy in an MCAO model using ANN for data analysis with a reduced animal sample size, demonstrating the potential for leveraging advanced statistical methods to minimize sample sizes in experimental biomedical research.