Behavioral outcome measures to improve experimental stroke research

Functional recovery after an experimental stroke can be assessed by multiple behavioural tests, however, there is no consensus about which test to use in long-term stroke recovery studies or whether the tests are affected by stroke surgery, post-operative care or behavioural compensation due to repeated testing. This review describes the tests most commonly used to assess motor and sensorimotor function, cognition and mood in stroke animals. Although it is difficult to predict the direction of future research, it may be possible to prevent false-positive results by selecting an appropriate task or a battery of tasks. It is also expected that the upcoming stroke recovery recommendations and the improved dialogue between academy, industry and healthcare professionals will further promote translational success

Conditional deletion of LRRC8A in the brain reduces stroke damage independently of swelling-activated glutamate release

The ubiquitous volume-regulated anion channels (VRACs) facilitate cell volume control and contribute to many other physiological processes. Treatment with non-specific VRAC blockers or brain-specific deletion of the essential VRAC subunit LRRC8A is highly protective in rodent models of stroke. Here, we tested the widely accepted idea that the harmful effects of VRACs are mediated by release of the excitatory neurotransmitter glutamate. We produced conditional LRRC8A knockout either exclusively in astrocytes or in the majority of brain cells. Genetically modified mice were subjected to an experimental stroke (middle cerebral artery occlusion). The astrocytic LRRC8A knockout yielded no protection. Conversely, the brain-wide LRRC8A deletion strongly reduced cerebral infarction in both heterozygous (Het) and full KO mice. Yet, despite identical protection, Het mice had full swelling-activated glutamate release, whereas KO animals showed its virtual absence. These findings suggest that LRRC8A contributes to ischemic brain injury via a mechanism other than VRAC-mediated glutamate release

Interaction of ARC and Daxx: a novel endogenous target to preserve motor function and cell loss after focal brain ischemia in mice

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenousARCprotein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD).TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30±8% (mean±SD; p=0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1ug intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20±7% (mean±SD; p˃0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX–ASK1–JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1–JNK activation. Our work identifies for the first time ARC–DAXX binding to block ASK1–JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy

Selective ROCK2 inhibition in focal cerebral ischemia

Objective: Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known. Methods: We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice. Results: KD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 h from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke. Interpretation Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation

Genetic relationships of European, Mediterranean, and SW Asian populations using a panel of 55 AISNPs

The set of 55 ancestry informative SNPs (AISNPs) originally developed by the Kidd Lab has been studied on a large number of populations and continues to be applied to new population samples. The existing reference database of population samples allows the relationships of new population samples to be inferred on a global level. Analyses show that these autosomal markers constitute one of the better panels of AISNPs. Continuing to build this reference database enhances its value. Because more than half of the 25 ethnic groups recently studied with these AISNPs are from Southwest Asia and the Mediterranean region, we present here various analyses focused on populations from these regions along with selected reference populations from nearby regions where genotype data are available. Many of these ethnic groups have not been previously studied for forensic markers. Data on populations from other world regions have also been added to the database but are not included in these focused analyses. The new population samples added to ALFRED and FROG-kb increase the total to 164 population samples that have been studied for all 55 AISNPs

Schlaganfall, Stress und Depression - Beweise eines Hirnischämiemodells einer Maus

Emerging evidence in the last decade points out an association between stress, depression and stroke. Clinical studies report that a considerable portion of stroke survivors develop depression after stroke. Also, an increasing number of clinical case-control studies indicate an association between stress levels and ischemic stroke. Experimental studies focusing on the bi-directional interaction between stroke and stress as well as depression are crucial to better understand the pathophysiology of stroke and depression. This PhD work focuses on three separate projects that investigate the nature and mechanisms of this relationship. In study 1 the effects of 30 minutes left and right middle cerebral artery occlusion (MCAo) on mouse “anxiety”-like and “depression”-like behavior were evaluated. Left MCAo but not right MCAo led to chronic depression and anxiety-like behavior; citalopram treatment was shown to reverse these effects. 30 min MCAo caused significant and chronic reduction of striatal dopamine (DA) levels. Striatal DA loss was normalized to a large degree by citalopram treatment. Further histological analysis revealed a significant loss of dopaminergic neurons in ipsilateral ventral tegmental area (VTA) and Substantio Nigra (SN), as assessed by Tyrosine Hydroxylase (TH) immunostaining and NeuN-immunostaining. Cell loss in SN and VTA, and effects of MCAo were also significantly attenuated by citalopram treatment. Our study indicate that it is possible to model Post stroke depression in mouse models and provide interesting findings regarding the possible mechanisms that result in the observed changes, such as sustained depletion of dopamine. Study 2 and 3 investigate the effects of chronic stress on stroke outcome, endothelial function and oxidative stress load. Over the course of four weeks animals were exposed to a chronic stress procedure. Animals of the treatment group were given the I(f)-channel inhibitor ivabradine or glucocorticoid antagonist mifepristone. In both studies stress exposure increased adrenal gland weight, overall corticosterone levels and increased the lesion size after MCAo. Heart rate was both acutely and chronically elevated in the stressed mice. Both studies confirmed that chronic stress exposure impairs endothelium function and down regulates eNOS mRNA levels in the brain and aorta along with increasing the markers of oxidative stress. Both Ivabradine and mifepristone treatment reduced lesion size, normalized endothelial dysfunction and reduced oxidative burden. Both studies indicate that a significant reduction in endothelial function along with markedly increased oxidative stress may be a possible pathway by which stress can facilitate stroke.Neu entdeckte Anzeichen im letzten Jahrzehnt weisen auf einen Zusammenhang von Stress, Depression und Schlaganfällen hin. Klinische Studien berichten, dass ein erheblicher Teil von Schlaganfall-Überlebenden im Anschluss Depressionen entwickeln. Außerdem zeigt eine zunehmende Anzahl von klinischen Fall- Kontroll-Studien einen Zusammenhang zwischen Stress und Schlaganfällen. Experimentelle Studien über die bidirektionale Interaktion zwischen Schlaganfällen und Stress als auch Depression sind ausschlaggebend zum Verständnis der Pathophysiologie von Stress und Depression. Diese Doktorarbeit konzentriert sich auf drei separate Projekte, welche die Natur und Mechanismen dieser Beziehung untersucht. In Studie 1 wurden die Effekte von 30 Minuten linken und rechten Verschluss der mittleren Zerebralarterie (MCAo) von "Angst" und "Depression" ähnlichem Verhalten an einer Maus ausgewertet. Nur die Linke MCAo führte zu chronischer Depression und ängstlichem Verhalten, eine Behandlung mit Citalopram hat den Effekt umgekehrt. 30 Minuten MCAo verursachte eine erhebliche und chronische Reduzierung des striatalen Dopaminspiegels (DA). Striataler DA Verlust wurde zum größten Teil durch die Behandlung mit Citalopram normalisiert. Eine weitere histologische Analyse zeigte einen großen Verlust von dopaminergen Neuronen im ipsilateralen ventralen tegmentalen Bereich, bewertet durch Tyrosinhydroxylase (TH) Immunfärbung und NeuN-Immunfärbung. Der Zellverlust in SN und VTA und die Effekte der MCAo wurden durch die Behandlung mit Citalopram erheblich geschwächt. Unsere Studie zeigt, dass es möglich ist Depression nach einem Schlaganfall in einem Mausmodell zu simulieren und eröffnete uns interessante Erkenntnisse über die möglichen Mechanismen, die in den beobachteten Veränderungen wie anhaltende Erschöpfung von Dopamin entstehen. Studie 2 und 3 untersuchen die Effekte von chronischem Stress auf einen Schlaganfall, Endothelfunktion und oxidative Stressbelastung. Über einen Zeitraum von vier Wochen wurden Tiere einer Prozedur von chronischem Stress ausgesetzt. Den Tieren in der Behandlungsgruppe wurden If-Kanal-Hemmer Ivabradin oder Glucocorticoide Antagonist Mifepristone verabreicht. In beiden Studien erhöhte Stress-Belastung das Gewischt der Nebenniere, den allgemeinen Corticosteron Spiegel und die Läsionsgröße nach der MCAo. Die Herzfrequenz der gestressten Mäuse war akut und chronisch erhöht. Beide Studien bestätigen, dass chronischer Stress die Endothelfunktion beeinträchtigt und die eNOS mRNA Spiegel im Gehirn und der Aorta herunterregelt und gleichzeitig die Marker für oxiditativen Stress erhöht. Die Behandlungen mit Ivabradin und Mifepristone reduzierten beide die Läsionsgröße, normalisierten die Endothelfunktion und reduzierten die oxidative Belastung. Beide Studien weisen darauf hin, dass eine erhebliche Reduzierung der Endothelfunktion zusammen mit deutlich erhöhtem oxidativen Stress zu einem Schlaganfall führen kann

Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice

Polyneuropathy is a frequent and potentially severe side effect of clinical tumor chemotherapy. The goal of this study was to characterize paclitaxel-, cisplatin-, vincristine- and bortezomib-induced neuropathy in C57BL/6 mice with a comparative approach. The phenotype of the animals was evaluated at four time points with behavioral and electrophysiological tests, followed by histology. Treatment protocols used in this study were well tolerated and induced a sensory and predominantly axonal polyneuropathy. Behavioral testing revealed normal motor coordination, whereas all mice receiving verum treatment developed mechanical allodynia and distinct gait alterations. Electrophysiological evaluation showed a significant decrease of the caudal sensory nerve action potential amplitude for all cytostatic agents and a moderate reduction of nerve conduction velocity for cisplatin and paclitaxel. This finding was confirmed by histological analysis of the sciatic nerve which showed predominantly axonal damage: Paclitaxel and vincristine affected mostly large myelinated fibers, bortezomib small myelinated fibers and cisplatin damaged all types of myelinated fibers to a similar degree. Neuropathic symptoms developed faster in paclitaxel and vincristine treated animals compared to cisplatin and bortezomib treatment. The animal models in this study can be used to elucidate pathomechanisms underlying chemotherapy-induced polyneuropathy and for the development of novel therapeutic and preventative strategies