Abstract Background and aims Ischemic stroke causes significant long-term residual deficits that are poorly modelled and understood. Here, we build on our previous concept of “chronic mouse stroke model” to study 6-months´ clinical and tissue changes in young and old mice. Methods Adult male C57BL/6N young (n=31, 3 months old) and old (n= 30, 14 months old) mice were subjected to 1 hour transient middle cerebral artery occlusion with filament (fMCAo), were optimally supported with the “mouse Stroke Unit” protocol to increase survival for long-term studies, and were followed behaviorally (the open-field, tail suspension, cylinder, Corner and the horizontal ladder-rung test) and clinically (temperature, body weight, detailed neurological status) up to 6 months. At 6 months, brains and spinal cords were cryosectioned and studied for local and remote atrophy and cellular changes. Results Old mice had significantly higher acute mortality versus young, primarily due to edema-related herniation. Survivors exhibited prolonged (>1 month) and age-dependent weight loss (p<0.05). Both age groups developed severe focal neurological deficits with partial spontaneous recovery but persistent sensorimotor impairments up to 6 months. Ιpsilesional hemispheric atrophy (approximately 30% in both groups) was accompanied by ventricular enlargement and remote atrophy of sensory cortex, corpus callosum and thalamus, associated with ultra-chronic, region- and age-dependent astrocytic and microglial activation in brain. Spinal cord architecture showed preserved neuronal populations with affected contralateral corticospinal tracts and corresponding chronic residual microglial activation. Conclusions Mice can translationally model core long-term ischemic stroke parameters, revealing multiple chronic targets for drug discovery and treatment in stroke. Conflict of interest CP, IM and AL were supported by Unipharma S.A. with Research Grants. NK: nothing to disclose, APav: nothing to disclose, NZ: nothing to disclose, MG: nothing to disclose, ES-P: nothing to disclose, CPet: nothing to disclose, NP:nothing to disclose, AKot: nothing to disclose, AC: nothing to disclose, AKok: nothing to disclose

Abstract Background and aims Data on the role of thyroid hormone (TH) signaling in stroke have been contradictory between studies. Here, we aim to study the acute effects after TH-agonist/-antagonist treatment in experimental stroke. Methods C57BL/6 mice (n=90) underwent transient focal middle cerebral artery occlusion (fMCAO) for 1 hour, followed clinically up to day 3. Animals were randomized blindly to groups receiving either T3 (at doses of 25 μg/kg or 200 μg/kg), Desethyl-amiodarone (DEA) or Desbutyl-dronedarone (DBD) (at doses 25mg/kg) at reperfusion and on day 1; Sobetirome at 1mg/kg at reperfusion and daily up to day 3; controls received the corresponding vehicle. Histopathological brain studies on cryosections included infarct and hemispheric edema volumetry, as well as astrocytosis and neuronal death using a novel developed FluoroJadeB method for histological detection of penumbra from core and healthy tissue. Results Acute high-dose of T3 did not improve brain edema, infarct volume or neuronal survival compared to controls (p>0.1). Low-dose T3 worsened penumbral neuronal survival (p=0.0086). TH-receptors’ antagonism with DEA induced a clinical worsening with corresponding increased infarct size and neuronal death at day 3 (p<0.05); DBD conferred no effects (p>0.1). TRβ agonism with Sobetirome did not show any clinical effect or penumbral neuronal benefit (p>0.1). Conclusions Acute post-ischemic TH-receptor activation with low- and high- T3 doses or sobetirome confers mixed effects that need further clarification. However, TH-receptors antagonism with DEA (the active metabolite of amiodaron) is detrimental for stroke and should probably be avoided, at least during the first 3 days post-ischemia. Conflict of interest APav and NZ were supported by Tsetis Foundation, CP, IM and AL were supported by Uni-pharma S.A. with Research Grants. AK: nothing to disclose, IT: nothing to disclose, VA: nothing to disclose, EK: nothing to disclose, APap: nothing to disclose, MG: nothing to disclose, APap: nothing to disclose

Abstract Background and aims Stroke quantification in rodents sections remains subjective and labor-intensive. Therefore, we advanced our machine-learning based “StrokeAnalyst-1” (SA1) to the deep-learning based “StrokeAnalyst-2” (SA2). Methods We used mouse brain coronal sections with stroke and compiled one training (n=1200, Nissl-stained) and 4 validation datasets (n=77: MAP2-, 2x Nissl- and TTC-stained). Manual annotation of all datasets served as ground-truth. We built SA2 to integrate deep-learning based image segmentation selecting -after several tests- a visual-transformer (ViT) network. SA2 preprocesses images of brain sections, performs background segmentation (BSG) and corrects for rotation. User-defined prompts to its ViT enable hemisphere separation and stroke detection (SSG) with corresponding output areas. We fine-tuned ViT with increasing numbers of sections and ViT-parametrization. DICE-coefficients for BSG and SSG evaluated segmentation accuracy of SA2 or common available methods for stroke analysis (manual, thresholding and hybrid ImageJ approaches, InfarctSizer and SA1) on validation sets. Results Existing manual and semi-automated ImageJ or InfarctSizer segmentation methods are limited by low accuracy, time-consuming, bias or non-generalizability (DICE range 0.0 - 0.97 for BSG and 0.0 - 0.91 for SSG). SA1 achieved 0.87-0.99 for BSG for all validation sets and 0.0 - 0.92 for SSG, TTC-specific as expected. Untrained ViT models achieved DICE 0.0 - 0.97 for BSG and 0.0 - 0.82 for SSG. Fine-tuning lead to plateaus with consistent DICE of 0.84 - 0.97 for BSG and variable 0.36 - 0.89 for SSG, in all validation sets, with a processing time of 2 seconds. Conclusions SA2 enables automated, ultra-fast, unbiased and staining-independent detection of stroke on brain sections. Conflict of interest All authors: nothing to disclose

The mouse is the species most commonly used in preclinical research, but protein analytics of murine cerebrospinal fluid (CSF) remains challenging because of low sample volumes (often <10 μl) and frequent contaminations with blood. We developed an improved CSF sampling method that allows routine collection of larger volumes (20-30 μl) of pure CSF from individual mice, enabling multiple protein analytical assays from a single sample. Based on cell counts and hemoglobin ELISAs, we provide an easy quality control workflow for obtaining cell- and blood-free murine CSF. Through mass spectrometry-based proteomics using an absolutely quantified external standard, we estimated concentrations for hundreds of mouse CSF proteins. While repeated CSF sampling from the same mouse was possible, it induced CSF proteome changes. Applying the improved method, we found that the mouse CSF proteome remains largely stable over time in wild-type mice, but that amyloid pathology in the 5xFAD mouse model of Alzheimer's disease massively changes the CSF proteome. Neurofilament light chain and TREM2, markers of neurodegeneration and activated microglia, respectively, were strongly upregulated and validated using immunoassays. In conclusion, our refined murine CSF collection method overcomes previous limitations, allowing multiple quantitative protein analyses for applications in biomedicine.

The filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with intravascular reperfusion/recanalization. However, it lacks alignment with current clinical advances for stroke care (e.g., Stroke Units), usually employs subjective or vague neurological scoring among laboratories, and exhibits high acute-phase mortality. Here, we address these limitations with validated video-guided protocols. We present the mouse Stroke Unit (mSU) protocol with instructional videos and a decision algorithm (Risk Stratification Score), bridging the gap between clinical and mouse stroke modeling. To increase accuracy and sensitivity of stroke neurological scoring, we present for the first time a video-standardized format of the focal Experimental Stroke Scale (fESS) and prove its value up to 6 months post-stroke. Additionally, protocols for mice Ladder-rung test, as well as the known Cylinder test, for unbiased, quantitative assessment of limbs´ motor function are presented. Results highlight mSU's translational efficacy. Focal ESS (fESS) excels over other known scales in detecting focal stroke deficits, capturing recovery, and maintaining sensitivity for up to 6 months post-stroke. Ladder-rung and Cylinder tests objectively quantify and monitor fore- and hind-limb motor deficits, long-term. In summary, integrating mSU, fESS, and motor function tests provides a robust framework for clinically relevant stroke investigations. Our protocols improve the translational value in mouse stroke research.

Mice are the most commonly used preclinical animal model, but protein analytics of murine cerebrospinal fluid (CSF) remains challenging because of low CSF volume (often <10 µl) and frequent blood contaminations. We developed an improved CSF sampling method that allows routine collection of increased volumes (20-30 µl) of pure CSF from individual mice, enabling multiple protein analytical assays from a single sample. Based on cell counts and hemoglobin ELISAs, we provide an easy quality control workflow for obtaining cell- and blood-free murine CSF. Through mass spectrometry-based proteomics using an absolutely quantified external standard, we estimated concentrations for hundreds of mouse CSF proteins. While repeated CSF sampling from the same mouse was possible, it induced CSF proteome changes. Applying the improved method, we found that the mouse CSF proteome remains largely stable over time in wild-type mice, but that amyloid pathology in the 5xFAD mouse model of Alzheimer’s disease massively changes the CSF proteome. Neurofilament light chain and TREM2, markers of neurodegeneration and activated microglia, respectively, were strongly upregulated and validated using immunoassays. In conclusion, our refined murine CSF collection method overcomes previous limitations, allowing multiple quantitative protein analyses for applications in biomedicine. ### Competing Interest Statement The authors have declared no competing interest.

BackgroundSince the first report of fatal Borna virus-1 (BoDV-1) encephalitis in 2018, cases gradually increased. There is a lack of diagnostic algorithm, and there is no effective treatment so far.Case presentationWe report an acute BoDV-1 encephalitis in a 77-year-old female with flu-like onset, rapid progression to word-finding difficulties, personality changes, global disorientation, diffuse cognitive slowness, and gait ataxia and further deterioration with fever, meningism, severe hyponatremia, epileptic seizures, cognitive decline, and focal cortical and cerebellar symptoms/signs. The extensive diagnostic workup (cerebrovascular fluid, serum, and MRI) for (meningo-)encephalitis was negative for known causes. Our empirical common antiviral, antimicrobial, and immunosuppressive treatment efforts failed. The patient fell into coma 5 days after admission, lost all brainstem reflexes on day 18, remained fully dependent on invasive mechanical ventilation thereafter and died on day 42. Brain and spinal cord autopsy confirmed an extensive, diffuse, and severe non-purulent, lymphocytic sclerosing panencephalomyelitis due to BoDV-1, affecting neocortical, subcortical, cerebellar, neurohypophysis, and spinal cord areas. Along with our case, we critically reviewed all reported BoDV-1 encephalitis cases.ConclusionThe diagnosis of acute BoDV-1 encephalitis is challenging and delayed, while it progresses to fatal. In this study, we list all tried and failed treatments so far for future reference and propose a diagnostic algorithm for prompt suspicion and diagnosis.

Abstract Background The first case of fatal Borna Virus-1 encephalitis (BoDV-1) was reported in 2018. Here, we report another fatal case of BoDV-1 encephalitis with early severe hyponatremia, indicative of neurohypophysial dysfunction. Case presentation: A 77-year-old female living in southern Germany was admitted to hospital in 2020 due to rapidly progressing word-finding difficulties, personality changes, global disorientation, diffuse cognitive slowness, and gait ataxia, initially without fever. After a rapid deterioration with fever, gait instability and ataxia, rapid cognitive decline, meningism, epileptic seizures, aphasia, and signs of latent right hemiparesis, the suspicion of a (meningo-)encephalitis was set. Furthermore, an unexplained, severe hyponatremia had been present since admission. Laboratory workup in cerebrovascular fluid (CSF) and serum as well as brain imaging was negative. Despite extensive empirical antiviral, antimicrobial, and immunosuppressive treatment efforts, the patient fell into coma (day 5), lost all brainstem functions (day 18), and remained fully dependent on invasive mechanical ventilation. Finally, she clinically developed a status of brain death and died 42 days after initial admission. Brain autopsy confirmed an extensive, diffuse, and severe affection of neocortical, subcortical and cerebellar structures as well as the neurohypophysis due to infection with BoDV-1. In light of the autopsy results, the hyponatremia could imply an early basal brain involvement, which could narrow down the initial differential diagnosis. Conclusion The diagnosis of BoDV-1 encephalitis remains clinically challenging. The disease progresses quickly to irreversible brain damage. An early, unexplained, hyponatremia in the presence of severe and rapidly evolving encephalitis may narrow down the diagnosis.

Heart transplantation remains the conventional treatment in end-stage heart failure, with static cold storage (SCS) being the standard technique used for donor preservation. Nevertheless, prolonged cold ischemic storage is associated with the increased risk of early graft dysfunction attributed to residual ischemia, reperfusion, and rewarming damage. In addition, the demand for the use of marginal grafts requires the development of new methods for organ preservation and repair. In this review, we focus on current knowledge and novel methods of donor preservation in heart transplantation. Hypothermic or normothermic machine perfusion may be a promising novel method of donor preservation based on the administration of cardioprotective agents. Machine perfusion seems to be comparable to cold cardioplegia regarding donor preservation and allows potential repair treatments to be employed and the assessment of graft function before implantation. It is also a promising platform for using marginal organs and increasing donor pool. New pharmacological cardiac repair treatments, as well as cardioprotective interventions have emerged and could allow for the optimization of this modality, making it more practical and cost-effective for the real world of transplantation. Recently, the use of triiodothyronine during normothermic perfusion has shown a favorable profile on cardiac function and microvascular dysfunction, likely by suppressing pro-apoptotic signaling and increasing the expression of cardioprotective molecules.

The present study investigated the effects of triiodothyronine (T3) administration in ex vivo model of rat heart normothermic perfusion. T3 is cardioprotective and has the potential to repair the injured myocardium. Isolated hearts were subjected to normothermic perfusion (NP) with Krebs-Henseleit for 4 h with vehicle (NP) or 60 nM T3 in the perfusate (NP + T3). Left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), perfusion pressure (PP) and percentage of change of these parameters from the baseline values were measured. Activation of stress induced kinase signaling was assessed in tissue samples. Baseline parameters were similar between groups. LVEDP was increased from the baseline by 13% (70) for NP + T3 vs. 139% (160) for NP group, p = 0.048. LVDP was reduced by 18.2% (5) for NP + T3 vs. 25.3% (19) for NP group, p = 0.01. PP was increased by 41% (19) for NP + T3 vs.91% (56) for NP group, p = 0.024. T3 increased activation of pro-survival Akt by 1.85 fold (p = 0.047) and AMPK by 2.25 fold (p = 0.01) and reduced activation of pro-apoptotic p38 MAPK by 3fold (p = 0.04) and p54 JNK by 4.0 fold (p = 0.04). Administration of T3 in normothermic perfusion had favorable effects on cardiac function and perfusion pressure and switched death to pro-survival kinase signaling.

Small animals stroke models have widely been used to study the mechanisms of ischemic brain damage in controllable experimental settings. The evaluation of stroke lesions mainly relies on visual inspection of tissue samples collected after brain sectioning, slice staining and scanning, a procedure that is highly subjective and prone to human error. In this study we developed a machine-learning based methodology for automatic segmentation of lesions in mouse brain tissue samples, stained with Triphenyltetrazolium chloride (2% TTC). Our approach relies on the creation of a statistical mouse brain atlas of healthy TTC slices that was lacking in the literature. For this purpose we applied tissue clustering and Markov Random Fields (MRF) for brain tissue detection followed by deformable image registration for spatial normalization. The obtained statistical atlas is then exploited by outlier detection techniques and Random Forest classification to extract lesion probability maps in new slices. The good agreement between our segmentation results and expert-based lesion delineation on 12 mouse brains highlights the potential of the proposed approach to automate stroke volumetry analysis, thereby contributing to increased translational capacity of experimental stroke.

An unbiased, automated and reliable method for analysis of brain lesions in tissue after ischemic stroke is missing. Manual infarct volumetry or by threshold-based semi-automated approaches is laborious, and biased to human error or biased by many false -positive and -negative data, respectively. Thereby, we developed a novel machine learning, atlas-based method for fully automated stroke analysis in mouse brain slices stained with 2% Triphenyltetrazolium-chloride (2% TTC), named "StrokeAnalyst", which runs on a user-friendly graphical interface. StrokeAnalyst registers subject images on a common spatial domain (a novel mouse TTC- brain atlas of 80 average mathematical images), calculates pixel-based, tissue-intensity statistics (z-scores), applies outlier-detection and machine learning (Random-Forest) models to increase accuracy of lesion detection, and produces volumetry data and detailed neuroanatomical information per lesion. We validated StrokeAnalyst in two separate experimental sets using the filament stroke model. StrokeAnalyst detects stroke lesions in a rater-independent and reproducible way, correctly detects hemispheric volumes even in presence of post-stroke edema and significantly minimizes false-positive errors compared to threshold-based approaches (false-positive rate 1.2-2.3%, p < 0.05). It can process scanner-acquired, and even smartphone-captured or pdf-retrieved images. Overall, StrokeAnalyst surpasses all previous TTC-volumetry approaches and increases quality, reproducibility and reliability of stroke detection in relevant preclinical models.

Tissue hypoxia occurs in various conditions such as myocardial or brain ischemia and infarction, sepsis, and trauma, and induces cellular damage and tissue remodeling with recapitulation of fetal-like reprogramming, which eventually results in organ failure. Analogies seem to exist between the damaged hypoxic and developing organs, indicating that a regulatory network which drives embryonic organ development may control aspects of heart (or tissue) repair. In this context, thyroid hormone (TH), which is a critical regulator of organ maturation, physiologic angiogenesis, and mitochondrial biogenesis during fetal development, may be of important physiological relevance upon stress (hypoxia)-induced fetal reprogramming. TH signaling has been implicated in hypoxic tissue remodeling after myocardial infarction and T3 prevents remodeling of the postinfarcted heart. Similarly, preliminary experimental evidence suggests that T3 can prevent early tissue hypoxia during sepsis with important physiological consequences. Thus, based on common pathways between different paradigms, we propose a possible role of TH in tissue hypoxia after sepsis with the potential to reduce secondary organ failure.

Why can we still not translate preclinical research to clinical treatments for acute strokes? Despite > 1000 successful preclinical studies, drugs, and concepts for acute stroke, only two have reached clinical translation. This is the translational block. Yet, we continue to routinely model strokes using almost the same concepts we have used for over 30 years. Methodological improvements and criteria from the last decade have shed some light but have not solved the problem. In this conceptual analysis, we review the current status and reappraise it by thinking “out-of-the-box” and over the edges. As such, we query why other scientific fields have also faced the same translational failures, to find common denominators. In parallel, we query how migraine, multiple sclerosis, and hypothermia in hypoxic encephalopathy have achieved significant translation successes. Should we view ischemic stroke as a “chronic, relapsing, vascular” disease, then secondary prevention strategies are also a successful translation. Finally, based on the lessons learned, we propose how stroke should be modeled, and how preclinical and clinical scientists, editors, grant reviewers, and industry should reconsider their routine way of conducting research. Translational success for stroke treatments may eventually require a bold change with solutions that are outside of the box.

CO2-reactivity and neurovascular coupling are sequentially lost within the first 24 h after subarachnoid hemorrhage (SAH). Whether and when these impairments recover is not known. Therefore, we investigated the reactivity of pial and intraparenchymal vessels by in vivo two-photon microscopy one month after experimental SAH. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. One month later, cerebral blood flow following CO2-challenge and forepaw stimulation was assessed by laser Doppler fluxmetry. Diameters of pial and intraparenchymal arterioles were quantified by in vivo two-photon microscopy. One month after SAH, pial and parenchymal vessels dilated in response to CO2. Neurovascular coupling was almost completely absent after SAH: vessel diameter did not change upon forepaw stimulation compared to a 20% increase in sham-operated mice. The current results demonstrate that neurovascular function differentially recovers after SAH: while CO2-reactivity normalizes within one month after SAH, neurovascular coupling is still absent. These findings show an acute and persistent loss of neurovascular coupling after SAH that may serve as a link between early brain injury and delayed cerebral ischemia, two distinct pathophysiological phenomena after SAH that were so far believed not to be directly related.

We report the novel synthesis of cyclic PLP139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H147 replaces W144 and F151-K150 replace H147 as TCR contacts, which may explain the difference on each peptide's response.

EAE is induced to susceptible mice using linear peptides of myelin proteins of the central nervous system. Specific peptide motifs within the peptide-binding groove of the MHC peptide-complex determines the affinity of the peptide in each animal and the consequent T-cell receptor recognition and activation of the cell. Altered peptide ligand (APL) vaccination is a novel approach based on an effort to induce T-cell tolerance or alter cytokine profile from pro-inflammatory to anti-inflammatory. In the present study we synthesized the MOG35-55 peptide and altered its 3-dimensional conformation to make it a cyclic one (c-MOG35-55). EAE was induced in C57BL/6 mice and pathology was studied on acute and chronic phase of the disease. Our data indicates that c-MOG35-55 peptide alone induces a mild transient acute phase without chronic axonopathy. Administration of the c-MOG35-55 peptide at a 1:1 ratio during disease induction significantly ameliorates clinical disease and underlying pathology, such as demyelination and axonopathy in the acute and chronic phases. Binding and structural studies revealed milder interactions between the c-MOG35-55 and mouse or human MHC class II alleles (H2-IAb and HLA-DR2). Collectively, we provide data supporting for the first time the concept that the cyclic modification of an established encephalitogenic peptide ameliorates the clinical outcomes and underlying pathological processes of EAE. Such a cyclic modification of linear peptides could provide a novel treatment approach for future, patient-selective, immunomodulative treatments of multiple sclerosis.

Objective: To assess the potential effect of variants in genes encoding molecules that are implicated in leukocyte trafficking into the CNS on the clinical phenotype of multiple sclerosis (MS). Methods: A total of 389 Greek MS cases and 336 controls were recruited in 3 MS centers from Cyprus and Greece. We genotyped 147 tagging single nucleotide polymorphisms (SNPs) in 9 genes encoding for P-selectin (SELP), integrins (ITGA4, ITGB1, and ITGB7), adhesion molecules (ICAM1, VCAM1, and MADCAM1), fibronectin 1 (FN1), and osteopontin (SPP1) involved in lymphocyte adhesion and trafficking into the CNS. Clinical end points of the study were age at MS onset and MS severity as measured by the Multiple Sclerosis Severity Score. Permutation testing was applied to all analyses. Results: SNPs rs6721763 of the ITGA4 and rs6532040 of the SPP1 were found to significantly influence disease severity (permutation p values: 3.00e-06 and 0.009884, respectively). SNP rs1250249 of the FN1 had a dose-dependent effect on age at disease onset (permutation p value: 0.0002). Conclusions: This study provides evidence implicating variants encoding adhesion molecules, responsible for lymphocyte adhesion and trafficking within the CNS, as modifiers of MS disease severity. These genetic biomarkers, which can be available at the time of diagnosis, may be used to assess the biological aggressiveness of the disease and thus guide decisions on treatment.