Effects of T3 Administration on Ex Vivo Rat Hearts Subjected to Normothermic Perfusion: Therapeutic Implications in Donor Heart Preservation and Repair.

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.

Donor Heart Preservation: Current Knowledge and the New Era of Machine Perfusion.

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.

Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis.

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.

Cyclic MOG35-55 ameliorates clinical and neuropathological features of experimental autoimmune encephalomyelitis.

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.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

The Impact of Methylprednisolone Pulses during Relapses of Multiple Sclerosis on the Kinetics of Anti-Interferon-Beta Antibodies.

BACKGROUND/AIMS: We assessed the, hitherto unknown, impact of intravenous methylprednisolone (ivMP) pulses during relapses of multiple sclerosis (MS) on the kinetics of anti-interferon-beta neutralizing antibodies (Nabs) and binding antibodies (Babs). METHODS: Babs (ELISA) and Nabs (antiviral cytopathic effect assay) titers were evaluated before, immediately after and at 1 month following ivMP in 60 MS patients. RESULTS: ivMP reduces Nabs and Babs titers for at least 1 month. Baseline titers determine Nabs and Babs seronegativity at the end of ivMP. Clinical response to ivMP tends to be better in Nabs(+) patients. CONCLUSION: Sampling for Nabs/Babs should be avoided during or shortly after ivMP to avoid transient positive or negative results that may obscure the decision to switch treatment.

Connexin43 and connexin47 alterations after neural precursor cells transplantation in experimental autoimmune encephalomyelitis.

Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune-modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG-induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post-induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP(+) NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP(+) cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP(+) NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue.

Case report: Fatal Borna virus encephalitis manifesting with basal brain and brainstem symptoms.

Background: Since 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 presentation: We 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. Conclusion: The 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.

Machine learning based analysis of stroke lesions on mouse tissue sections.

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.

Translational Block in Stroke: A Constructive and "Out-of-the-Box" Reappraisal.

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.

Effects of Thyroid Hormone on Tissue Hypoxia: Relevance to Sepsis Therapy.

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.

Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury.

Despite efforts aimed at developing novel therapeutics for traumatic brain injury (TBI), no specific pharmacological agent is currently clinically available. Here, we show that the pan-histone deacetylase (HDAC) inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h postinjury. Using a well-characterized, clinically relevant mouse model of closed head injury (CHI), we demonstrate that a single dose of ITF2357 administered 24 h postinjury improves neurobehavioral recovery from d 6 up to 14 d postinjury (improved neurological score vs. vehicle; P< or =0.05), and that this functional benefit is accompanied by decreased neuronal degeneration, reduced lesion volume (22% reduction vs. vehicle; P< or =0.01), and is preceded by increased acetylated histone H3 levels and attenuation of injury-induced decreases in cytoprotective heat-shock protein 70 kDa and phosphorylated Akt. Moreover, reduced glial accumulation and activation were observed 3 d postinjury, and total p53 levels at the area of injury and caspase-3 immunoreactivity within microglia/macrophages at the trauma area were elevated, suggesting enhanced clearance of these cells via apoptosis following treatment. Hence, our findings underscore the relevance of HDAC inhibitors for ameliorating trauma-induced functional deficits and warrant consideration of applying ITF2357 for this indication.

Long-term impairment of neurovascular coupling following experimental subarachnoid hemorrhage.

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.

Altered peptide ligands of myelin basic protein ( MBP87-99 ) conjugated to reduced mannan modulate immune responses in mice.

Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87-99 (MBP(87-99)), an immunodominant peptide epitope identified in MS. Mutations of residues K(91) and P(96), known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R(91), A(96)]MBP(87-99) and [A(91), A(96)]MBP(87-99). Immunization of mice with these altered peptide ligands emulsified in complete Freund's adjuvant induced both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) responses compared with only IFN-gamma responses induced to the native MBP(87-99) peptide. It was of interest that [R(91), A(96)]MBP(87-99) conjugated to reduced mannan induced 70% less IFN-gamma compared with the native MBP(87-99) peptide. However, [A(91), A(96)]MBP(87-99) conjugated to reduced mannan did not induce IFN-gamma-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A(91), A(96)]MBP(87-99) peptide conjugated to reduced mannan did not cross-react with the native MBP(87-99) peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-A(s), novel interactions were noted. It is clear that the double-mutant peptide analogue [A(91), A(96)]MBP(87-99) conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS.

Effectiveness of a new modified intraluminal suture for temporary middle cerebral artery occlusion in rats of various weight.

Intraluminal temporary middle cerebral artery occlusion (MCAO) is a common model of ischemic stroke in the rat with significant, suture and weight-dependent variability along with increased risk of subarachnoid haemorrhage (SAH). Our purpose was to increase reproducibility and decrease SAH using a modification of the Koizumi suture. We compared a Koizumi 5/0 Ethilon poly-l-lysine-coated suture (s-2, group B) to an identical, uncoated one (s-1, group A) and the Belayev's 3/0 suture (s-3, group C), in the 2-h MCAO model in Wistar rats of varying weight (310-527 g). Assessment included successful infarction rates, the modified neurological stroke scale (mNSS), a modified Bederson's scale (mBS), the grid-walking test (GWT), infarction volume (with rostrocaudal subanalysis and analysis of cortical/striatal involvement) and hemispheric edema. The s-2 suture increased the successful MCAO from 61.1% and 66.6% (groups A and C) to 97.5% in group B and induced a more severe clinical stroke (P<0.05) irrespective of animal's weight, with no incidence of SAH. Infarction volume and ipsilateral hemispheric edema significantly (P<0.05) increased and well correlated with the mNSS (P

Inadequate food and water intake determine mortality following stroke in mice.

Experimental stroke models producing clinically relevant functional deficits are often associated with high mortality. Because the mechanisms that underlie post-stroke mortality are largely unknown, results obtained using these models are often difficult to interpret, thereby limiting their translational potential. Given that specific forms of post-stroke care reduce mortality in patients, we hypothesized that inadequate food and water intake may underlie mortality following experimental stroke. C57BL/6 mice were subjected to 1 h of intraluminal filament middle cerebral artery occlusion. Nutritional support beginning on the second day after filament middle cerebral artery occlusion reduced the 14-day mortality rate from 59% to 15%. The surviving mice in the post-stroke support group had the same infarct size as non-surviving control mice, suggesting that post-stroke care was not neuroprotective and that inadequate food and/or water intake are the main reasons for filament middle cerebral artery occlusion-induced mortality. This notion was supported by the presence of significant hypoglycemia, ketonemia, and dehydration in control mice. Taken together, these data suggest that post-filament middle cerebral artery occlusion mortality in mice is not primarily caused by ischemic brain damage, but secondarily by inadequate food and/or water intake. Thus, providing nutritional support following filament middle cerebral artery occlusion greatly minimizes mortality bias and allows the study of long-term morphological and functional sequelae of stroke in mice.

Gene variants of adhesion molecules act as modifiers of disease severity in MS.

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.