Lipocalin-2 as an Infection-Related Biomarker to Predict Clinical Outcome in Ischemic Stroke

Objectives From previous data in animal models of cerebral ischemia, lipocalin-2 (LCN2), a protein related to neutrophil function and cellular iron homeostasis, is supposed to have a value as a biomarker in ischemic stroke patients. Therefore, we examined LCN2 expression in the ischemic brain in an animal model and measured plasma levels of LCN2 in ischemic stroke patients. Methods In the mouse model of transient middle cerebral artery occlusion (tMCAO), LCN2 expression in the brain was analyzed by immunohistochemistry and correlated to cellular nonheme iron deposition up to 42 days after tMCAO. In human stroke patients, plasma levels of LCN2 were determined one week after ischemic stroke. In addition to established predictive parameters such as age, National Institutes of Health Stroke Scale and thrombolytic therapy, LCN2 was included into linear logistic regression modeling to predict clinical outcome at 90 days after stroke. Results Immunohistochemistry revealed expression of LCN2 in the mouse brain already at one day following tMCAO, and the amount of LCN2 subsequently increased with a maximum at 2 weeks after tMCAO. Accumulation of cellular nonheme iron was detectable one week post tMCAO and continued to increase. In ischemic stroke patients, higher plasma levels of LCN2 were associated with a worse clinical outcome at 90 days and with the occurrence of post-stroke infections. Conclusions LCN2 is expressed in the ischemic brain after temporary experimental ischemia and paralleled by the accumulation of cellular nonheme iron. Plasma levels of LCN2 measured in patients one week after ischemic stroke contribute to the prediction of clinical outcome at 90 days and reflect the systemic response to post-stroke infections

Microglial autophagy-associated phagocytosis is essential for recovery from neuroinflammation

Multiple sclerosis (MS) is a leading cause of incurable progressive disability in young adults caused by inflammation and neurodegeneration in the central nervous system (CNS). The capacity of microglia to clear tissue debris is essential for maintaining and restoring CNS homeostasis. This capacity diminishes with age, and age strongly associates with MS disease progression, although the underlying mechanisms are still largely elusive. Here, we demonstrate that the recovery from CNS inflammation in a murine model of MS is dependent on the ability of microglia to clear tissue debris. Microglia-specific deletion of the autophagy regulator Atg7, but not the canonical macroautophagy protein Ulk1, led to increased intracellular accumulation of phagocytosed myelin and progressive MS-like disease. This impairment correlated with a microglial phenotype previously associated with neurodegenerative pathologies. Moreover, Atg7-deficient microglia showed notable transcriptional and functional similarities to microglia from aged wild-type mice that were also unable to clear myelin and recover from disease. In contrast, induction of autophagy in aged mice using the disaccharide trehalose found in plants and fungi led to functional myelin clearance and disease remission. Our results demonstrate that a noncanonical form of autophagy in microglia is responsible for myelin degradation and clearance leading to recovery from MS-like disease and that boosting this process has a therapeutic potential for age-related neuroinflammatory conditions.Swedish Research CouncilSwedish Brain FoundationSwedish Association for Persons with Neurological DisabilitiesStockholm County Council (ALF project)AstraZeneca (AstraZeneca-Science for Life Laboratory collaboration)European Union Horizon 2020/European Research Council Consolidator Grant (Epi4MS)Knut and Alice Wallenbergs FoundationMargeretha af Ugglas FoundationAlltid Litt SterkereFoundation of Swedish MS researchNEURO SwedenKarolinska InstitutetAccepte

A Call to Use the Multicomponent Exercise Tai Chi to Improve Recovery From COVID-19 and Long COVID

Approximately 10% of all COVID patients develop long COVID symptoms, which may persist from 1 month up to longer than 1 year. Long COVID may affect any organ/system and manifest in a broad range of symptoms such as shortness of breath, post-exercise malaise, cognitive decline, chronic fatigue, gastrointestinal disorders, musculoskeletal pain and deterioration of mental health. In this context, health institutions struggle with resources to keep up with the prolonged rehabilitation for the increasing number of individuals affected by long COVID. Tai Chi is a multicomponent rehabilitation approach comprising correct breathing technique, balance and neuromuscular training as well as stress- and emotional management. In addition, practicing Tai Chi elicits the relaxation response and balances the autonomic nervous system thus regulating respiration, heart rate, blood pressure and vitality in general. Moreover, Tai Chi has been shown to increase lung capacity, improve cognitive status and mental health, and thereby even the quality of life in diseases such as chronic obstructive pulmonary disease (COPD). Hence, we advocate Tai Chi as potent and suitable rehabilitation tool for post-COVID-19-affected individuals

Efficacy of vitamin D in treating multiple sclerosis-like neuroinflammation depends on developmental stage

AbstractThe association of vitamin D deficiency with higher prevalence, relapse rate and progression of multiple sclerosis (MS) has stimulated great interest in using vitamin D supplementation as a preventative measure and even a therapy for established MS. However, there is a considerable lack of evidence when it comes to an age/developmental stage-dependent efficacy of vitamin D action and a time-window for the most effective prophylactic treatment remains unclear.We studied the effect of vitamin D supplementation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS, at three different developmental stages in rats. Supplementation treatment was initiated: i) prior to gestation and maintained throughout pre- and early postnatal development (gestation and lactation); ii) after weaning, throughout juvenile/adolescence period and iii) in adult age. We observed a marked attenuation of EAE in juvenile/adolescent rats reflected in a less severe CNS inflammation and demyelination, accompanied by a lower amount of IFN-γ producing MOG-specific T cells. Moreover, the cytokine expression pattern in these rats reflected a more anti-inflammatory phenotype of their peripheral immune response. However, the same supplementation regimen failed to improve the disease outcome both in adult rats and in rats treated during pre- and early post-natal development.Our data demonstrate a developmental stage-dependent efficiency of vitamin D to ameliorate neuroinflammation, suggesting that childhood and adolescence should be the target for the most effective preventive treatment

Imatinib Ameliorates Neuroinflammation in a Rat Model of Multiple Sclerosis by Enhancing Blood-Brain Barrier Integrity and by Modulating the Peripheral Immune Response

<div><p>Central nervous system (CNS) disorders such as ischemic stroke, multiple sclerosis (MS) or Alzheimeŕs disease are characterized by the loss of blood-brain barrier (BBB) integrity. Here we demonstrate that the small tyrosine kinase inhibitor imatinib enhances BBB integrity in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). Treatment was accompanied by decreased CNS inflammation and demyelination and especially reduced T-cell recruitment. This was supported by downregulation of the chemokine receptor (CCR) 2 in CNS and lymph nodes, and by modulation of the peripheral immune response towards an anti-inflammatory phenotype. Interestingly, imatinib ameliorated neuroinflammation, even when the treatment was initiated after the clinical manifestation of the disease. We have previously shown that imatinib reduces BBB disruption and stroke volume after experimentally induced ischemic stroke by targeting platelet-derived growth factor receptor -α (PDGFR-α) signaling. Here we demonstrate that PDGFR-α signaling is a central regulator of BBB integrity during neuroinflammation and therefore imatinib should be considered as a potentially effective treatment for MS.</p> </div

Imatinib reduces infiltration of immune cells and attenuates microglia activation.

<p>IHC analysis on paraffin embedded spinal cord tissue cross-sections on day 10 p.i. (A–D; n = 8 rats/experimental group; representative images shown) and 14 p.i. (E–H; n = 5 rats/experimental group; representative images shown). Although showing no sign of CNS inflammation and demyelination, control animals already started recruiting W3/13⁺ T-cells (B) and ED1⁺ macrophages (D) to the meninges and in the perivascular space, whereas the imatinib-treated group showed a delay in recruitment of inflammatory cells to the CNS. On day 14 p.i., spinal cords of the imatinib-treated rats exhibiting demyelinated lesions recruited lower amounts of W3/13⁺ T-cells (G, H) while ED1⁺ macrophages infiltration was similar to the controls (E, F). Scale bar, 200 µm (A–H). (I–Z) IF performed on spinal cord cross-sections of rats injected with fluorescent tracer (dextran, red) on day 14 p.i. α-Ox-42, ED1, Ox-6, Ox-22, CD45RA and W3/13 antibody staining (all in green) in imatinib- (I, L, O, R, U, X) and PBS-treated rats (J, M, P, S, V, Y). (I–K) Microglia activation was significantly decreased in the imatinib-treated rats, while Ox-42⁺ cells were detectable around leaky blood vessels (asterix) in the control tissue. (L–N) The amount of macrophages/activated microglia cells was significantly decreased in the spinal cords of the imatinib-treated rats. (O–Q) Significantly lower amounts of MHC class II⁺ cells were found in the meninges and parenchyma of the imatinib-treated rats vs. PBS controls. (R–Z) Significantly lower amounts of Ox-22⁺, CD45RA⁺ and W3/13⁺ cells were found in the meninges and parenchyma of the imatinib-treated rats vs. PBS controls (R, U, X vs. S, V, Y). Quantifications of Ox-42, ED1, Ox-6, Ox-22, CD45RA and W3/13 expression based on green fluorescent pixel area quantifications from spinal cord cross-sections (K, N, Q, T, W, Z). n = 5 rats/experimental group. Scale bar, 50 µm. Error bars, S.E.M. Statistics were calculated using t-test and <i>P</i> values <0.05 were considered significant (<i>P</i><0.05 = *, <i>P</i><0.01 = **, <i>P</i><0.001 = ***). Imatinib or PBS oral gavage was performed from day 5 p.i until the end of the experiment.</p

Imatinib enhances BBB integrity during EAE.

<p>(A, B) Extravasation of 70 kDa dextran in wholemount thoracic and lumbar spinal cord portions on day 10 and 14 p.i. Imatinib-treated rats exhibited less extravasation on both time-points, compared to PBS controls. Tracer-injected naive rat is shown as negative control. (C) Dextran extravasation in spinal cord cross-sections. PBS-treated rats showed distinct meningeal permeability on day 10 p.i., which was not observed in the imatinib-treated animals. On day 14 p.i., imatinib-treated rats showed less signs of BBB disruption in contrast to profound extravasation in the meninges and perivascular space in both gray and white matter of the control spinal cords (white arrows). (D) Quantification of vascular permeability on day 10 and 14 p.i. based on red fluorescent pixel area recorded in spinal cord sections and wholemounts (n = 5 rats/experimental group/time-point). Scale bars, 1 mm (A, B) and 50 µm (C). Error bars, S.E.M. Statistics were calculated using t-test and <i>P</i> values <0.05 were considered significant. <i>P</i><0.05 = *, <i>P</i><0.01 = **, <i>P</i><0.001 = ***. IHC analysis of paraffin embedded spinal cord tissue sections on day 10 p.i. (E–H) and 14 p.i. (I–P). α-dysferlin was used for detecting permeable CNS vasculature and α-occludin for detecting the tight junction components. (E–H) Healthy animals from both groups were compared on day 10 p.i. (n = 8 rats/experimental group; representative images shown). Almost total absence of dysferlin⁺ blood vessels observed in the spinal cord gray matter of the imatinib group (E), while PBS controls exhibited dysferlin⁺ vessels more frequently (F). Occludin⁺ blood vessels were rarely detectable in both groups (G, H). (I–P) On day 14 p.i. (n = 5 rats/experimental group; representative images shown), spinal cord tissues from the same anatomical positions undergoing EAE from both groups were compared. Demyelinated lesions and lesion-associated blood vessels in the imatinib-treated rats expressed predominantly occludin (K, O), while dysferlin upregulation prevailed in the lesions of the PBS controls (J, N). Scale bars, 100 µm (E–H, M–P), 250 µm (I–L). Imatinib or PBS oral gavage was performed from day 5 p.i until the end of the experiment (A–P).</p

Imatinib inhibits migration of T-cells into the CNS by downregulating chemokine expression in endothelial cells and not by altering recruitment of naïve T-cells into draining lymph nodes.

<p>(A) Gene expression profiling in inguinal lymph nodes day 2 p.i. by qPCR. Neither mRNA transcript levels of CD34 and Glycam-1, adhesion markers expressed at the HEV nor CCR7 and L-selectin, homing markers on naïve T-cells were differentially expressed in imatinib vs. control treated mice (n = 4 mice/experimental group, both inguinal lymph nodes/animal). Imatinib or PBS oral gavage was performed from the immunization day until the end of the experiment (day 2 p.i.). (B) Endothelial vessel fragments (EVF) were biochemically isolated from the spinal cord of imatinib or PBS treated mice day 13 p.i. Gene expression profiling by qPCR revealed that P-selectin, CCL2, CCL19 and CXCL2 but not VCAM-1 and ICAM-1 were downregulated in imatinib-treated mice (n = 5 mice/experimental group). Imatinib or PBS oral gavage was performed from day 2 p.i. until day 13 p.i. (C–D). Evaluation of different T-cell subsets in spinal cords from imatinib-treated and control rats revealed less overall infiltration of both CD3⁺/CD8⁺ as well as CD3⁺/CD8⁻ cells in response to imatinib. The relative proportion of CD3⁺/CD8⁺ and CD3⁺/CD8⁻ cells was equal between the treatments. Scale bar, 50 µm. Imatinib or PBS oral gavage was performed from day 5 p.i until the end of the experiment. Error bars, S.E.M., Statistics were calculated using t-test and <i>P</i> values <0.05 were considered significant (<i>P</i><0.05 = *, <i>P</i><0.01 = **, <i>P</i><0.001 = ***).</p

Imatinib suppresses the peripheral immune response.

<p>(A–D) Genome wide expression array analysis performed on inguinal lymph node cells harvested from imatinib-treated and control rats on day 10 p.i. (Affymetrix 1.0 ST. 3' arrays; n = 6 arrays/experimental group). (A) Functional annotations differentially regulated between imatinib and PBS-treated rats. Immune cell trafficking was profoundly downregulated in the imatinib group (red pie-chart), as well as numerous immune functions (blue pie-chart). Numbers indicate the amount of molecules differentially expressed in the certain biological function. (B–D) Canonical pathways most significantly affected by imatinib treatment. Leucocyte extravasation was downregulated in the imatinib-treated rats, especially matrix metalloproteinases and CXCR3 (B). The anti-inflammatory interleukine response is also downregulated in the imatinib group in contrast to controls (C). The communication between the innate and adaptive immune response, especially Toll-like receptor (Tlr) signaling is generally downregulated in the imatinib group (D). Statistics are calculated using t-test and calculated <i>P</i> values indicated high significance for each presented molecule <i>P</i><0.00001 = ***). Error bars (not visible), S.E.M. (E) Gene expression profiling in inguinal lymph nodes day 10 p.i. by qPCR. mRNA transcript levels for Th2-cell lineage proliferation: <i>IL4</i> and <i>STAT6</i> are higher in imatinib-treated rats., whereas control rats showed elevated mRNA levels for <i>TLR2</i> and <i>CD4</i> transcripts (n = 8 rats/experimental group, both inguinal lymph nodes/animal). (F) MOG-induced IFNγ Elispot analysis on imatinib-treated and control rat spleenocytes harvested on day 10 p.i. ConA used as a positive control, MBP as an unspecific antigen (n = 4 rats/experimental group). Imatinib-treated rats had significantly lower number of proliferating MOG specific T-cells comparing to the controls. (G–H) MOG re-stimulation assay with spleenocytes harvested from imatinib-treated or control mice on day 7 p.i. (n = 4 mice/experimental group). Levels of Th1/Th2 specific cytokines measured after three days <i>in vitro</i> culturing in the presence of MOG, MBP or ConA. (A–F) Imatinib or PBS oral gavage was performed from day 5 p.i until the end of the experiment. (G–H) Imatinib or PBS oral gavage was performed from day 2 p.i until the end of the experiment Error bars, S.E.M. Statistics were calculated using t-test and <i>P</i> values <0.05 were considered significant (<i>P</i><0.05 = *, <i>P</i><0.01 = **, <i>P</i><0.001 = ***).</p