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 Foxp3EGFP 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.
BACKGROUND AND PURPOSE—Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain–gut microbiota axis was recently suggested as a modulator of ...nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke.
METHODS—We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry.
RESULTS—We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups.
CONCLUSIONS—Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.
Background
Noise phobia is a common behavior problem in dogs for which there are limited treatment options.
Objective
To evaluate the efficacy and safety of imepitoin in comparison to placebo for the ...control of anxiety and fear associated with noise phobia in dogs.
Animals
Two hundred thirty‐eight client‐owned dogs with noise phobia were recruited in veterinary clinics.
Methods
This placebo‐controlled, randomized, double‐blinded, clinical trial used a predictable noise event as eliciting context, the traditional New Year's Eve fireworks in Germany and the Netherlands. Owners began treatment 2 days before the anticipated noise event with administration of either imepitoin 30 mg/kg body weight Q12h or placebo for 3 consecutive days. On New Year's Eve, owners noted their observations of their dog's fear and anxiety behavior at 1600, 2200, 0020, and 0100 hours and scored the overall treatment effect on the following day.
Results
In the 16‐item owner report of fear and anxiety signs, fear and anxiety behaviors were significantly reduced under imepitoin treatment compared to placebo (delta −6.1 scoring points; P < .0001). A significantly higher proportion of owners reported a good or excellent overall treatment effect in the imepitoin group compared to placebo (odds ratio 4.689; 95% CI, 2.79‐7.89; P < .0001).
Conclusion
Imepitoin effectively controls fear and anxiety associated with noise phobia in dogs.
Despite its limited regenerative capacity, the central nervous system (CNS) shares more repair mechanisms with peripheral tissues than previously recognized. Scar formation is a ubiquitous healing ...mechanism aimed at patching tissue defects via the generation of fibrous extracellular matrix (ECM). This process, orchestrated by stromal cells, can unfavorably affect the capacity of tissues to restore function. Vascular mural cells have been found to contribute to scarring after spinal cord injury. In the case of stroke, little is known about the responses of pericytes (PCs) and stromal cells. Here, we show that capillary PCs are rapidly lost after cerebral ischemia in both experimental and human stroke. Coincident with this loss is a massive proliferation of resident platelet-derived growth factor receptor beta (PDGFRβ)+ and CD105+ stromal cells, which originate from the neurovascular unit and deposit ECM in the ischemic mouse brain. The presence of PDGFRβ+ stromal cells demarcates a fibrotic, contracted, and macrophage-laden lesion core from the rim of hypertrophic astroglia in both experimental and human stroke. We suggest that a previously unrecognized population of CNS-resident stromal cells drives a dynamic process of scarring after cerebral ischemia, which appears distinct from the glial scar and represents a novel target for regenerative stroke therapies.
Temporary immunosuppression has been identified as a major risk factor for the development of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous ...system was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive.
The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy, α7 nicotinic acetylcholine receptor-deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and modulate parasympathetic activity.
Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor-deficient mice reversed pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-inflammatory signaling in the lung.
Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central nervous system injury.
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.
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.
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.
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.
Imepitoin is a low affinity partial agonist for the benzodiazepine binding site of γ-aminobutyric acid (GABA
) receptors, and is currently used as an antiepileptic in dogs. Here we tested imepitoin ...for anxiolytic properties. In an
model, imepitoin was capable of preventing the effect of corticotrophin releasing factor (CRF) on locus coeruleus neurons without suppressing the basal activity of these cells, an activity which is suggestive for an anti-stress effect of imepitoin. In addition, we applied a battery of standard rodent preclinical tests for anxiety behavior including elevated plus mazes in mice and rats, light-dark-box in mice and rats, social interaction test in rats, or the Vogel conflict test in rats. In all models, the observed profile of imepitoin appeared similar to benzodiazepines and typical for anxiolytic drugs. We also observed anxiolytic activity in dogs in a provoked open field sound-induced fear model, where reactions to noises were elicited by a sound recording of thunderstorms. Imepitoin caused an increase in locomotion measured in distance traveled and an ameliorating effect on cortisol levels in response to thunderstorm noises. For comparison, dexmedetomidine caused a decrease in locomotion and had no effect on cortisol. In all animal models the doses needed for an anxiolytic effect were not associated with sedation. In rodents, there was at least a factor of 10 between anxiolytic doses and doses with mild signs of sedation. In summary, imepitoin showed similar anxiolytic activities as benzodiazepines but without producing the known adverse reactions of benzodiazepines such as sedation.
Epilepsy in the cat is a serious medical condition. To date there are no licensed treatments for feline epilepsy and no well-controlled clinical studies on the efficacy or safety of antiepileptic ...drugs in cats. The aim of this study was to collect tolerability data and first exploratory efficacy data of imepitoin in both healthy and epileptic cats.
In two tolerability studies, 30 healthy cats received imepition twice daily in doses of 0, 30, 40 or 80 mg/kg bodyweight for 30 days. No serious adverse events were observed in any of the dose groups. In the imepitoin treated groups, emesis was observed in some animals temporarily and intermittently mainly in the second and third weeks of treatment. In a small, single-arm, open label, uncontrolled clinical trial eight cats suffering from idiopathic epilepsy were treated with imepitoin twice daily at doses of 30 mg/kg bodyweight for 30 days. Four of these cats (50%) achieved seizure freedom for at least 8 weeks under treatment. Adverse events, mostly lethargy, decreased appetite and emesis, were often mild and transient.
In summary, imepitoin was well tolerated in healthy and epileptic cats and showed in a pilot trial indication for efficacy in treating feline epilepsy.
Anxiety in dogs, especially in relation to certain noises, is a common issue which can lead to clinically significant problems like noise phobias. While several scales have been used to assess sound ...sensitivity and reactivity, clinical monitoring has tended to depend on unvalidated methods, general assessment, and/or historical comparison with owners' recall of previous episodes. Therefore, we aimed to develop and validate a scale to assess canine anxiety.
We used the data from 226 dogs from a previously reported double blind placebo controlled study in order to determine the validity of the 16 item "Lincoln Canine Anxiety Scale." Unidimensionality was assessed through correlation between individual item scores and total score, with internal consistency assessed using Cronbach's alpha. Factor analysis was used to determine the dimensionality of the scale. Item response theory (IRT) was used to gain insight into the value of single items to the overall scale scores. To characterize the score characteristics in an anxiety-eliciting context we analyzed the behaviors of placebo treated dogs assessed at 00:20 h, the time point of maximum noise stimulus during New Year's Eve fireworks. Sensitivity of the scale to treatment effects was determined from its performance in the wider study.
The majority of correlations between individual items and total score were >0.48, with Cronbach's alpha equalling 0.88, indicating good internal consistency. Principal Component Analysis (PCA) confirmed a unidimensional structure. IRT indicated that the scale could be reduced to 11 items without significantly reducing its value. The scale showed good treatment and stimulus sensitivity, with a score change of ~20 points differentiating "no/worse" effect from an "excellent" effect and a 30% difference between treatment (imepitoin) and placebo.
In our initial validation the Lincoln Canine Anxiety Scale appears to provide a reliable method for determining anxiety and fear responses by dogs and monitoring the effects of treatment.
Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel ...therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats. Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans. In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.
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