Role of dihydrotestosterone in post-stroke peripheral immunosuppression after cerebral ischemia

Stroke is a sexually dimorphic disease with male gender considered a disadvantage in terms of risk and disease outcome. In intact males, stroke induces peripheral immunosuppression, characterized by decreased splenocyte numbers and proliferation and altered percentages of viable T, B, and CD11b⁺ cells. To investigate whether the potent androgen and known immunomodulator, dihydrotestosterone (DHT), exacerbates post-stroke immunosuppression in castrated male mice after focal stroke, we evaluated the effect of middle cerebral artery occlusion (MCAO) on peripheral and central nervous system (CNS) immune responses in castrated mice with or without controlled levels of DHT. MCAO reduced spleen cell numbers in both groups, but altered T cell and B cell percentages in remaining splenocytes and concomitantly increased the percentage of CD11b⁺ blood cells solely in DHT-replaced animals at 24 h. Furthermore, DHT-replacement reduced splenocyte proliferation which was accompanied by an increased percentage of immunosuppressive regulatory T cells relative to castrates 96 h post-MCAO. In brain, the percentages of immune cell populations in the ischemic hemisphere relative to the non-ischemic hemisphere were similar between castrated and DHT-replaced mice after MCAO. These data suggest DHT modulates peripheral immunosuppression after MCAO but with relatively little effect on early immune response of the recovering CNS.     

Targeted mutation of Fas ligand gene attenuates brain inflammation in experimental stroke

Inflammation is an important contributing mechanism in ischemic brain injury. The current study elucidates a previously unexplored role of Fas ligand (FasL) in post-stroke inflammatory responses that is independent of its well-known effect in triggering apoptosis. Focal cerebral ischemia was induced for 2 h by right middle cerebral artery occlusion (MCAO) in FasL mutant (gld) and wild-type mice. FasL mutation profoundly reduced brain damage and improved neurological performance from 6 to 72 h after ischemic stroke. The production of inflammatory cytokines in the brain was attenuated in gld mice after ischemia in the absence of dramatic change in inflammatory cell apoptosis. FasL mutation attenuated the recruitment of peripheral inflammatory cells (neutrophil) and inhibited the activation of residential glial cells (microglia and astrocyte). FasL mutation reduced CD8⁺ T cells and turned the Th1/Th2 balance towards Th2 in the brain and peripheral blood after cerebral ischemia. In contrast to cerebral ischemia, the molecular and cellular inflammatory changes induced by intracerebroventricular injection of lipopolysaccharide (LPS) were also attenuated in gld mice. Moreover, the soluble FasL (sFasL) and phospho-SAPK/JNK were decreased in gld mice, suggesting that the inflammatory role of FasL in experimental stroke might relate to sFasL and the c-Jun N-terminal kinase (JNK) signaling pathway. Taken together, our data suggest a novel role of FasL in the damaging inflammatory responses associated with cerebral ischemia. Neutralization of FasL may be a novel therapeutic strategy to suppress post-stroke inflammation and improve the long-term outcomes of stroke.     

SB 234551 selective ET(A) receptor antagonism: perfusion/diffusion MRI used to define treatable stroke model, time to treatment and mechanism of protection

Mismatches between tissue perfusion-weighted imaging (PWI; an index of blood flow deficit) and cellular diffusion-weighted imaging (DWI; an index of tissue injury) provide information on potentially salvageable penumbra tissue in focal stroke and can identify “treatable” stroke patients. The present pre-clinical studies were conducted to: a.) Determine PWI (using perfusion delay) and DWI measurements in two experimental stroke models, b.) Utilize these measurements to characterize selective ET_A receptor antagonism (i.e., determine efficacy, time-to-treatment and susceptibility to treatment in the different stroke models), and c.) Determine if increasing the reduced blood flow following a stroke is a mechanism of protection. Permanent middle cerebral artery occlusion (MCAO) or sham surgeries were produced in Sprague Dawley rats (SD; proximal MCAO; hypothesized to be a model of slowly evolving brain injury with a significant penumbra) and in spontaneously hypertensive rats (SHR; distal MCAO; hypothesized to be a model of rapidly evolving brain injury with little penumbra). Infusions of vehicle or SB 234551 (3, 10, or 30 µg/kg/min) were initiated at 0, 75, and/or 180 min post-surgery and maintained for the remainder of 24 h post-surgery. Hyper-intense areas of perfusion delay (PWI) in the forebrain were measured using Gadolinium (Gd) bolus contrast. DWI hyper-intense areas were also measured, and the degree of forebrain DWI-PWI mismatch was determined. Region specific analyses (ROI) were also conducted in the core ischemic and low perfusion/penumbra areas to provide indices of perfusion and changes in the degree of tissue perfusion due to SB 234551 treatment. At 24 h post-surgery, final infarct volume was measured by DWI and by staining forebrain slices. Following SD proximal MCAO, there was a significant mismatch in the ischemic forebrain PWI compared to DWI (PWI > DWI) at 60 min which was maintained up to 150 min (all p < 0.05). By 24 h post-stroke, infarct volume was identical to the area of early perfusion deficit/PWI, suggesting a slow progression of infarct development that expanded into the significant, earlier cortical penumbra (i.e., model with salvageable tissue with potential for intervention). When SB 234551 was administered within the period of peak mismatch (i.e., at 75 min post-stroke), SB 234551 provided significant dose-related reductions in cortical (penumbral) progression to infarction (p < 0.05). Cortical protection was related to an increased/normalization of the stroke-induced decrease in tissue perfusion in cortical penumbra areas (p < 0.05). No SB 234551-induced changes in reduced tissue perfusion were observed in the striatum core ischemic area. Also, when SB-234551 was administered beyond the time of mismatch, no effect on cortical penumbra progression to infarct was observed. In comparison and strikingly different, following SHR distal MCAO there was no mismatch between PWI and DWI (PWI = DWI) as early as 60 min post-stroke, with this early change in SHR DWI being identical to the final infarct volume at 24 h, suggesting a rapidly occurring brain injury with little cortical penumbra (i.e., model with little salvageable tissue or potential for intervention). In distal MCAO, SB 234551 administered immediately at the time of stroke did not have any effect on infarct volume in SHR. These data demonstrate that selective blockade of ET_A receptors is protective following proximal MCAO in SD (i.e. a model similar to “treatable” clinical patients). The protective mechanism appears to be due to enhanced collateral blood flow and salvage of penumbra. Therefore, the use of PWI-DWI mismatch signatures can identify treatable stroke models characterized by a salvageable penumbra and can define appropriate time to treatment protocols. In addition, tissue perfusion information obtained under these conditions might clarify mechanism of protection in the evaluation of protective compounds for focal stroke.     

The sympathetic nervous system modulates CD4+Foxp3+ regulatory T cells via noradrenaline-dependent apoptosis in a murine model of lymphoproliferative disease

The sympathetic nervous system (SNS) plays a crucial role in the course and development of autoimmune disease in Fas-deficient lpr/lpr mice. As regulatory T cells (Tregs) are considered important modulators of autoimmune processes, we analyzed the interaction between the SNS and Tregs in this murine model of lymphoproliferative disease. We found that the percentage of Tregs among CD4⁺ T cells is increased in the spleen, lymph nodes, and thymus of lpr/lpr mice as compared to age-matched C57Bl/6J (B6) mice. Furthermore, noradrenaline (NA), the main sympathetic neurotransmitter, induced apoptosis in B6- and lpr/lpr-derived Tregs. NA also reduced the frequency of Foxp3⁺ cells and Foxp3 mRNA expression via β₂-adrenoceptor (β₂-AR)-mediated mechanisms in a concentration and time-dependent manner. Destruction of peripheral sympathetic nerves by 6-hydroxydopamine significantly increased the percentage of Tregs in B6 control mice to an extent comparable to aged-matched lpr/lpr mice. The concentration of splenic NA negatively correlated with the frequency of CD4⁺Foxp3⁺ Tregs. Additionally, 60 days after sympathectomy, a partial recovery of NA concentrations led to Treg percentages comparable to those of intact, vehicle-treated controls. Immunohistochemical analysis of the spleen revealed localization of single Foxp3⁺ Tregs in proximity to NA-producing nerve fibers, providing an interface between Tregs and the SNS. Taken together, our data suggest a relation between the degree of splenic sympathetic innervation and the size of the Treg compartment. While there are few examples of endogenous substances capable of affecting Tregs, our results provide a possible explanation of how the magnitude of the Treg compartment in the spleen can be regulated by the SNS.     

Higher mean platelet volume determined shortly after the symptom onset in acute ischemic stroke patients is associated with a larger infarct volume on CT brain scans and with worse clinical outcome

Objective

Mean platelet volume (MPV) determined shortly after the onset of acute ischemic stroke represents the pre-stroke values. Data on its relationship to stroke severity/outcome have been conflicting. We related MPV to infarct volume on CT brain scans and risk of death/dependence 7 days and 3 months post-stroke.

Methods

MPV (within 30 h since stroke onset), infarct volume (13–83 h since stroke onset) and clinical outcomes were evaluated in 81 consecutive patients (32 men, age 52–91 years, 10 small artery occlusion, 10 large artery atherosclerosis, 29 cardioembolic, 32 multiple probable/possible etiology).

Results

Higher MPV was independently associated with larger ln-infarct volume [estimate 0.259, 95% confidence interval (CI) 0.004–0.513, P = 0.046], greater risk of death/dependence 7 days post-stroke [relative risk (RR) = 1.077, 95% CI 1.005–1.115, P = 0.036], and greater risk of death/dependence 3 months post-stroke (RR = 1.077, 95% CI 1.001–1.158, P = 0.048). Considered covariates: stroke etiology, CT scan timing, platelet count and other hematological parameters, demographic variables, history of cerebrovascular, cardiac or cardiovascular diseases, diabetes, serum chemistry, previous antiplatelet and statin use and treatments delivered after the index event.

Conclusions

Data support the view about MPV as a determinant of severity/outcome of the acute ischemic stroke.     

Total oligomeric flavonoids of Cyperus rotundus ameliorates neurological deficits, excitotoxicity and behavioral alterations induced by cerebral ischemic-reperfusion injury in rats

Interactions between neurons and astrocytes play a critical role in the central nervous system homeostasis. Cyperus rotundus (family: Cyperaceae), a traditional Indian medicinal herb, used as nervine tonic and nootropic in the Ayurvedic system of medicine. The present study was undertaken to investigate the neuroprotective effect of total oligomeric flavonoids (TOFs), prepared from C. rotundus, in rat model of cerebral ischemia and reperfusion. Male Sprague Dawley rats (290-340 g) were subjected to middle cerebral artery occlusion (MCAO) for 2 h and reperfusion for 70 h. Experimental animals were divided into four groups: Group I - sham operated (n = 7); Group II - vehicle treated ischemic-reperfusion (IR) (n = 9), and Group III and IV - TOFs treated (100 and 200 mg/kg body weight, p.o., respectively; n = 7 in each group). Vehicle or TOFs were pretreated for four days before the induction of ischemia and continued for next three days after the ischemia i.e. treatment was scheduled totally for a period of 7 days. MCAO surgery was performed on day 4, 1 h after TOFs administration. Neuroprotective effect of TOFs was substantiated in terms of neurological deficits, excitotoxicity (glutamate, glutamine synthetase and Na⁺K⁺ATPase levels), oxidative stress (malondialdehyde, super oxide dismutase, and glutathione) and neurobehavioral functions in the experimental animals. TOFs decreased glutamate, glutamine synthetase (GS) and increased Na⁺K⁺ATPase activity in a dose dependent manner when compared to the IR rats. Treatment with TOFs significantly reduced the neurological deficits and reversed the anxiogenic behavior in rats. Further, it also significantly decreased MDA and increased superoxide dismutase (SOD) and glutathione content in brains of experimental rats. Histopathological examination using cresyl violet staining revealed the attenuation of neuronal loss by TOFs in stroke rats. The present study demonstrates the unswerving involvement of TOFs on ischemia-reperfusion triggered biochemical alterations in MCAO/R rats. Hence, TOFs might be an attractive candidate for further studies in the development of new drugs for cerebral stroke treatment.     

Involvement of levels of Toll like receptor-4 in monocytes,CD4 + T-lymphocyte subsets,and cytokines in patients with immune thrombocytopenic purpura

Introduction

Toll-like receptors have been found to be associated with immune-mediated diseases but it is still not clear whether they play a role in immune thrombocytopenic purpura (ITP), especially TLR4. CD4 + T-lymphocyte abnormalities, including Th17, Th1, Th2, and regulator T cell (Treg), are considered important in ITP. There have been few studies regarding the expression of TLR4 and the relationships between TLR4 and Th17 levels in ITP.

Materials and Methods

In this study, we evaluated the expression of TLR4 in monocytes, the plasma concentrations of IL-23, IL-17 and the profiles of Th17, Th1, Th2 cells in 70 patients with ITP and 31 healthy controls. In addition, we evaluated IL-2 and Treg cells in 46 cases of 70 patients with ITP and the same 31 controls.

Results

Higher levels of TLR4 expression, higher relative numbers of Th17 and Th1 cells and lower levels of Treg cells were observed in patients when compared with controls (p = 0.001 for TLR4; p < 0.001 for Th17; p = 0.014 for Th1; p = 0.001 for Treg). The levels of IL-23 and IL-2 were increased (p = 0.022 for IL-23; p = 0.025 for IL-2), the relative levels of Th2 and concentrations of IL-17 were similar across both groups (p = 0.446 for Th2; p = 0.316 for IL-17). A significant negative correlation was observed between levels of TLR4 and Treg(r = -0.544, p < 0.001), but a significantly positive correlation was observed between IL-2 and IL-23 concentration in patients (r = 0.441, p = 0.004). Neither the correlation between TLR4 and the other CD4⁺ T cells and cytokines nor the correlation between the three cytokines and CD4+ T cells was found to be statistically significant.

Conclusions

Our data showed that TLR4, CD4 + T cells (Th1, Th17 and Treg cells) and related cytokines (IL-23, IL-2) may take part in the pathogenesis of ITP. TLR4 may play a role through the TLR4-cytokine-CD4+ T lymphocyte cell pathway.     

Monocytes are essential for the neuroprotective effect of human cord blood cells following middle cerebral artery occlusion in rat

Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 h later by intravenous administration of HUCB MNC preparations depleted of either CD14 ⁺ monocytes, CD133 ⁺ stem cells, CD2 ⁺ T-cells or CD19 ⁺ B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14 ⁺ monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery.     

Stroke affects intestinal immune cell trafficking to the central nervous system

Stroke is an acute neurological disease with a strong inflammatory component that can be regulated by the intestinal microbiota and intestinal immune cells. Although stroke has been shown to alter immune cell populations in the gut, the dynamics of cell trafficking have not been elucidated. To study the trafficking of gut-derived immune cells after stroke, we used mice expressing the photoconvertible protein Kikume Green-Red, which turns form green to red when exposed to violet light. Mice underwent laparotomy and the small intestine was exposed to violet laser light. Immune cells were isolated from the small intestine immediately after photoconversion and 2 days later. Percentage of immune cells (CD45⁺KikR⁺) that expressed the red variant of the protein (KikR) was higher immediately after photoconversion than 2 days later, indicating cell egress from the small intestine. To investigate whether intestinal immune cells traffic to the periphery and/or the central nervous system (CNS) after stroke, we analyzed KikR⁺ immune cells (2 days after photoconversion) in peripheral lymphoid organs, meninges and brain, 3 and 14 days after transient occlusion of the middle cerebral artery (tMCAo) or sham-surgery. Although migration was observed in naïve and sham animals, stroke induced a higher mobilization of gut KikR⁺ immune cells, especially at 3 days after stroke, to all the organs analyzed. Notably, we detected a significant migration of CD45^hi immune cells from the gut to the brain and meninges at 3 days after stroke. Comparison of cell trafficking between organs revealed a significant preference of intestinal CD11c⁺ cells to migrate from the small intestine to brain and meninges after stroke. We conclude that stroke increases immune cell trafficking from the small intestine to peripheral lymphoid organs and the CNS where they might contribute to post-stroke inflammation.     

The asparaginyl endopeptidase legumain after experimental stroke

Various proteases in the brain contribute to ischemic brain injury. We investigated the involvement of the asparaginyl endopeptidase legumain after experimental stroke. On the basis of gene array studies and in situ hybridizations, we observed an increase of legumain expression in the peri-infarct area of rats after transient occlusion of the middle cerebral artery (MCAO) for 120 mins with a maximum expression at 24 and 48 h. Immunohistochemical analyses revealed the expression of legumain in Iba1⁺ microglial cells and glial fibrillary acidic protein-positive astrocytes of the peri-infarct area in mice after MCAO. Post-stroke recovery was also studied in aged legumain-deficient mice (45 to 58 weeks old). Legumain-deficient mice did not show any differences in physiologic parameters compared with respective littermates before, during MCAO (45 mins), and the subsequent recovery period of 8 days. Moreover, legumain deficiency had no effect on mortality, infarct volume, and the neurologic deficit determined by the rotating pole test, a standardized grip strength test, and the pole test. However, a reduced number of invading CD74⁺ cells in the ischemic hemisphere indicates an involvement in post-stroke inflammation. We conclude that legumain is not essential for the functional deficit after MCAO but may be involved in mechanisms of immune cell invasion.     

CD4+ T cells promote delayed B cell responses in the ischemic brain after experimental stroke

CD4⁺ T lymphocytes are key mediators of tissue damage after ischemic stroke. However, their infiltration kinetics and interactions with other immune cells in the delayed phase of ischemia remain elusive. We hypothesized that CD4⁺ T cells facilitate delayed autoreactive B cell responses in the brain, which have been previously linked to post-stroke cognitive impairment (PSCI). Therefore, we treated myelin oligodendrocyte glycoprotein T cell receptor transgenic 2D2 mice of both sexes with anti-CD4 antibody following 60-minute middle cerebral artery occlusion and assessed lymphocyte infiltration for up to 72 days. Anti-CD4-treatment eliminated CD4⁺ T cells from the circulation and ischemic brain for 28 days and inhibited B cell infiltration into the brain, particularly in animals with large infarcts. Absence of CD4⁺ T cells did not influence infarct maturation or survival. Once the CD4⁺ population recovered in the periphery, both CD4⁺ T and B lymphocytes entered the infarct site forming follicle-like structures. Additionally, we provide further evidence for PSCI that could be attenuated by CD4 depletion. Our findings demonstrate that CD4⁺ T cells are essential in delayed B cell infiltration into the ischemic brain after stroke. Importantly, lymphocyte infiltration after stroke is a long-lasting process. As CD4 depletion improved cognitive functions in an experimental set-up, these findings set the stage to elaborate more specific immune modulating therapies in treating PSCI.     

Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

Repeated bouts of aerobic exercise enhance regulatory T cell responses in a murine asthma model

We have reported previously that moderate intensity aerobic exercise training attenuates airway inflammation in a murine asthma model. Recent studies implicate regulatory T (Treg) cells in decreasing asthma-related airway inflammation; as such, the current study examined the effect of exercise on Treg cell function in a murine asthma model. Mice were sensitized with ovalbumin (OVA) prior to the start of exercise training at a moderate intensity 3×/week for 4 weeks; exercise was performed as treadmill running (13.5 m/min, 0% grade). Mice were OVA challenged repeatedly throughout the exercise protocol. At protocol completion, mice were analyzed for changes in the number and suppressive function of CD4⁺CD25⁺Foxp3⁺ cells isolated from lungs, mediastinal lymph nodes, and spleens. Results show that exercise increased significantly the number of Foxp3⁺ cells within the lungs and mediastinal lymph nodes, but not the spleens, of OVA-treated mice as compared with sedentary controls. Exercise also enhanced the suppression function of CD4⁺CD25⁺Foxp3⁺ Treg cells derived from OVA-treated mice as compared with sedentary controls. Specifically, Treg cells from exercised, OVA-treated mice more effectively suppressed CD4⁺CD25^? cell proliferation and Th2 cytokine production in vitro. Enhanced suppression was associated with increased protein levels of TGF-β and lesser amounts of IL-10 and IL-17; however, blocking TGF-β had no effect on suppressive functions. These data demonstrate that exercise-mediated increases in Treg cell function may play a role in the attenuation of airway inflammation. Further, these results indicate that moderate intensity aerobic exercise training may alter the Treg cell function within the asthmatic airway.     

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).     

Intranasal Dexamethasone Reduces Mortality and Brain Damage in a Mouse Experimental Ischemic Stroke Model

Neuroinflammation triggered by the expression of damaged-associated molecular patterns released from dying cells plays a critical role in the pathogenesis of ischemic stroke. However, the benefits from the control of neuroinflammation in the clinical outcome have not been established. In this study, the effectiveness of intranasal, a highly efficient route to reach the central nervous system, and intraperitoneal dexamethasone administration in the treatment of neuroinflammation was evaluated in a 60-min middle cerebral artery occlusion (MCAO) model in C57BL/6 male mice. We performed a side-by-side comparison using intranasal versus intraperitoneal dexamethasone, a timecourse including immediate (0 h) or 4 or 12 h poststroke intranasal administration, as well as 4 intranasal doses of dexamethasone beginning 12 h after the MCAO versus a single dose at 12 h to identify the most effective conditions to treat neuroinflammation in MCAO mice. The best results were obtained 12 h after MCAO and when mice received a single dose of dexamethasone (0.25 mg/kg) intranasally. This treatment significantly reduced mortality, neurological deficits, infarct volume size, blood–brain barrier permeability in the somatosensory cortex, inflammatory cell infiltration, and glial activation. Our results demonstrate that a single low dose of intranasal dexamethasone has neuroprotective therapeutic effects in the MCAO model, showing a better clinical outcome than the intraperitoneal administration. Based on these results, we propose a new therapeutic approach for the treatment of the damage process that accompanies ischemic stroke.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00884-9) contains supplementary material, which is available to authorized users.     

Social interaction plays a critical role in neurogenesis and recovery after stroke

Stroke survivors often experience social isolation. Social interaction improves quality of life and decreases mortality after stroke. Male mice (20–25 g; C57BL/6N), all initially pair housed, were subjected to middle cerebral artery occlusion (MCAO). Mice were subsequently assigned into one of three housing conditions: (1) Isolated (SI); (2) Paired with their original cage mate who was also subjected to stroke (stroke partner (PH-SP)); or (3) Paired with their original cage mate who underwent sham surgery (healthy partner (PH-HP)). Infarct analysis was performed 72 h after stroke and chronic survival was assessed at day 30. Immediate post-stroke isolation led to a significant increase in infarct size and mortality. Interestingly, mice paired with a healthy partner had significantly lower mortality than mice paired with a stroke partner, despite equivalent infarct damage. To control for changes in infarct size induced by immediate post-stroke isolation, additional cohorts were assessed that remained pair housed for three days after stroke prior to randomization. Levels of brain-derived neurotrophic factor (BDNF) were assessed at 90 days and cell proliferation (in cohorts injected with 5-bromo-2′-deoxyuridine, BrdU) was evaluated at 8 and 90 days after stroke. All mice in the delayed housing protocol had equivalent infarct volumes (SI, PH-HP and PH-SP). Mice paired with a healthy partner showed enhanced behavioral recovery compared with either isolated mice or mice paired with a stroke partner. Behavioral improvements paralleled changes in BDNF levels and neurogenesis. These findings suggest that the social environment has an important role in recovery after ischemic brain injury.     

IgA natural antibodies are produced following T-cell independent B-cell activation following stroke

Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA + cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII^-/-, CD4^-/-, and MyD88^-/- mice to determine if B-lymphocytes mature into IgA + PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA + PCs develops independently of CD4 + helper T-lymphocytes and MyD88 signaling. Subsequent sequencing of immunoglobulin genes of individual IgA + PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA + PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.     

Delayed diapedesis of CD8 T cells contributes to long-term pathology after ischemic stroke in male mice

ObjectiveStroke is a debilitating disorder with significant annual mortality and morbidity rates worldwide. Immune cells are recruited to the injured brain within hours after stroke onset and can exhibit either protective or detrimental effects on recovery. However, immune cells, including CD8 T cells, persist in the injured brain for weeks, suggesting a longer-term role for the adaptive immune system during functional recovery. The aim of this study was to determine if the delayed secondary diapedesis of CD8 T cells into the ischemic brain negatively impacts functional recovery after transient ischemic stroke in male mice.ResultsMice exhibited an increased number of leukocytes in the ipsilesional hemispheres at 14 days (3-fold; p < 0.001) and 30 days (2.2-fold; p = 0.02) after transient middle cerebral artery occlusion (tMCAo) compared to 8 days post-tMCAo, at which time acute neuroinflammation predominantly resolves. Moreover, mice with higher ipsilesional CD8 T cells at 30 days (R² = 0.52, p < 0.01) exhibited worse functional recovery. To confirm a detrimental role of chronic CD8 T cell diapedesis on recovery, peripheral CD8 T cells were depleted beginning 10 days post-tMCAo. Delayed CD8 T cell depletion improved motor recovery on the rotarod (F_(1,28) = 4.264; p = 0.048) compared to isotype control-treated mice. CD8 T cell-depleted mice also exhibited 2-fold (p < 0.001) reduced leukocyte infiltration at 30 days post-tMCAo. Specifically, macrophage, neutrophil, and CD4 T cell numbers were reduced in the ipsilesional hemisphere of the CD8 T cell-depleted mice independent of inflammatory status of the post-stroke CNS (e.g. microglial phenotype and cytokine production). RNAseq identified a unique profile for brain infiltrating CD8 T cells at 30 days post-tMCAo, with 46 genes differentially expressed relative to CD8 T cells at 3 days post-tMCAo.ConclusionOur data reveal a role for CD8 T cells in the chronic phase post-stroke that can be therapeutically targeted. We demonstrate long-term CD8 T cell recruitment into the ipsilesional hemisphere that affects both immune cell numbers present in the injured brain and functional recovery through one month after stroke onset.     

Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3^EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3⁺ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4⁺ cells depleted of Foxp3⁺ Tregs into RAG1^−/− mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25⁺ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.