N-acetyl aspartate levels early after ischemic stroke accurately reflect long-term brain damage.

Background: Estimation of brain damage following an ischemic stroke is most often performed within the first few days after the insult, where large amounts of oedematous fluid have accumulated. This can potentially hamper correct measurement of infarcted area, since oedema formation poorly reflects infarct size. This study presents a non-invasive, easily applicable and reliable method to accurately predict long-term evolution and late-stage infarction. Objective: We performed a longitudinal analysis of brain infarct evolution after MCAO in mice, in order to determine whether water-compensated N-Acetylaspartate (NAA) levels in the infarct area, measured 24 h after the insult, is a suitable marker for late-stage infarction and thereby prognosis. Methods: Twenty mice were divided into 4 groups and scanned longitudinally at different time-points after MCAO, followed by euthanisation for histology: Group 1) MRI/MRS at day 1 after MCAO (n = 4), Group 2) MRI/MRS at days 1 and 7 after MCAO (n = 5), Group 3) MRI/MRS at days 1, 7, and 14 after MCAO (n = 3), and Group 4) MRI/MRS at days 1, 7, 14, and 28 after MCAO (n = 4). At days 1, 7, 14, and 28, NAA levels were correlated with histological determination of neuronal death based on Nissl and H&E stainings. Results: Twenty-four hours after the insult, NAA levels in the infarcted area decreased by 35 %, but steadily returned to normal after 28 days. In the acute phases, NAA levels strongly correlated with loss of Nissl substance (r2 = -0.874, p = 0.002), whereas NAA levels in later stages reflect glial metabolism and tissue reorganisation. Most importantly, NAA levels 24 h after MCAO was highly correlated with late stage infarction at days 14 and 28 (r2 = 0.73, p = 0.01), in contrast to T2 (r2 = 0.06, p = 0.59). Conclusions: By using a fixed voxel, which is easily positioned in the affected area, it is possible to obtain reliable measures of the extent of neuronal loss at early time points independent of oedema and brain deformation. Importantly, NAA levels 24 h after MCAO accurately reflects late-stage infarction, suggesting that NAA is a useful prognostic biomarker early after an ischemic stroke.

Long-term immune cell profiling in stroke patients with or without infections.

PURPOSE: Infections are frequent complications in acute ischemic stroke and may be caused by an altered immune response influencing brain damage. We compared long-term immune responses in stroke patients with or without infections during the recovery period by performing a long-term profiling of clinically relevant inflammatory parameters from stroke onset until day 49. MATERIALS AND METHODS: Thirty-four stroke patients were retrospectively included and divided into two groups depending on infection status. Group 1 had no infections (N = 17) and group 2 had post-admission infection (N = 17). The patients were evaluated carefully for infections and evolution of the peripheral inflammatory response. Neutrophils, monocytes, lymphocytes, total leukocytes and C-reactive protein were evaluated in relation to the occurrence and development of infections. In both patient groups, an acute boost in neutrophils and monocytes were observed whereas the opposite was true for lymphocytes. RESULTS: In Group 1, neutrophils and monocytes approached normal levels after 20-30 days, but remained elevated in Group 2. We found an increase in neutrophils (p = 0.01) and leukocytes (p < 0.01) as well as C-reactive protein (p < 0.01) among infected patients. Lymphocytes remained depressed in Group 2, while Group 1 slowly approached baseline levels. In both groups, CRP levels initially increased with a slow return to baseline levels. From day 0 to 49 after stroke, uninfected patients generally experienced a decline in leukocytes, neutrophils and monocytes (all p < 0.05), while no similar changes happened among infected patients. CONCLUSIONS: Our study provides an overview of general immune cell kinetics after stroke related to infection status. Immune cell numbers were severely disturbed for weeks after the insult, independent of infection status, although infected patients achieved the highest cell counts of neutrophils, leukocytes and for C-reactive protein. The sustained depression of lymphocytes, especially and paradoxically among infected patients, warrants future studies into the mechanisms behind this, with potential for future therapies aimed at restoring normal immunity and thereby improving patient outcome.

Multiple Soluble Components of the Glycocalyx Are Increased in Patient Plasma After Ischemic Stroke.

Background and Purpose- The GLX (glycocalyx) is a protein/polysaccharide meshwork at the cellular surface. Consisting largely of glycosaminoglycans and proteoglycans, the GLX can shed in response to stress. In this study, we assay 11 components of the GLX in plasma from patients with ischemic stroke from a longitudinal cohort. Methods- Plasma samples from healthy individuals (N=8), and patients with ischemic stroke day ≥3, day 7, and day 90 (N=9-14) were immunoassayed for diverse components of the GLX. Results- Median stroke severity was mild (National Institutes of Health Stroke Scale 2.0 (range, 0-6) at day ≤3). Three (keratan-chondroitin-heparan-sulfate) of 4 glycosaminoglycans and CD44 (proteoglycan) were increased at day 7 and returned to baseline at day 90. Proteoglycan syndecan (Syn)-3 increased and Syn-2 levels decreased, significantly. Conclusions- Individual GLX components are often assayed as stand-alone biomarkers for endothelial health. This study suggests a full assessment of GLX components is more indicative of the endothelial health of an individual and represents a complex GLX signature that may be valuable as a composite biomarker of disease.

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor.

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

Statin treatment before stroke reduces pro-inflammatory cytokine levels after stroke.

OBJECTIVE: In this clinical case-control study, we investigated statin treatment in stroke patients on a range of inflammatory effectors in peripheral blood. We focus on RhoA GTPase and its downstream effectors as a future inflammatory target in stroke treatment. METHODS: Data from 10 patients already on statins at stroke onset (Pre-S group) was compared with data from both 29 patients starting statin treatment right after stroke onset (Post-S group) and with 8 healthy controls. In T-cells isolated from stroke patients, we analyzed the activity of the main cytoskeletal regulator RhoA GTPase and its downstream effectors: rho-associated protein kinase (ROCK), myosin phosphatase targeting protein subunit 1 (pMYPT1), myosin light chain kinase (pMLC) and cofilin. In the blood samples, we further determined levels of 12 key plasma cytokines as well as C-reactive protein (CRP) and kallikrein. RESULTS: Compared to healthy controls, the Post-S group achieved significantly higher RhoA and ROCK activities, while the Pre-S did not differ from controls. Levels of pMYPT1, pMLC and cofilin did not differ from controls in the Pre-S and Post-S groups. At day 90 after stroke, interferon γ and IL-18 were significantly increased in the Post-S group compared to the Pre-S group. We found a positive correlation between CRP and NIHSS, whereas kallikrein levels showed no correlation with NIHSS at any of the days. CONCLUSION: Stroke induces changes in the RhoA-ROCK pathway in T-cells. CRP and NIHSS score correlated positively in the study. Statins may have an anti-inflammatory effect as statin treatment before stroke reduces post-stroke pro-inflammatory levels. RhoA GTPase and its downstream effectors are possibly the key to improve statin treatment in stroke.

RhoA Drives T-Cell Activation and Encephalitogenic Potential in an Animal Model of Multiple Sclerosis.

T-cells are known to be intimately involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). T-cell activation is controlled by a range of intracellular signaling pathways regulating cellular responses such as proliferation, cytokine production, integrin expression, and migration. These processes are crucial for the T-cells' ability to mediate inflammatory processes in autoimmune diseases such as MS. RhoA is a ubiquitously expressed small GTPase well described as a regulator of the actin cytoskeleton. It is essential for embryonic development and together with other Rho GTPases controls various cellular processes such as cell development, shaping, proliferation, and locomotion. However, the specific contribution of RhoA to these processes in T-cells in general, and in autoreactive T-cells in particular, has not been fully characterized. Using mice with a T-cell specific deletion of the RhoA gene (RhoAfl/flLckCre+), we investigated the role of RhoA in T-cell development, functionality, and encephalitogenic potential in EAE. We show that lack of RhoA specifically in T-cells results in reduced numbers of mature T-cells in thymus and spleen but normal counts in peripheral blood. EAE induction in RhoAfl/flLckCre+ mice results in significantly reduced disease incidence and severity, which coincides with a reduced CNS T-cell infiltration. Besides presenting reduced migratory capacity, both naïve and autoreactive effector T-cells from RhoAfl/flLckCre+ mice show decreased viability, proliferative capacity, and an activation profile associated with reduced production of Th1 pro-inflammatory cytokines. Our study demonstrates that RhoA is a central regulator of several archetypical T-cell responses, and furthermore points toward RhoA as a new potential therapeutic target in diseases such as MS, where T-cell activity plays a central role.

Detection of Glycan Shedding in the Blood: New Class of Multiple Sclerosis Biomarkers?

Introduction: Multiple sclerosis (MS) is a devastating autoimmune disease, afflicting people in the prime of their lives. Presently, after initial clinical presentation, there are no reliable markers for whether a patient will develop MS, or whether their prognosis will be aggressive or relapsing-remitting. Furthermore, many MS patients do not respond to treatment. Thus, markers for diagnosis, prognosis, and treatment-responsiveness are lacking for a disease, where a precision medicine approach would be valuable. The glycocalyx (GLX) is the carbohydrate-rich outer surface of the blood vessel wall and is the first interaction between the blood and the vessel. We hypothesized that cleavage of the GLX may be an early stage predictor of immune attack, blood-brain barrier (BBB) breakdown, and disease severity in MS. Methods: Two experimental models of MS, experimental autoimmune encephalitis (EAE), were included in this study. EAE was induced in C57BL/6J mice and Lewis rats, which were monitored for weight loss and clinical presentation in comparison to healthy controls. Plasma samples were obtained longitudinally from mice until peak disease severity and at peak disease severity in rats. Soluble GLX-associated glycosaminoglycans (GAG) and proteoglycans (PG) were detected in plasma samples. Results: All animals receiving EAE emulsion developed fulminant EAE (100% penetrance). Increased plasma levels of chondroitin sulfate were detected before the onset of clinical symptoms and remained elevated at peak disease severity. Hyaluronic acid was increased at the height of the disease, whereas heparan sulfate was transiently increased during early stages only. By contrast, syndecans 1, 3, and 4 were detected in EAE samples as well as healthy controls, with no significant differences between the two groups. Discussion: In this study, we present data supporting the shedding of the GLX as a new class of biomarker for MS. In particular, soluble, sugar-based GLX components are associated with disease severity in two models of MS, molecules that would not be detected in proteomics-based screens of MS patient samples. Patient studies are presently underway.

T-cells and macrophages peak weeks after experimental stroke: Spatial and temporal characteristics.

The activities of the central and peripheral immune systems impact neurological outcome after ischemic stroke. However, studies investigating the temporal profile of leukocyte infiltration, especially T-cell recruitment, are sparse. Our aim was to investigate leukocyte infiltration at different time points after experimental stroke in mice. Permanent middle cerebral artery occlusion was performed on 11 weeks old C57BL/6J mice, allowed to survive for 1, 3, 8, 14 or 28 days. In addition to infarct size measurements, detailed immunohistochemical analyses of T-cell and macrophage influx were performed. A recently introduced F-19 MR probe (V-sense), designed to track macrophages, was furthermore tested. Fourteen and 28 days after permanent middle cerebral artery occlusion a significant increase in CD3+ T-cells was found within the ipsilateral hemisphere compared to controls, especially within the infarct core and the corpus callosum. The number of CD68+ cells within the infarct core was significantly increased at days 8, 14 and 28. This temporal pattern was also seen in MRI. After experimental stroke within the infarcted cortex we found a delayed (day 14) infiltration of T-cells and macrophages. Furthermore, our data show that T-cells are present in higher numbers in the corpus callosum compared to the rest of the brain (except from the infarct core where they were highest).

Trichuris suis secrete products that reduce disease severity in a multiple sclerosis model.

Multiple sclerosis is a chronic inflammatory central nervous system (CNS) disease, which affects about 1 in 1000 individuals in the western world. It has been suggested that this relatively high prevalence is linked to a high level of hygiene, i.e. a reduced exposure to various microorganisms, including parasites. Parasites are known to employ different immunomodulatory and antiinflammatory strategies, which enable them to evade destruction by the immune system. We have investigated the immunomodulation by the swine whipworm, Trichuris suis, by measuring the impact of oral administration of T. suis ova as well as of intraperitoneal administration of T. suis excretory/secretory products on the development and progression of experimental autoimmune encephalomyelitis - an animal model that shares clinical and pathological characteristics with multiple sclerosis. Intraperitoneal administration of excretory/secretory products before disease onset, resulted in a significant decrease in disease severity as well as markedly reduced TH1 and TH17 T-cell responses, centrally in the spinal cord as well as in the periphery, i.e. the spleen. Thus, parenteral administration of T. suis-derived products results in a skewing of the immune response with a significant impact on disease severity in a CNS inflammatory disease model.

Oxidative damage and chemokine production dominate days before immune cell infiltration and EAE disease debut.

BACKGROUND: Multiple sclerosis is widely accepted as an inflammatory disease. However, studies indicate that degenerative processes in the CNS occur prior to inflammation. In the widely used animal model experimental autoimmune encephalomyelitis (EAE), we investigated the significance of degenerative processes from mitochondrial membrane potentials, reactive oxidative species, cell death markers, chemokines, and inflammatory cell types in brain, spinal cord, and optic nerve tissue during the effector phase of the disease, before clinical disease was evident. METHODS: Sixty-two rats were placed in eight groups, n = 6 to 10. Four groups were immunized with spinal cord homogenate emulsified in complete Freund's adjuvant (one served as EAE group), three groups were immunized with complete Freund's adjuvant only, and a control group was injected with phosphate buffered saline only. Groups were sacrificed 3, 5, 7, or 12-13 days after the intervention and analyzed for early signs of CNS degeneration. RESULTS: Loss of mitochondrial membrane potential and oxidative changes was observed days before clinical disease debut at day 9.75 ± 0.89. The early mitochondrial changes were not associated with cytochrome C release, cleavage of caspases 9 (38/40 kDa) and 3 (17/19 kDa), and cleavage of PARP (89 kDa) or spectrin (120/150 kDa), and apoptosis was not initiated. Axonal degeneration was only present at disease onset. Increases in a range of cytokines and chemokines were observed systemically as a consequence of immunization with complete Freund's adjuvant, whereas the encephalitogenic emulsion induced an upregulation of the chemokines Ccl2, Ccl20, and Cxcl1, specifically in brain tissue, 7 days after immunization. CONCLUSION: Five to seven days after immunization, subtle decreases in the mitochondrial membrane potential and an increased reactive oxygen species burden in brain tissue were observed. No cell death was detected at these time-points, but a specific expression pattern of chemokines indicates activity in the CNS, several days before clinical disease debut.

Systematic review of survival time in experimental mouse stroke with impact on reliability of infarct estimation.

BACKGROUND: Stroke is the second most common cause of death worldwide. Only one treatment for acute ischemic stroke is currently available, thrombolysis with rt-PA, but it is limited in its use. Many efforts have been invested in order to find additive treatments, without success. A multitude of reasons for the translational problems from mouse experimental stroke to clinical trials probably exists, including infarct size estimations around the peak time of edema formation. Furthermore, edema is a more prominent feature of stroke in mice than in humans, because of the tendency to produce larger infarcts with more substantial edema. PURPOSE: This paper will give an overview of previous studies of experimental mouse stroke, and correlate survival time to peak time of edema formation. Furthermore, investigations of whether the included studies corrected the infarct measurements for edema and a comparison of correction methods will be discussed. METHOD: Relevant terms were searched in the National Library of Medicine PubMed database. A method for classification of infarct measurement methods was made using a naming convention. CONCLUSION: Our study shows that infarct size estimations are often performed around the peak time of edema, with a median of 24h. Most studies do consider edema formation, however, there is no consensus on what method to use to correct for edema. Furthermore, investigations into neuroprotective drugs should use longer survival times to ensure completion of the investigated process. Our findings indicate a need for more research in this area, and establishment of common correction methodology.

The Hypothermic Influence on CHOP and Ero1-α in an Endoplasmic Reticulum Stress Model of Cerebral Ischemia.

Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response, resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP) activation. In an in vitro and in vivo model of ischemic stroke, we investigated whether hypothermia regulates the unfolded protein response of CHOP and Endoplasmic reticulum oxidoreductin-α (Ero1-α), because Ero1-α is suggested to be a downstream CHOP target. The gene expression of CHOP and Ero1-α was measured using Quantitative-PCR (Q-PCR) in rat hippocampi following global cerebral ischemia, and in hypoxic pheochromocytoma cells during normothermic (37 °C) and hypothermic (31 °C) conditions. As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia. A stable increase in CHOP expression was observed throughout the time course (p < 0.01, p < 0.0001), whereas Ero1-α expression peaked at three to six hours (p < 0.0001). Induced hypothermia in hypoxia stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours (p < 0.0001). On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001). Hypothermia attenuated the expression of CHOP, supporting that hypothermia suppress endoplasmic reticulum stress induced apoptosis in stroke. As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.

Drug-induced hypothermia by 5HT1A agonists provide neuroprotection in experimental stroke: new perspectives for acute patient treatment.

BACKGROUND: Drug-induced hypothermia reduces brain damage in animal stroke models and is an undiscovered potential in human stroke treatment. We studied hypothermia induced by the serotonergic agonists S14671 (1-[2-(2-thenoylamino)ethyl]-4[1-(7- methoxynaphtyl)]piperazine) and ipsapirone in a rat stroke model and in man by literature meta-analysis. METHODS: Rats had 60 minutes of middle cerebral artery occlusion (MCAO) and then 7 days of survival. Body temperatures were monitored for 22 hours. Thirty minutes after MCAO, 1 group (n = 9) received bolus of S14671 (.75 mg/kg) and continuous infusion of .06 mg/kg hour(-1) S14671 for 20 hours. Other MCAO rats (n = 7) had bolus of ipsapirone (.75 mg/kg) and continuous infusion of .25 mg/kg hour(-1) ipsapirone for 3 hours. Controls (n = 9; n = 5) received similar amounts of vehicle as bolus and continuous infusion for 20 hours/3 hours. Additional controls of the S14761 effect in MCAO were performed as previously mentioned (n = 10) but with rats kept normothermic by a heating lamp for 22 hours. Finally, a meta-analysis of ipsapirone-induced hypothermia in man was included. RESULTS: Infarct volumes were reduced by 50% in hypothermic rats versus controls (P < .05). S14671 rats kept normothermic did not show infarct reduction (P > .05). The body temperature after stroke was reduced 1.0-3.0°C compared with controls for 20 hours with S14671 treatment and for 6 hours with ipsapirone treatment. In humans, ipsapirone reduced temperature in average with .55 °C ranging between .1-1.4 °C. CONCLUSIONS: 5-hydroxytryptamine receptor 1A (5HT(1A)) agonists significantly reduce infarct volumes in MCAO rats primarily because of the hypothermic drug effect. 5HT(1A) agonists may be introduced to reduce body temperatures rapidly and prepare patients for further therapeutic hypothermia.

Dexamethasone enhances necrosis-like neuronal death in ischemic rat hippocampus involving µ-calpain activation.

Transient forebrain ischemia (TFI) leads to hippocampal CA1 pyramidal cell death which is aggravated by glucocorticoids (GC). It is unknown how GC affect apoptosis and necrosis in cerebral ischemia. We therefore investigated the co-localization of activated caspase-3 (casp-3) with apoptosis- and necrosis-like cell death morphologies in CA1 of rats treated with dexamethasone prior to TFI (DPTI). In addition, apoptosis- (casp-9, casp-3, casp-3-cleaved PARP and cleaved α-spectrin 145/150 and 120kDa) and necrosis-related (calpain-specific casp-9 cleavage, µ-calpain upregulation and cleaved α-spectrin 145/150kDa) cell death mechanisms were investigated by Western blot analysis. DPTI expedited CA1 neuronal death from day 4 to day 1 and increased the magnitude of CA1 neuronal death from 66.2% to 91.3% at day 7. Furthermore, DPTI decreased the overall (days 1-7) percentage of dying neurons displaying apoptosis-like morphology from 4.7% to 0.3% and, conversely, increased the percentage of neurons with necrosis-like morphology from 95.3% to 99.7%. In animals subjected to TFI without dexamethasone (ischemia-only), 7.4% of all dying CA1 neurons were casp-3-immunoreactive (IR), of which 3.1% co-localized with apoptosis-like and 4.3% with necrosis-like changes. By contrast, DPTI decreased the percentage of dying neurons with casp-3 IR to 1.4%, of which 0.3% co-localized with apoptosis-like changes and 1.1% with necrosis-like changes. Western blot analysis from DPTI animals showed a significant elevation of µ-calpain, a calpain-produced necrosis-related casp-9 fragment (25kDa) and cleavage of α-spectrin into 145/150kDa fragments at day 4, whereas in ischemia-only animals a significant increase of casp-3-cleaved PARP, cleavage of α-spectrin into 145/150 and 120kDa fragments was detected at day 7. We conclude that DPTI, in addition to augmenting and expediting CA1 neuronal death, causes a shift from apoptosis-like cell death to necrosis involving µ-calpain activation.

Drug-induced hypothermia as beneficial treatment before and after cerebral ischemia.

OBJECTIVES: Hypothermia is still unproven as beneficial treatment in human stroke, although in animal models, conditioning the brain with hypothermia has induced tolerance to insults. Here, we delineate the feasibility of drug-induced mild hypothermia in reducing ischemic brain damage when conditioning before (preconditioning) and after (postconditioning) experimental stroke. METHODS: Hypothermia was induced in rats with a bolus of 6 mg/kg talipexole followed by 20 h continuous talipexole infusion of 6 mg/kg in total. Controls received similar treatment with saline. The core body temperature was continuously monitored. In preconditioning, hypothermia was terminated before either reversible occlusion of the middle cerebral artery (MCAO) for 60 min or global ischemia for 10 min with 2-vessel occlusion and hypotension. In postconditioning, rats experienced 60 min of MCAO before hypothermia was induced either immediately or with 3 h delay. Rats survived ischemia for 2, 7 or 90 days. Infarct volumes were quantified by stereology. Additional experiments of methodological relevance were included in the study. RESULTS: Talipexole induced mild hypothermia (35.1±1.1 to 36.0±0.5°C) for <20 h. Hypothermic pre- and postconditioning reduced infarct sizes by more than 60% as monitored during the first 90 days after experimental stroke (p<0.05). CONCLUSION: Talipexole is registered for use as a dopamine substitute in humans with Parkinson's disease. Although dosages cannot be directly translated to patients, our study exemplifies in an animal model that drug-induced hypothermia in a clinical setting might reduce cerebral ischemic damage before neuro- and cardiac surgical procedures and after stroke.

Global and 3D spatial assessment of neuroinflammation in rodent models of Multiple Sclerosis.

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.

Leukocyte infiltration in experimental stroke.

Stroke is one of the leading causes of death worldwide. At present, the only available treatment is thrombolysis, which should be initiated no later than 4.5 hours after onset of symptoms. Several studies have shown that an attenuation of the inflammatory response in relation to stroke could widen the therapeutic window. However, the immune system has important functions following infarction, such as removal of dead cells and the subsequent astrocytosis as well as prevention of post-ischemic infection. Hence, detailed knowledge concerning the temporal profile of leukocyte infiltration is necessary in order to develop new and effective treatments.The purpose of this review is to determine the temporal profile of leukocyte (neutrophil granulocytes, macrophages and T-cells) infiltration following experimental stroke. We found that the number of neutrophil granulocytes peaks between day 1 and 3 after experimental stroke, with short occlusion times (30 and 60 minutes of middle cerebral artery occlusion (MCAO)) leading to a later peak in response (P <0.001). Macrophages/microglia were found to peak later than day 3 and stay in the infarcted area for longer time periods, whereas duration of occlusion had no influence on the temporal infiltration (P = 0.475). Studies on T-cell infiltration are few; however, a tendency towards infiltration peak at later time points (from day 4 onwards) was seen.This review provides a framework for the instigation of post-stroke anti-inflammatory treatment, which could prove beneficial and widen the therapeutic window compared to current treatment options.

Remote post-conditioning reduces hypoxic damage early after experimental stroke.

OBJECTIVES: Given that reliable markers for early ischemic brain damage are lacking, we set out to test whether pimonidazole can be used as a reliable tool in the quantification of hypoxic insults, at early time points following experimental stroke. METHODS: We have used semi-quantitative Western blotting detection of pimonidazole adducts in a rat model of reversible middle cerebral artery occlusion (MCAO), treated with remote post-conditioning. RESULTS: First, we demonstrated that a linear relationship exist between pimonidazole binding in the ischemic hemisphere and duration of ischemia, in animals subjected to 5, 15, 30, or 60 minutes of occlusion followed by 120 minutes of reflow. Then we showed a significant reduction in pimonidazole binding in the infarcted hemisphere, when rats with 60 minutes of MCAO, immediately after establishment of cerebral reflow, had 3×15 minutes intermittent hind limb ischemia followed by 24-hour survival. We analysed the middle cerebral arteries from animals with 60 minutes of MCAO and early remote post-conditioning, followed by 30 minutes, 24, or 48 hours of reflow. At 24 hours of reflow increases in phosphorylated protein kinase C-alpha with concomitantly increased levels of p38 phosphorylation were observed. CONCLUSIONS: Our investigation demonstrates that pimonidazole can be used for quantifying ischemic impact in stroke, even after very short survival times. It furthermore shows that early remote post-conditioning reduces ischemic damage, probably through hyperpolarization and reduced reflow vasospasm in the conduit middle cerebral arteries.

Cannabinoid treatment renders neurons less vulnerable than oligodendrocytes in Experimental Autoimmune Encephalomyelitis.

Using the rat model Experimental Autoimmune Encephalomyelitis (EAE), we have investigated the cytokinetical and cellular events of axonal degeneration and demyelination following treatment with 5 mg/kg/24h R(+)WIN55,212-2 or 10 mg/kg/24h R(+)WIN55,212-2, which have immunosuppressive effects. EAE was induced using MOG(1-125) in Dark Agouti rats and treatment was initiated at symptom debut and continued until first relapse culminated. The central nervous system (CNS) cell death including caspase and calpain activation, axonal degeneration and demyelination as well as a wide range of immunological parameters were quantified. We found a significant reduction in axonal degeneration associated with reduced calpain 1 following treatment with 5 mg/kg/24h R(+)WIN55,212-2. Treatment with 10 mg/kg/24h resulted furthermore in an improved clinical performance and a reduction in inflammatory activity and demyelination. Furthermore, the cytokines IL-2, IL-6, IL-10, RANTES, and TGF-ß were significantly reduced as were the cellular infiltration with regulatory T cells. We suggest that cannabinoids in low doses are neuroprotective through a reduction in calpain 1 expression. Our study implies that long-term low-dose cannabinoid administration to multiple sclerosis (MS) patients could result in some degree of neuroprotection, and thereby slow down the atrophy associated with this disease.

Neuroprotection without immunomodulation is not sufficient to reduce first relapse severity in experimental autoimmune encephalomyelitis.

OBJECTIVES: Multiple sclerosis can be characterized by a strong neuroinflammatory and progressive neurodegenerative component leading to prolonged disability. The synthetic compound R(+)WIN55,212-2 is reported to be neuroprotective at moderate doses and both neuroprotective and immunomodulatory at high doses, most likely due to differences in receptor affinities. In order to investigate the effects of neuroprotection and immunomodulation in an animal model of multiple sclerosis, we examined the impact of increasing concentrations of R(+)WIN55,212-2 on the inflammatory profile in CNS during first relapse and related this to demyelination, axonal degeneration and relapse severity. METHODS: Experimental autoimmune encephalomyelitis was induced in Dark Agouti rats and treatment with R(+)WIN55,212-2 was initiated at symptom debut. The animals were scored clinically throughout the experiment, and axonal degeneration, demyelination, T cells, microglia/macrophages, TNF-alpha, IL-12, IFN-gamma, IL-10 and the T(H)17 response were estimated at the peak of the first relapse. RESULTS: Treatment with high-dose R(+)WIN55,212-2 (10 and 20 mg/kg) significantly improved the clinical performance of the animals during relapse. Interestingly, treatment at any dosage did not affect the brain levels of TNF-alpha, IL-12 and IFN-gamma (T(H)1 response), whereas high-dose cannabinoid treatment reduced the number of T cells and microglia/macrophages in addition to the T(H)17 response. At the same time, we observed a significant reduction in axonal degeneration in all treatment groups whereas only high-dose treatment resulted in reduced demyelination. CONCLUSION: High-dose R(+)WIN55,212-2 treatment reduces demyelination and axonal degeneration and has immunomodulatory effects which significantly improve clinical performance, whereas a reduction in axonal degeneration on its own, induced by 5 mg/kg R(+)WIN55,212-2, has no impact on first relapse severity.