Hemoglobin-albumin-lymphocyte-platelet score and early neurological deterioration in acute ischemic stroke: a single-center retrospective cohort study

 Describing a problem or predictions and 'associations'  DO NOTHING TOWARDS RECOVERY! You're all fired!

Hemoglobin-albumin-lymphocyte-platelet score and early neurological deterioration in acute ischemic stroke: a single-center retrospective cohort study

• 

  Ci Zhang 1,2†

• 

  Wei Zhang 1†

• Y

  Yinyang Zhang 3

• 

  Guosong Xue 1*

• 1. Department of Neurology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China

• 2. Department of Neurology, Suqian Hospital of Nanjing Drum Tower Hospital Group, Suqian, Jiangsu, China

Abstract

Purpose: 

To investigate the association between the hemoglobin-albumin-lymphocyte-platelet (HALP) score and early neurological deterioration (END) in individuals with stroke and to develop an exploratory prediction model for END.

Patients and methods: 

Clinical data from 595 patients with acute ischemic stroke (AIS) admitted to the Affiliated Hospital of Xuzhou Medical University from April 2022 to April 2024 were retrospectively analyzed. Patients were randomly divided into a training set and a validation set in a 7:3 ratio. Multivariable logistic regression analysis was utilized in the training data to examine END risk variables and build a corresponding predictive model. Model performance was evaluated using receiver operating characteristic (ROC) curves to assess discrimination and the Hosmer–Lemeshow goodness-of-fit test to evaluate calibration. Clinical decision curve analysis (DCA) was further applied to determine the model’s clinical value. To improve interpretability, the importance of included predictors was assessed using the SHapley Additive exPlanation (SHAP) method.

Results: 

Of the 595 patients, 186 (31.3%) developed early neurological deterioration (END), while the remaining 409 (68.7%) comprised the non-END group. The training cohort consisted of 416 randomly assigned participants. Multivariate logistic regression showed that the large artery atherosclerosis (LAA) subtype, elevated baseline National Institutes of Health Stroke Scale (NIHSS) score, and lower HALP score constituted independent determinants for END development (p < 0.05). In further stepwise-adjusted analyses, the association between HALP and END remained statistically significant after controlling for demographic characteristics, vascular risk factors, and treatment-related variables. A nomogram incorporating HALP score, LAA subtype, and baseline NIHSS score showed moderate discrimination in both the training and validation sets.

Conclusion: 

A lower HALP score was associated with END in patients with AIS. A nomogram incorporating HALP score, LAA subtype, and baseline NIHSS score demonstrated moderate discrimination and may provide useful information for early risk stratification in AIS. This prediction model requires validation in a larger, prospective clinical study.

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What Happens To Your Immune System After One Sauna Session by mindbodygreen

 

I'm just got a personal sauna from NuRecover but will use a cold shower instead of a chiller bucket for the cold shock proteins. Still to be put together.

What Happens To Your Immune System After One Sauna Session

 There’s a reason sauna use keeps showing up in longevity research. Regular exposure has been associated with lower risks of cardiovascular disease¹ ,respiratory illness²,dementia³, and even overall mortality⁴ .But those outcomes don’t tell us much about the immediate effects. What actually changes in the body during a single session? And how quickly do those changes happen? A new study⁵set out to answer that by tracking immune responses before, during, and after a 30-minute sauna session, offering a closer look at the body’s short-term reaction to heat. 

What happens to your immune system during a 30-minute sauna

Researchers studied 51 adults, both men and women, during a standard Finnish sauna session set to about 73°C (roughly 163°F). Each participant spent 30 minutes in the sauna, with blood samples taken before, immediately after, and 30 minutes post-session. The goal was to track how immune cells and inflammatory signals changed in response to heat stress. Participants were allowed to drink water throughout, which helped control for dehydration and made the setup closer to a typical real-world sauna experience. Instead of just looking at overall immune activity, the researchers zoomed in on specific white blood cells and a wide range of cytokines, which are signaling molecules involved in inflammation and immune response. This gave a more detailed picture of how the body reacts in the short term. 

Sauna’s immediate effect on immune cells

The biggest shift came down to your white blood cells, which are a key part of your immune system. After the sauna session, those levels went up, including important types like neutrophils and lymphocytes. These are the cells that help your body spot and respond to anything that doesn’t belong, whether that’s a virus or another kind of threat. What stands out is how quickly this happens. The increase shows up right after the sauna, then goes back down within about 30 minutes. So this isn’t a long-lasting spike; it’s more like a short window where your immune system is a bit more alert. This pattern mirrors what happens during exercise. When you work out, immune cells move out of tissues and into your bloodstream, where they’re more ready to respond if needed. A sauna seems to trigger a comparable response, just through heat instead of movement. 

What about inflammation & body temperature?

One thing the researchers looked at closely was inflammation, since that’s often what people think of when they hear the body is under “stress.” But in this case, there weren’t big changes across most inflammatory markers. Out of dozens of signals they measured, only a few shifted in a meaningful way. That suggests your body isn’t going into a full inflammatory response during a sauna. Instead, the response seems more about mobilization than inflammation. Your immune cells are being redistributed and activated, not necessarily pushed into an inflammatory state. Body temperature did play a role, though. On average, people’s temperature rose by about 2°C (roughly 3.6°F) during the session. And the more it increased, the more certain immune-related signals shifted alongside it. 

Adding sauna use to your routine

 So what does this mean for your day-to-day routine? It doesn’t mean a single sauna session will prevent illness or replace other foundational habits. But it does suggest that sauna use can act as a short-term stimulus for your immune system, similar to a workout.If you already use a sauna, this adds another layer of context. That post-sauna feeling isn’t just relaxation. Your body is actively responding, mobilizing immune cells in a way that may support overall immune surveillance over time. 

If you’re considering adding it in, consistency matters more than intensity. This study looked at one session, but previous research has linked regular sauna use to broader health benefits, including a lower risk of certain chronic conditions.

It’s also worth paying attention to how you personally respond. Hydration, heat tolerance, and recovery all play a role in how beneficial the experience feels.

The takeaway

This study doesn’t suggest that sauna use is a cure-all. But it does offer a clearer picture of what’s happening in the body during a session. Your immune system isn’t passive in that environment. It’s responding, adapting, and briefly shifting into a more active state.

Relationship Between Lymphocyte-Associated Inflammatory Markers and Post-Stroke Cognitive Impairment

 ABSOLUTELY USELESS!  Nothing here helps survivors recover. You're all fired for not knowing that the only goal in stroke is 100% recovery

Relationship Between Lymphocyte-Associated Inflammatory Markers and Post-Stroke Cognitive Impairment

Authors Hu QY, Liu J, Cui CH, Guo MF, Shi YT, Zhang XM, Jia BF, Li XY, Sun SJ

Received 10 June 2025

Accepted for publication 6 September 2025

Published 25 September 2025 Volume 2025:18 Pages 13347—13358

DOI https://doi.org/10.2147/JIR.S545953

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Ning Quan

Qian-Ying Hu,^1,* Juan Liu,^1,* Cai-Hong Cui,¹ Mei-Fang Guo,² Yu-Tong Shi,² Xiao-Man Zhang,² Bing-Fei Jia,² Xin-Yu Li,² Su-Juan Sun<sup>3

1</sup>Department of Rehabilitation Medicine, Affiliated Hospital of Hebei University, Baoding, Hebei, 071000, People’s Republic of China; ²Department of Basic Medical Sciences, Hebei University, Baoding, Hebei, 071000, People’s Republic of China; ³Department of Nursing, Hebei General Hospital, Shijiazhuang, Hebei, 050000, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Su-Juan Sun, Department of Nursing, Hebei General Hospital, No. 348 Heping West Road, Xinhua District, Shijiazhuang, Hebei, 050000, People’s Republic of China, Tel +86 13933093071, Email sujuansunssjm@126.com Cai-Hong Cui, Department of Rehabilitation Medicine, Affiliated Hospital of Hebei University, No. 212 of Yuhua East Road, Lianchi District, Baoding, Hebei, 071000, People’s Republic of China, Tel +86 13463236473, Email caihongcuicchk@126.com

Objective: To determine whether differences in lymphocyte-related inflammatory markers in the ultra-early phase of stroke (within 24 hours of onset) are associated with post-stroke cognitive impairment in the early recovery phase (within 30 days of stroke onset), and to further assess the predictive value of these markers.
Methods: The study population consisted of patients who underwent rehabilitation treatment at the Rehabilitation Department of Hebei University Affiliated Hospital between December 2024 and June 2025, within 30 days of stroke onset, ie, during the early recovery phase of stroke. Patients were grouped based on whether they developed cognitive impairment. A retrospective analysis was conducted of patients’ blood markers and neurological deficit scores within 24 hours of stroke onset to examine the relationship between ultra-early blood markers and neurological deficits and post-stroke cognitive impairment.
Results: There were no significant differences in baseline data between the two groups. However, the proportion of hemorrhagic stroke patients was significantly higher in the PSCI group than in the non-PSCI group (39.7% vs 18.8%, P=0.026< 0.05). NLR and NIHSS scores showed significant differences between the two groups. Multivariate analysis indicated that NIHSS (OR=1.297, 95% CI: 1.167– 1.442, p< 0.001) was independently associated with PSCI, while NLR (OR=1.107, 95% CI: 0.995– 1.231, p=0.063) showed a borderline association with PSCI. MLR showed differences between the two groups in univariate analysis (P=0.018) but was excluded in multivariate analysis. ULR did not show significant differences.
Conclusion: NIHSS is a strong predictive factor (P < 0.05), with a cut of value of 12 calculated by the ROC curve. NLR is at the threshold for an independent risk factor. Subsequent ROC curves indicate that NLR has low diagnostic sensitivity but high specificity, making it more suitable for screening rather than diagnostic use. MLR and ULR did not demonstrate high predictive value; further studies should be conducted to expand the sample size, perform subgroup analyses, and increase follow-up.

Keywords: post-stroke cognitive impairment, NIHSS, NLR, MLR, ULR

Background

The 2020 China Stroke Report shows that the prevalence of stroke in China is 1,114.8 per 100,000 people, the annual incidence rate is 246.8 per 100,000 people, and the mortality rate is 149.49 per 100,000 people. China has become the country with the highest lifetime risk of stroke and the heaviest disease burden globally.¹ Post-stroke cognitive impairment (PSCI) refers to a clinical syndrome characterized by cognitive impairment that occurs after a stroke event and persists for up to six months. According to literature reports, approximately one-third of stroke patients experience.² The diagnosis of PSCI is typically based on clinical assessment, neuropsychological assessment, and neuroimaging. The Montreal Cognitive Assessment (MoCA) and the Mini-Mental State Examination (MMSE) are the most widely used cognitive tests in PSCI research.³ However, both are subject to copyright restrictions, which pose challenges for research. Meanwhile, the new cognitive test Mini-Cog, which is simple to administer and time-efficient, can aid in detecting the early stages of cognitive impairment. In a post-hoc analysis of a population-based study of older adults in the United States, the Mini-Cog scores classified as “possibly impaired” or “possibly normal” using the algorithm were comparable to those of the MMSE with a cutoff point of 25 in terms of sensitivity (76% vs 79%) and specificity (89% vs 88%) for dementia.-Cog, which was scored as “possibly impaired” or “possibly normal” by the algorithm, showed similar sensitivity (76% vs 79%) and specificity (89% vs 88%) for dementia compared to the MMSE with a cutoff point of 25. These results were comparable to those obtained using traditional neuropsychological assessments (sensitivity 75%, specificity 90%).⁴ A meta-analysis indicated that for cognitive impairment (including dementia and mild cognitive impairment) in primary care settings, Mini-Cog showed 73% sensitivity and 84% specificity. In secondary care settings, Mini-Cog showed 73% sensitivity and 76% specificity⁵ All performed well.

In addition to clinical examinations, neuropsychological assessments, and imaging studies, various blood biomarkers have become a focal point of research. By measuring indicators associated with brain injury in the early stages of stroke, it is possible to predict the occurrence of PSCI, which aids in the early identification of PSCI and facilitates further rehabilitation interventions. Inflammatory factors are closely associated with cognitive impairment. Clinically, lymphocyte-related inflammatory markers, such as the neutrophil-lymphocyte ratio (NLR), have been shown to have predictive value in a prospective cohort study of cognitive impairment following ischemic stroke. Peripheral NLR levels are significantly elevated in PSCI patients.³

Another inflammatory marker closely related to lymphocytes—the monocyte-to-lymphocyte ratio (MLR)—is more commonly used in the prediction of cardiovascular disease.⁶ The serum uric acid to lymphocyte ratio (ULR) has been validated as a new lymphocyte-related inflammatory marker with superior predictive value for hemorrhagic stroke compared to uric acid or lymphocyte levels alone in a large prospective cohort study conducted in China.⁷ This study was conducted to further clarify whether differences in inflammatory markers, especially lymphocyte-related inflammatory markers, in the ultra-early stage of stroke (within 24 hours of onset), including hemorrhagic stroke, are associated with post-stroke cognitive impairment in the early recovery stage (within 30 days of stroke onset).⁸

Research Methods

This study is a retrospective study targeting patients who underwent rehabilitation therapy at the Rehabilitation Department of Hebei University Affiliated Hospital between December 2024 and June 2025, specifically those within 30 days post-stroke, ie, in the early recovery phase of stroke. The study population was divided into an observation group (PSCI group) and a control group (non-PSCI group) based on the occurrence of cognitive impairment. A retrospective analysis was conducted of blood markers and neurological deficit scores within 24 hours of stroke onset, to investigate the relationship between ultra-early blood markers and neurological deficits and the occurrence of cognitive impairment after stroke.

Refer to previous literature,³ the inclusion criteria are as follows: (1) age ≥ 18 years; (2) confirmed by cranial computed tomography (CT) or magnetic resonance imaging (MRI);² (3) Patients who have experienced a stroke within the past 30 days, are in the early stages of recovery, have stable conditions, are conscious, can speak fluently, and are able to cooperate with rehabilitation assessments and treatments; (4) Patients who visited the Department of Neurology, Department of Neurosurgery, Department of Critical Care Medicine, or other relevant departments at Hebei University Affiliated Hospital within 24 hours of experiencing a stroke, with complete clinical data and traceable medical records from within 24 hours of the onset of symptoms.

Refer to previous literature,³ the exclusion criteria are as follows: (1) Patients who had been diagnosed with cognitive impairment disorders prior to the stroke, including Alzheimer’s disease, frontotemporal dementia, Parkinson’s disease dementia, Lewy body dementia, mixed dementia, and other types of cognitive impairment or dementia caused by various reasons; (2) Patients unable to cooperate with cognitive function assessments, including those with severe aphasia, hearing impairments, dysarthria, impaired consciousness, visual impairments, or those unable to write normally due to limb dysfunction caused by stroke; (3) Patients with unstable conditions who are unable to cooperate with assessments and rehabilitation therapy, including but not limited to those with severe illnesses such as cardiovascular diseases (eg, severe heart failure, severe arrhythmias), renal diseases (eg, renal failure), pulmonary diseases (eg, severe pneumonia, respiratory failure), etc.; (4) Patients who had conditions prior to the stroke that could trigger inflammatory and immune responses (eg, acute infections, tumors, blood disorders, autoimmune diseases, recent major surgeries, or trauma); (5) Patients who had been taking medications that could interfere with the inflammatory and immune systems prior to the stroke (eg, antibiotics, corticosteroids, immunosuppressants, targeted therapies, etc).; (6) Patients with a history of recurrent strokes.

Based on the Mini-Cog score, patients were divided into a post-stroke cognitive impairment (PSCI) group and a group without post-stroke cognitive impairment. This study complies with the World Medical Association’s Declaration of Helsinki and has been approved by the Ethics Committee of Hebei University Affiliated Hospital (the Ethics Committee of Affiliated Hospital of Hebei University:HDFYLL - KY - 2024 - 120).

Data Collection

Baseline Characteristics

Age, sex, body mass index (BMI), educational attainment (elementary, middle school, university),smoking and alcohol use,were collected.

Clinical Variables

This included past medical history (including coronary heart disease, diabetes, and hypertension, the definition criteria are a previous clear diagnosis of a related disease and/or long-term use of therapeutic drugs prior to onset, stroke type (hemorrhagic or ischemic).

Observation Indicators

Extract and analyze medical records from the acute phase within 24 hours of onset, extracting data on neutrophils, lymphocytes, monocytes, and uric acid, and calculate NLR, MLR, and ULR. Uric acid levels were analyzed using the Fusion biochemical analyzer from Johnson & Johnson, and blood cell counts were measured using the XN-L™ Series blood analyzer from Sysmex Corporation. Stroke severity was assessed using the National Institute of Health Stroke Scale (NIHSS) to evaluate neurological deficit scores.⁹

Cognitive Function Assessment and Grouping

The official Chinese version of the Mini-Cog official website was used to assess the cognitive function of the patients, which was truncated into a control group (no cognitive impairment group) and an observation group (cognitive impairment group) with 4 points.

The sample size calculation formula is:  where the expected prevalence rate (P) is 1/3 (33%) as described in the background section above, the confidence interval is 95%, and the allowable error is 0.1. The minimum sample size calculated is 85 cases, which will be assessed by therapists who are trained and proficient in using the Mini-Cog. A total of 127 cases were ultimately included in this study, 69 in the control group and 58 in the observation group.

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Role of Immune Cells Migrating to the Ischemic Brain

I got nothing out of this that could help any survivor recover better. But since I'm not medically trained I obviously know nothing. 

Role of Immune Cells Migrating to the Ischemic Brain

Anna M. Planas

Originally published27 Aug 2018Stroke. 2018;49:2261-2267

The central nervous system and the immune system are tightly interconnected through complex communicating networks.¹ Immune cells are distributed in specific central nervous system compartments. Microglia are the innate immune cells resident in the brain parenchyma. Macrophages surround the blood vessels and also line the leptomeninges and the choroid plexus together with dendritic cells and lymphocytes, among other immune cells, where they play immunosurveillance functions. Therefore, the immune system keeps a close watch on brain function and reacts when brain homeostasis is lost because of injuries or diseases. Sterile organ damage may turn immune cells into harmful agents and for this reason they are regarded as targets for therapeutic intervention in acute stroke.² Stroke induces strong inflammatory reactions involving the local production of cytokines, such as TNF-α (tumor necrosis factor-α) by various brain cells, including human neurons,³ activation of glial and endothelial cells, blood-brain barrier damage, and infiltration of different types of leukocytes after an orchestrated time course.⁴ Given the variety of leukocyte subsets trafficking to the ischemic brain tissue, this review will focus on neutrophils, monocyte/macrophages, and T and NK (natural killer) lymphocytes. For further information, the readers are addressed to previous reviews on dendritic cells⁵ and B lymphocytes.⁶ The different immune cells are considered separately in the next sections, but leukocyte infiltration surely comprises intercellular crosstalks by mechanisms that are not entirely known.

Neutrophils

Neutrophils are among the first cells attracted to the brain after ischemic stroke where they are detected in the microvessels within the first hour⁷ and peak at 1 to 3 days.^4,7,8 Neutrophils are short-lived innate immune cells containing different types of granules with antimicrobial pro-oxidant and proteolytic enzymes that can damage the tissues. Accordingly, neutrophils are regarded as detrimental following compelling evidence associating these cells with blood-brain barrier breakdown and brain injury.^7,9 Also, higher blood neutrophil counts are associated with larger infarct volumes in acute ischemic stroke patients.¹⁰ Nevertheless, the pathogenic role of neutrophils in ischemic stroke is still not conclusive. For instance, there are conflicting results in the literature on the potential benefit of neutrophil depletion in experimental ischemia models.^9,11 Furthermore, we lack entire demonstration that neutrophils reach the ischemic tissue before substantial neuronal death has occurred.¹¹ Nonetheless, neutrophils can exert detrimental effects already from the vessel wall. Adhesion of neutrophils to the inflamed endothelium after ischemia/reperfusion is involved in the no-reflow phenomenon, obstructing blood flow in precapillary arterioles, postcapillary venules, and the capillary bed.^7,12 In addition, neutrophils in the vessel lumen and at perivascular locations can damage the blood-brain barrier by releasing proteolytic enzymes and pro-oxidant molecules (Figure).⁹ Moreover, neutrophils can produce NETs (neutrophil extracellular traps) promoting clot formation.¹³ NETs can precipitate thrombotic events and impair tPA (tissue-type plasminogen activator)-induced thrombolysis.¹⁴ In turn, thrombolysis may exacerbate detrimental effects of neutrophils because tPA promotes neutrophil transmigration to the reperfused tissue by proteolytic activation of plasmin and matrix metalloproteinases.¹⁵ These effects might contribute to explain why neutrophilia and high neutrophil-to-lymphocyte ratio are associated with the risk of hemorrhagic transformation in ischemic stroke patients treated with tPA.¹⁶ After permanent middle cerebral artery occlusion (MCAo) in mice, we observed the formation of intravascular NETs and found NETs in perivascular locations and in the brain parenchyma.¹⁷

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Figure. Schematic representation of leukocyte infiltration to the ischemic brain tissue. Neutrophils are attracted to the activated endothelium and reach perivascular spaces after extravasation from intracerebral venules and leptomeningeal vessels. Activated neutrophils are prothrombotic and can damage the blood-brain barrier (BBB). The presence of neutrophils in the brain parenchyma is observed only under certain circumstances, but the conditions determining that neutrophils remain in perivascular spaces or reach the parenchyma are still poorly defined. Attracted by certain chemokines, immature proinflammatory monocytes infiltrate the ischemic tissue where they mature to macrophages, acquire signs of alternative polarization, and seem to be involved in tissue repair. Current experimental evidence suggests that lymphocytes, in particular T cells and γδ T cells, play detrimental roles in the acute phase of stroke by promoting thromboinflammation and tissue damage. Natural killer (NK) cells are attracted to the ischemic tissue, but their function is not fully clear. RBC indicates red blood cells.

There is also some controversy on whether neutrophils actually reach the ischemic brain parenchyma at all. An elegant study by Enzmann et al¹⁸ noticed the massive accumulation of neutrophils in perivascular spaces surrounding venules within the ischemic tissue after ischemia/reperfusion in mice. Most neutrophils remained perivascular, and only a few were detected in the brain parenchyma.¹⁸ This is an important observation because it highlights that perivascular spaces are a niche for neutrophils where they accumulate after transient MCAo. We also detected neutrophils in perivascular locations and leptomeningeal spaces in the mouse after permanent MCAo.¹⁷ These results suggest that, besides extravasating from intracerebral venules, neutrophils extravasate from leptomeningeal vessels and migrate from the subpial space along the vessels penetrating the cortex.¹⁷ However, our study¹⁷ and previous studies⁷ found neutrophils in the ischemic brain parenchyma using models of permanent MCAo. Ischemic conditions involving severe endothelial damage, vessel rupture, and microbleeds or hemorrhagic transformation, are expected to facilitate the presence of neutrophils in the brain parenchyma. Other conditions, such as high blood glucose, also promote neutrophil infiltration.¹⁹ A recent study²⁰ analyzed the postmortem brain of 16 ischemic stroke patients and confirmed the presence of neutrophils in the leptomeninges and perivascular spaces, but neutrophils were rare in the infarcted parenchyma with the exception of 1 patient deceased 3 days after stroke with no signs of infection. Interestingly, the time to death of this series of patients was 1 day in 2 cases, 3 days in the case above mentioned, and then times ranged from 8 days to 240 days poststroke.²⁰ Neutrophils display a specific time-window of attraction to the damaged tissues after acute injuries, and they have a short life in tissues. Therefore, the time to death of ischemic stroke patients is critical to look for the presence of neutrophils in the brain parenchyma. More studies of human tissue within the first days poststroke are necessary to understand under which conditions neutrophils might gain access to the infarcted brain parenchyma.
Despite many advances, there are aspects of neutrophil behavior in stroke that are still difficult to interpret. For instance, neutrophils with anti-inflammatory and repair phenotypes were found in the ischemic brain tissue of experimental animals,²¹ neutrophils of ischemic stroke patients show a reduced oxidative burst and NET formation,²² and microglia surrounding blood vessels phagocyte neutrophils.²³ The possibility that neutrophils were passive bystanders under some circumstances but active players in others depending on specific features of the ischemic lesion needs further consideration.

Monocyte/Macrophages

After brain ischemia, microglia acquire a reactive morphology resembling macrophages. Classically, immunohistochemical studies have described the presence of reactive microglia/macrophages peaking at ≈4 days postischemia in rats or mice, but it was not possible to distinguish whether these cells derived from resident microglia or they infiltrated from the periphery. Nowadays, flow cytometry, cell type-specific fluorescent reporter mice, adoptive transfer of fluorescent cells, generation of chimeras, and recently identified specific microglia markers, allow differentiating resident reactive microglia from infiltrating macrophages. Monocyte infiltration is detected within the first 24 hours postischemia, peak at 4 days, and some of these cells persist for weeks and acquire features of tissue macrophages. Immature CCR2⁺Ly6C^hi proinflammatory monocytes are the subset of monocytes first attracted to the ischemic brain tissue.^24–26 These cells might be released by the bone marrow, but a study reported that monocytes reaching the ischemic brain originate in the spleen.²⁵
Infiltrating macrophages were classically associated with inflammation and brain damage after ischemic stroke. In mice, monocyte infiltration is largely dependent on CCR2 (C-C motif chemokine receptor type 2), the receptor of the chemokine CCL2 (C-C motif chemokine ligand 2), also known as MCP1 (monocyte chemoattractant protein 1). To investigate the role of monocytes, several studies used CCR2-deficient mice or CCR2 inhibitors, with the limitation that besides the subset of Ly6C^hi monocytes other cells, like some T cells, also express CCR2. CCR2-deficiency reduced the ischemic brain lesions in mice.²⁷ Challenging this view, CCR2 drug inhibitors exacerbated the brain lesion.²⁸ Furthermore, anti-CCR2 blocking antibodies impaired spontaneous long-term functional recovery,²⁹ depletion of monocytes/macrophages worsened the ischemic lesion,³⁰ and infiltrating macrophages prevented hemorrhagic transformation of the ischemic lesion.²⁴ By systemic injection of fluorescent monocytes after brain ischemia, we observed fluorescent cells in the subpial space, and along the vessels penetrating the cortex,²⁶ supporting the view that a subset of infiltrating macrophages establish persistent interactions with the blood vessels (Figure).
The phenotype of activated macrophages depends on the environmental stimuli. The M1 and M2 phenotypes are prototypical states of macrophage polarization achieved in culture after exposure to certain cytokines. The M1 phenotype is proinflammatory whereas the M2 phenotype promotes resolution of inflammation and repair. Macrophages infiltrating the ischemic tissue, including the Ly6C^lo population and some of the Ly6C^hi monocytes, acquire features of alternatively polarized M2 macrophages during the first week postischemia.^26,28–30 Studies of human ischemic infarcts reported that macrophages initially showed proinflammatory features that with lesion maturation transformed into anti-inflammatory phenotypes.²⁰ Interestingly, a study noticed that after ischemia in mice, the expression of M2 markers increased within the first week but then decreased, whereas proinflammatory markers persisted and predominated at week 2, suggesting a long-lasting inflammatory status.³¹
The factors that contribute to the time-dependent changes in macrophage phenotypes in the ischemic brain tissue are not entirely identified. Increased anaerobic glycolysis and activation of the hypoxia-inducible factor-1 are associated with proinflammatory M1 phenotypes, whereas energy production in M2 phenotypes rather relies on fatty acid oxidation.³² In M1 activated macrophages, arginine metabolism occurs through inducible nitric oxide synthase leading to generation of reactive oxygen and nitrogen species that damage proteins, lipids, and DNA. In contrast, M2 macrophages metabolize arginine through arginase-1 generating polyamines involved in cell division and collagen synthesis, among other functions.³² However, under pathological conditions 1 single phenotypic feature may not be sufficient to attribute any specific phenotype to the cells. Likely, cellular metabolic adaptations to ischemia and reperfusion together with the cytokine environment and phagocytic activity have an impact on the phenotype and function of macrophages and microglia.

Lymphocytes

T Cells

Severe stroke reduces the numbers of lymphocytes in the circulation and lymphoid organs.³³ In contrast, T-cell numbers increase in the ischemic brain within the first 24 hours and can persist for long times.⁴ During the first hours after ischemia/reperfusion, T cells facilitate adhesion of platelets and leukocytes to the vascular endothelium³⁴ causing a phenomenon called thromboinflammation³⁵ by which molecular and cellular players in thrombosis and coagulation promote proinflammatory pathways exacerbating the brain lesion.³⁶ However, the interaction of T cells with platelets may also have hemostatic effects preventing hemorrhagic transformation after severe ischemic stroke.³⁷ T cells are found in subpial and cortical vessels and infiltrating the ischemic lesion.^38,39 In addition, the choroid plexus is a gateway for T cells migrating to the periphery of cortical infarction.⁴⁰ Importantly, CD8⁺ cytotoxic T cells were detected in human ischemic infarcts.²⁰ Also, ischemic stroke patients show increased frequency of CD4⁺CD28^null cells in blood associated with stroke severity and serum levels of proinflammatory cytokines.⁴¹ CD4⁺CD28^null T cells are an interesting subset of T cells because they have enhanced effector functions, are associated with senescent T cells, and expand under inflammatory conditions.⁴² At later phases, CD4⁺ cells accumulate in the brain of mice peaking at day 14 and persisting at day 30 after ischemia/reperfusion.⁴³ Furthermore, emerging evidence suggests that antigen-mediated T-cell responses take place in subacute or chronic stages after stroke and may worsen stroke outcome.^43–47 However, in central nervous system trauma, protective autoimmunity mediated by T-cell responses is involved in promoting recovery.⁴⁸ Overall, T cells seem to play innate functions and interact with players in thrombosis and hemostasis in the acute phase of stroke, whereas at later stages they exert adaptive functions that could affect stroke outcome in the long term.

γδ T Cells

Subsets of unconventional innate T cells with invariant T-cell receptor could play a role in acute ischemic brain damage. Growing evidence supports that γδ T cells are pathogenic in experimental brain ischemia/reperfusion by secreting IL (interleukin)-17 and exacerbating the inflammatory response.^49–51 Moreover, IL-17A⁺ lymphocytes were detected in the postmortem brain of stroke patients.⁵⁰ Interestingly, γδ T cells are abundant in the gut from where they seem to traffic to the leptomeninges after brain ischemia.⁵² T helper 17 cells (Th17) and γδ T cells increase in the blood of stroke patients in association with increased levels of IL-17A, IL-23, IL-6, and IL-1β.⁵³ In spite of the fact that IL-17 producing cells are a small subset of cells, they seem to play a prominent role in orchestrating the inflammatory response in acute stroke and exacerbating the lesion (Figure).

Regulatory Lymphocytes

Regulatory lymphocytes exert immunomodulatory and immunosuppressor functions. Several lines of evidence support beneficial effects of regulatory T cells (Treg)⁵⁴ and regulatory B cells⁵⁵ in experimental brain ischemia. However, other studies found acute detrimental effects of Treg in brain ischemia/reperfusion⁵⁶ as previously reviewed.⁵⁷ Although the number of Treg found in the ischemic brain parenchyma during the first days poststroke is low, Treg strongly accumulate in the ischemic lesion 15 days poststroke where potentially they could inhibit autoimmune responses.⁴³ Increased apoptosis of Tregs, loss of Tregs in peripheral blood, and impaired suppressive function of the remaining Treg population has been reported in ischemic stroke patients.^53,58,59 However, other studies reported upregulation of Tregs in stroke patients in spite that decreased Treg function was observed, particularly in female patients.⁶⁰ Notably, an increased proportion of Treg cells was reported in the spleen of mice 4 days after transient MCAo.⁶¹

NK Cells

NK innate lymphocytes show a rapid and transient increase in the ischemic brain tissue.^4,8 A study reported no benefits of depleting NK cells in permanent or transient MCAo.⁴⁴ In contrast, another study suggested pathogenic actions of NK cells by promoting inflammation and neuronal cytotoxicity.⁶² This study reported infiltration of NK cells in the ischemic brain tissue of humans and mice where NK cell numbers peaked as soon as 3 hours postischemia and then declined.⁶² Interestingly, the β2-nACh-R (nicotinic acetylcholine) receptor seems to be involved in the NK cell decline observed in the ischemic tissue from 3 hours postischemia.⁶³ Induced-persistence of NK cells in the ischemic tissue achieved by interfering with this cholinergic receptor did not modify lesion size but increased systemic IFNγ (interferon γ), protected from bacterial infection, and enhanced poststroke survival.⁶³ More studies are needed to validate the putative capacity of brain infiltrating NK cells to prevent poststroke infection, the role of central acetylcholine in this process, as well as the suggested rapid pathogenic effect of NK cells worsening the acute ischemic brain lesion.

Therapeutic Intervention

Strategies designed to prevent negative actions of leukocytes have been taken to the clinic in acute ischemic stroke patients but with no success to date.⁶⁴ Several studies with drugs blocking the action of neutrophils were investigated.^11,64 As an example, the ASTIN trial (Acute Stroke Therapy by Inhibition of Neutrophils) investigated a compound known as UK-279 276, a recombinant neutrophil inhibitory factor that selectively binds the CD11b integrin of macrophage-1 antigen (CD11b/CD18).⁶⁵ The treatment did not improve recovery above placebo. This trial followed encouraging results of a few preclinical studies with UK-279 276 in experimental models of brain ischemia/reperfusion.⁶⁶ However, only a fifth of patients in the ASTIN trial received tPA.⁶⁵
Monocyte/macrophages may acutely exacerbate the inflammatory responses, but experimental studies have identified their involvement in resolution of inflammation, vascular protection, and recovery of function,^24,27–29 possibly linked to the phagocytic and vasculoprotective roles of these cells. These protective actions of monocytes are in line with the beneficial effects of administration of autologous bone marrow–derived mononuclear cells (MNC) after experimental ischemic stroke.⁶⁷ MNC contain myeloid and lymphoid cells, as well as hematopoietic and mesenchymal stem cells. MNC administration 24 hours after MCAo improved functional recovery, reduced lesion size and proinflammatory cytokines, and enhanced vessel density and neurogenesis,⁶⁸ and these benefits were long lasting.⁶⁹ Furthermore, MNC reduced blood-brain barrier permeability and decreased the severity of hemorrhagic transformation after tPA in an embolic stroke model.⁷⁰ However, MNC therapy did not improve outcome in hypertensive rats.⁷¹ MNC reach the periphery of brain infarction soon after administration,⁶⁸ and then the cells seem to differentiate into smooth muscle cells and endothelial cells, incorporate into vessel walls, and enhance the growth of leptomeningeal anastomoses, the circle of Willis, and basilar arteries.⁶⁹ Phase I trials administering MNC to ischemic stroke patients have shown safety, but the clinical efficacy of this cell therapy awaits demonstration.⁷²
Anti-inflammatory treatments, such as minocycline, have not been successful in the clinic.⁶⁴ Experimental evidences, including a cross-laboratory preclinical study in mouse models of brain ischemia,⁷³ support the therapeutic potential of the IL-1Ra (IL-1β receptor antagonist). A recent clinical trial with subcutaneous administration of IL-1Ra showed safety and reduction of plasma IL-6.⁷⁴ However, the analysis excluded a major clinical benefit of the treatment, and negative effects potentially attributable to interactions of IL-1Ra with tPA became apparent.⁷⁴
Experimental studies support damaging effects of T lymphocytes in the acute phase of stroke. Accordingly, fingolimod, a drug approved for remitting-relapsing multiple sclerosis that sequesters lymphocytes in the lymph nodes preventing lymphocyte access to the inflamed tissues, showed beneficial effects in preclinical studies and small clinical trials in acute ischemic stroke patients, including patients receiving thrombolysis.⁶⁴ By acting on S1P1 (sphingosine-1-phosphate receptor 1), fingolimod induces sustained lymphopenia, but current data do not show higher incidence of poststroke infection in patients receiving fingolimod. Fingolimod also acts on endothelial S1P1 receptor increasing vascular barrier function that might contribute to the observed benefits of this drug in ischemic stroke. Given that the benefits of fingolimod seem to be mediated by S1P1, whereas certain side effects are dependent on other S1P receptors, selective S1P1 agonists were studied in experimental stroke showing reduced lesion size after ischemia/reperfusion in mice.⁷⁵ In contrast to the benefits of blocking T-cell trafficking, systemic administration of regulatory T lymphocytes in rodent models of ischemic stroke reduced infarct size, ameliorated the neurological functions,⁷⁶ and reduced hemorrhagic transformation after tPA.⁷⁷
Leukocyte recruitment to inflammatory sites is attenuated by blocking α4β1 integrin (VLA-4 [very late antigen-4]) with natalizumab, an antibody in clinical use for multiple sclerosis treatment. Blockade of VLA-4 with CD49d antibody was investigated in a multicentric preclinical study in mice using 2 different models of cerebral ischemia.⁷⁸ CD49d antibody attenuated leukocyte infiltration and reduced infarct volume in small cortical lesions but not in large infarctions. Natalizumab was investigated in ischemic stroke patients in the ACTION trial (Effect of Natalizumab on Infarct Volume in Acute Ischemic Stroke).⁷⁹ Natalizumab did not meet the primary end point of the study, but secondary and exploratory end points suggested improvement of clinical outcomes,^64,79 encouraging the second ACTION2 trial. This phase-IIb trial was recently completed and the notes released by the sponsor (Biogen) state that natalizumab did not improve clinical outcomes compared with placebo.

Final Remarks

Experimental studies support detrimental effects of certain types of leukocytes in acute ischemic stroke. However, to date, this knowledge has not been translated into clinical treatments. No doubt immunomodulatory interventions in the acute phase of stroke need fine-tuning and long-term experimental studies to ensure that repair processes in subacute and chronic phases are not disturbed and the neurological deficits are attenuated. Cell therapies based on administration of autologous MNC have shown promising results in preclinical studies by promoting functional recovery, but clinical efficacy remains to be demonstrated. Results of various experimental models of brain ischemia suggest the possibility that the putative pathogenic contribution of certain leukocytes to the acute ischemic lesion might differ depending on lesion severity, regions affected, and degree of reperfusion. Importantly, most of the studies described above were obtained in young healthy male mice in spite of the fact that aging, sex, and comorbidities influence the phenotype and function of immune cells. Identification of the ischemic conditions where leukocytes might have a meaningful contribution to the brain lesion, the relevant subsets of leukocytes, and the time-window for intervention, requires more investigation. Combining immunomodulatory strategies with reperfusion therapies offer the opportunity to attenuate negative responses of the immune system that might impair reperfusion at the microvascular bed or trigger detrimental effects on the brain tissue.

Acknowledgments

I acknowledge relevant studies used to prepare this article that could not be cited because of word count restriction.

Sources of Funding

Supported by the Spanish Ministerio de Economía y Competitividad (SAF2017-87459-R).

Disclosures

None.

Footnotes

Correspondence to Anna M. Planas, PhD, Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Rosselló 161, Planta 6, 08036-Barcelona, Spain. Email anna.planas@iibb.csic.es